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Differential expression and processing of transforming growth factor beta induced protein (TGFBIp) in the normal human cornea during postnatal development and aging
Abstract
Transforming growth factor beta induced protein (TGFBIp, also named keratoepithelin) is an extracellular matrix protein abundant in the cornea. The purpose of this study was to determine the expression and processing of TGFBIp in the normal human cornea during postnatal development and aging. TGFBIp in corneas from individuals ranging from six months to 86 years of age was detected and quantified by immunoblotting. The level of TGFBIp in the cornea increases about 30% between 6 and 14 years of age, and adult corneas contain 0.7-0.8 microg TGFBIp per mg wet tissue. Two-dimensional (2-D) immunoblots of the corneal extracts showed a characteristic "zig-zag" pattern formed by different lower-molecular mass TGFBIp isoforms (30-60 kDa). However, the relative abundance of the different isoforms was different between infant corneas (<1 year) and the child/adult corneas (>6 years). Matrix-assisted laser desorption/ionization-time-of-flight mass spectrometry (MALDI-TOF MS) data of TGFBIp isoforms separated on large 2-D gels show that TGFBIp is proteolytically processed from the N-terminus. This observation was supported by in silico 2-D gel electrophoresis showing that sequential proteolytical trimming events from the N-terminus of mature TGFBIp generate TGFBIp isoforms which form a similar "zig-zag" pattern when separated by 2-D polyacrylamide gel electrophoresis (PAGE). This study shows that in humans TGFBIp is more abundant in mature corneas than in the developing cornea and that the processing of TGFBIp changes during postnatal development of the cornea. In addition, TGFBIp appears to be degraded in a highly orchestrated manner in the normal human cornea with the resulting C-terminal fragments being retained in the cornea. The age-related changes in the expression and processing of corneal TGFBIp suggests that TGFBIp may play a role in the postnatal development and maturation of the cornea. Furthermore, these observations may be relevant to the age at which mutant TGFBIp deposits in the cornea in those dystrophies caused by mutations in the transforming growth factor beta induced gene (TGFBI) as well as the mechanisms of corneal protein deposition.
... is currently unknown whether this proteolytic event occurs before or after secretion, but its proximity to the RGD motif indicates that it participates in regulating the cell binding properties of TGFBIp (Andersen et al., 2004). Analysis of corneal extracts by two-dimensional gel electrophoresis followed by immunoblotting revealed several TGFBIp isoforms with a molecular weight in the 30-60 kDa range and an isoelectric point between 5.5 and 6.2, forming a "zigzag" pattern (Karring et al., 2010). Mass spectrometry analysis revealed that all the isoforms were N-terminal truncated versions of the mature protein lacking 210-375 residues corresponding to the CROPT, FAS1-1, and FAS1-2 domains. ...
... Tissues were handled and analyzed as described in (Poulsen et al., 2019). posttranslational N-terminal trimming of TGFBIp, of which the smallest degradation products span the FAS1-4 region of TGFBIp (Karring et al., 2010). Therefore, the full-length protein and the FAS1-4 domain appear to be good model systems for studying TGFBI-linked corneal dystrophies, as they are the major start and end products in the cornea. ...
Transforming growth factor-β-induced protein (TGFBIp), an extracellular matrix protein, is the second most abundant protein in the corneal stroma. In this review, we summarize the current knowledge concerning the expression, molecular structure, binding partners, and functions of human TGFBIp. To date, 74 mutations in the transforming growth factor-β-induced gene (TGFBI) are associated with amyloid and amorphous protein deposition in TGFBI-linked corneal dystrophies. We discuss the current understanding of the biochemical mechanisms of TGFBI-linked corneal dystrophies and propose that mutations leading to granular corneal dystrophy (GCD) decrease the solubility of TGFBIp and affect the interactions between TGFBIp and components of the corneal stroma, whereas mutations associated with lattice corneal dystrophy (LCD) lead to a destabilization of the protein that disrupts proteolytic turnover, especially by the serine protease HtrA1. Future research should focus on TGFBIp function in the cornea, confirmation of the biochemical mechanisms in vivo, and the development of disease models. Future therapies for TGFBI-linked corneal dystrophies might include topical agents that regulate protein aggregation or gene therapy that targets the mutant allele by CRISPR/Cas9 technology.
... However, recent studies of normal and mutant TGFBIp variants from human corneas suggest that proteolytic degradation of TGFBIp plays a significant role in the pathobiology of the TGFBI-linked cornea dystrophies. In the normal human cornea, TGFBIp is proteolytically processed in a highly orchestrated manner and the observed fragments strongly suggest that proteolysis of the FAS1-4 domain (residues Val505-Leu632) plays a key role in the turnover of TGFBIp in the cornea [10]. However, in TGFBI-linked corneal dystrophies the processing of TGFBIp is altered and different mutations are associated with specific changes in the degradation pattern [11] and proteolytic cleavage sites [12]. ...
... Combining the present data with previous studies may provide important indications of the biological significance in corneal dystrophy; Karring et al. [10] showed that the C-terminal part of TGFBIp (including the FAS1-4 domain) is the last to be turned over in the normal human cornea. Whether this is due to a very specific turnover of TGFBIp, or that the Cterminal region is relatively more resistant to proteolysis in vivo compared to the other TGFBIp domains remains to be elucidated. ...
Hereditary mutations in the transforming growth factor beta induced (TGFBI) gene cause phenotypically distinct corneal dystrophies characterized by protein deposition in cornea. We show here that the Arg555Trp mutant of the fourth fasciclin 1 (FAS1-4) domain of the protein (TGFBIp/keratoepithelin/βig-h3), associated with granular corneal dystrophy type 1, is significantly less susceptible to proteolysis by thermolysin and trypsin than the WT domain. High-resolution liquid-state NMR of the WT and Arg555Trp mutant FAS1-4 domains revealed very similar structures except for the region around position 555. The Arg555Trp substitution causes Trp555 to be buried in an otherwise empty hydrophobic cavity of the FAS1-4 domain. The first thermolysin cleavage in the core of the FAS1-4 domain occurs on the N-terminal side of Leu558 adjacent to the Arg555 mutation. MD simulations indicated that the C-terminal end of helix α3' containing this cleavage site is less flexible in the mutant domain, explaining the observed proteolytic resistance. This structural change also alters the electrostatic properties, which may explain increased propensity of the mutant to aggregate in vitro with 2,2,2-trifluoroethanol. Based on our results we propose that the Arg555Trp mutation disrupts the normal degradation/turnover of corneal TGFBIp, leading to accumulation and increased propensity to aggregate through electrostatic interactions.
... Some aggregates of mut-TGFBI are likely wrapped and form large multivesicular bodies that are trafficked to the plasma membrane, where, upon fusion with the plasma membrane, they release their contents into the extracellular space via the exosome pathways [30]. Mut-TGFBI in ECM is hydrolyzed by a series of proteolytic enzymes, such as MMPs, aminopeptidase [31], and HTRA1. Some TGFBI gene-related corneal dystrophies are caused by abnormities of the extracellular proteolytic process. ...
Thiel-Behnke corneal dystrophy (TBCD) is an epithelial-stromal TGFBI dystrophy caused by mutations in the TGFBI (transforming growth factor beta induced) gene, though the underlying mechanisms and pathogenesis of TBCD are still obscure. The study identifies a novel mutation in the TGFBI gene (p.Gly623_His626del) in a TBCD pedigree. Characteristics of the typical vacuole formation, irregular corneal epithelial thickening and thinning, deposition of eosinophilic substances beneath the epithelium, and involvement of the anterior stroma were observed in this pedigree via transmission electron microscopy (TEM) and histological staining. Tgfbi-p.Gly623_Tyr626del mouse models of TBCD were subsequently generated via CRISPR/Cas9 technology, and the above characteristics were further verified via TEM and histological staining. Lysosomal dysfunction and downregulation of differential expression protein CTSD (cathepsin D) were observed using LysoTracker Green DND-26 and proteomic analysis, respectively. Hence, lysosomal dysfunction probably leads to autophagic flux obstruction in TBCD; this was supported by enhanced LC3-II and SQSTM1 levels and decreased CTSD. TFEB (transcription factor EB) was prominently decreased in TBCD corneal fibroblasts and administration of ATP-competitive MTOR inhibitor torin 1 reversed this decline, resulting in the degradation of accumulated mut-TGFBI (mutant TGFBI protein) via the ameliorative lysosomal function and autophagic flux owing to elevated TFEB activity as measured by western blot, confocal microscopy, and flow cytometry. Transfected HEK 293 cells overexpressing human full-length WT-TGFBI and mut-TGFBI were generated to further verify the results obtained in human corneal fibroblasts. Amelioration of lysosome dysfunction may therefore have therapeutic efficacy in the treatment of TBCD.
... Transforming growth factor β-induced protein (TGFBIp), also known as Keratoepithelin or BIGH3/βigh3, is an extracellular matrix (ECM) protein and the second most abundant protein in the human cornea [4,13,14]. The mutant protein is highly prone to aggregation and is proteolytically processed differently compared to the wild type (WT) protein that may lead to protein aggregation and deposition in the cornea [15,16]. ...
Stromal corneal dystrophies are a group of hereditary disorders caused by mutations in the TGFBI gene. The mutant TGFBIp is prone to protein aggregation and the mutant protein gets deposited in the cornea, leading to severe visual impairment. The mutations lead to a corneal specific protein aggregation suggesting the involvement of tissue-specific factors. The exact molecular mechanism of the process of tissue-specific protein aggregation remains to be elucidated. Differential proteolysis of mutant TGFBIp is a critical component of the disease pathology. The differential proteolysis gives rise to shorter peptides that are highly aggregation-prone and initiate the aggregation cascade. Analyzing the proteolytic processing of the different TGFBIp mutant may provide insight to aid in understanding the amyloid aggregation mechanism. We developed a MALDI-MSI methodology to identify expression and spatial localization of TGFBIp peptides in the cornea. Corneal tissue samples were collected from both control and dystrophic patients (with 2 different mutations), embedded in OCT and sectioned. The sections were trypsin digested and subjected to mass spectrometry imaging using a targeted approach to detect TGFBIp. MALDI-MSI identified peptides from TGFBIp that co-localized with the amyloid corneal deposits. In addition to the relative abundance data, the specific location of the peptides across the corneal sections as molecular signatures was also identified. Spatial distribution and intensity of the TGFBIp peptides showed differences between diseased and control models but also between the two LCD phenotypes. The TGFBIp peptide with m/z of 787.474 and m/z of 1179.579 showed increased expression in both LCD mutants compared to the controls. The peptide with m/z of 929.5 showed increased expression in the LCD phenotype with H626R mutation while the peptide with m/z of 1315.802 was abundant in the sample with R124C mutation. This initial report of 2D spatial protein signature and localization of TGFBIp may be expanded to other mutations to understand the proteolytic patterns of TGFBIp in different mutations.
... TGFBIp consists of a secretory signal peptide, a domain rich in cysteine residues, a cell attachment tripeptide Arg-Gly-Asp (RGD) signal residue, and four Fasciclin 1 (FAS-1) domains 9,10 . The TGFBI gene has 74 mutations reported to date 11 , and the mutant protein is associated with modified protein stability, altered proteolytic processing, and deposition of insoluble aggregates in various layers of the cornea [12][13][14][15][16] . The aggregation and deposition of TGFBIp display different clinical phenotypes; the deposits range from amyloidogenic structures to amorphous granular deposits, or a combination of both 17 . ...
Protein aggregation has been one of the leading triggers of various disease conditions, such as Alzheimer’s, Parkinson’s and other amyloidosis. TGFBI-associated corneal dystrophies are protein aggregation disorders in which the mutant TGFBIp aggregates and accumulates in the cornea, leading to a reduction in visual acuity and blindness in severe cases. Currently, the only therapy available is invasive and there is a known recurrence after surgery. In this study, we tested the inhibitory and amyloid dissociation properties of four osmolytes in an in-vitro TGFBI peptide aggregation model. The 23-amino acid long peptide (TGFBIp 611–633 with the mutation c.623 G>R) from the 4th FAS-1 domain of TGFBIp that rapidly forms amyloid fibrils was used in the study. Several biophysical methods like Thioflavin T (ThT) fluorescence, Circular Dichroism (CD), fluorescence microscopy and Transmission electron microscopy (TEM) were used to study the inhibitory and amyloid disaggregation properties of the four osmolytes (Betaine, Raffinose, Sarcosine, and Taurine). The osmolytes were effective in both inhibiting and disaggregating the amyloid fibrils derived from TGFBIp 611–633 c.623 G>R peptide. The osmolytes did not have an adverse toxic effect on cultured human corneal fibroblast cells and could potentially be a useful therapeutic strategy for patients with TGFBIp corneal dystrophies.
... Among its body locations, TGFBIp is best known for being a major component of the human cornea (Escribano et al., 1994;Klintworth et al., 1994), where it is associated with post-natal corneal development and maturation (Karring et al., 2010). TGFBIp is normally produced by epithelial cells and keratocytes of the corneal epithelium (Han et al., 2016), and it may contribute to corneal layer adhesion (Escribano et al., 1994). ...
A major cause of visual impairment, corneal dystrophies result from accumulation of protein deposits in the cornea. One of the proteins involved is transforming growth factor β-induced protein (TGFBIp), an extracellular matrix component that interacts with integrins but also produces corneal deposits when mutated. Human TGFBIp is a multi-domain 683-residue protein, which contains one CROPT domain and four FAS1 domains. Its structure spans ∼120 Å and reveals that vicinal domains FAS1-1/FAS1-2 and FAS1-3/FAS1-4 tightly interact in an equivalent manner. The FAS1 domains are sandwiches of two orthogonal four-stranded β sheets decorated with two three-helix insertions. The N-terminal FAS1 dimer forms a compact moiety with the structurally novel CROPT domain, which is a five-stranded all-β cysteine-knot solely found in TGFBIp and periostin. The overall TGFBIp architecture discloses regions for integrin binding and that most dystrophic mutations cluster at both molecule ends, within domains FAS1-1 and FAS1-4.
... Transforming growth factor-β-induced protein (TGFBIp) is an extracellular matrix (ECM) protein and is expressed in various types of cells, including smooth muscle cells, chondrocytes, fibroblasts, tumor cells, and CECs. [19][20][21][22][23] As a component of the ECM, TGFBIp interacts with ECM proteins, such as laminin, collagen, fibronectin, and glycosaminoglycans, 19 and increases cell migration, differentiation, adhesion, wound healing, metastasis, spread, and proliferation through interactions with intergrins. [24][25][26][27][28][29] In cornea, mutation of TGFBI gene is responsible for 5q31linked autosomal dominant corneal dystrophies. ...
Purpose
Transforming growth factor-β-induced protein (TGFBIp) is highly expressed in the cornea, and mutant TGFBIp induces corneal diseases. However, the function of TGFBIp in cornea epithelium is not fully investigated. Here, we tested the importance of TGFBIp in regulation of gene expression and corneal epithelial cell (CEC) activity.
Materials and Methods
The effect of TGFBIp on CEC activity was analyzed by cell migration, adhesion, proliferation and wound healing assay. Analysis of gene expression was examined by western blot and quantitative reverse transcription PCR.
Results
The results demonstrated that TGFBIp increased adhesion, migration, proliferation, and wound healing of CECs. Analysis of gene expression presented that TGFBIp-stimulated CECs exhibited increased expression of mucin family genes, such as MUC1, -4, -5AC, and -16. Furthermore, TGFBIp treatment increased the expression of MUC1, -4, -5AC, -7, and -16 in conjunctival epithelial cells. TGFBIp also increased the activity of intracellular signaling molecules ERK and AKT in CECs. Using pharmacologic inhibitors of ERK and AKT, we showed that the expression of mucin genes by TGFBIp is mediated by the activation of ERK and AKT signaling.
Conclusion
Our findings demonstrate that the locally generated TGFBIp in the cornea may contribute to wound healing of CECs by enhancing the migration, adhesion, and proliferation of CECs. In addition, our results suggest that TGFBIp has a protective effect on ocular surfaces by inducing the expression of mucin genes in corneal and conjunctival epithelial cells. These data suggest that TGFBIp is a useful therapeutic target for patients with corneal wounds.
... 19 This leads to a proposed coupling between disease and mutation-specific protein stability. TGFBIp has no known post-translational modification in the cornea; 62 however, N-terminal proteolytic processing in the cornea leaves a mostly intact Fas1-4 domain, 63 and Fas1-4 fragments have been found in the protein plaques of LCD patients. 58,59 Together, these observations make the recombinant Fas1-4 domain a highly relevant model for the effects of TGFBIp mutations in the Fas1-4 domain. ...
Lattice Corneal Dystrophy is associated with painful recurrent corneal erosions and amyloid corneal opacities induced by transforming growth factor β induced protein (TGFBIp) that impairs vision. The exact mechanism of amyloid fibril formation in Corneal Dystrophy is unknown but has been associated with destabilizing mutations in the fourth fasciclin 1 (Fas1-4) domain of TGFBIp. The green tea compound Epigallo-catechin gallate (EGCG) has been found to inhibit fibril formation of various amyloidogenic proteins in vitro. In this study we investigated the effect of EGCG as a potential treatment in Lattice Corneal Dystrophy (LCD) using Fas1-4 with the naturally occurring LCD-inducing A546T mutation. A few molar excess of EGCG were found to inhibit fibril formation in vitro by directing Fas1-4 A546T into stable EGCG-bound protein oligomers. Incubation with two molar equivalent EGCG led to a 4-fold reduction in the aggregates' membrane disruptive potential, potentially indicative of significantly lower cytotoxicity with regards to corneal erosions. EGCG did not induce oligomer formation by wildtype Fas1-4, indicating that treatment with EGCG would not interfere with the native function of the wild-type protein. Addition of EGCG to 10-day old fibrils reduced fibril content in a dose-dependent manner. Proteinase K was found to reduce the light scattering of non-treated fibrils by 31%, but reduced that of fibrils treated with eight molar equivalents of EGCG by 85%. This suggests that EGCG remodeling of fibril structure can facilitate aggregate removal by endogenous proteases and thus alleviate the protein deposits' light scattering symptoms.
... While the 4 th _FAS1 domain is not known to bind to periostin, it is still distributed to lysosomes by the R124H mutation 31 . It is known that the proteolytic fragments in patient samples include the 4 th _FAS1 domain along with the 1 st FAS1 domain 44 . Acidic environments could probably induce the conversion to β -sheet in the 4 th _FAS1 domain. ...
Most stromal corneal dystrophies are associated with aggregation and deposition of the mutated transforming growth factor-β induced protein (TGFβIp). The 4th_FAS1 domain of TGFβIp harbors ~80% of the mutations that forms amyloidogenic and non-amyloidogenic aggregates. To understand the mechanism of aggregation and the differences between the amyloidogenic and non-amyloidogenic phenotypes, we expressed the 4th_FAS1 domains of TGFβIp carrying the mutations R555W (non-amyloidogenic) and H572R (amyloidogenic) along with the wild-type (WT). R555W was more susceptible to acidic pH compared to H572R and displayed varying chemical stabilities with decreasing pH. Thermal denaturation studies at acidic pH showed that while WT did not undergo any conformational transition, the mutants exhibited a clear pH-dependent irreversible conversion from αβ conformation to β-sheet oligomers. The β-oligomers of both mutants were stable at physiological temperature and pH. Electron microscopy and dynamic light scattering studies showed that β-oligomers of H572R were larger compared to R555W. The β-oligomers of both mutants were cytotoxic to primary human corneal stromal fibroblast (pHCSF) cells. The β-oligomers of both mutants exhibit variations in their morphologies, sizes, thermal and chemical stabilities, aggregation patterns and cytotoxicities.
... The authors showed that the proteolytic processing of TGFBIp is altered, and the degradation patterns are specific for each mutation. These data are confirmed by Karring et al. (2010) who found that the c-terminal fragments were retained in the cornea after highly orchestrated N-terminal proteolytic processing. ...
Transforming growth factor beta-induced (TGFBI) corneal dystrophies are a group of inherited progressive corneal diseases. Accumulation of transforming growth factor beta-induced protein (TGFBIp) is involved in the pathogenesis of TGFBI corneal dystrophies; however, the exact molecular mechanisms are not fully elucidated. In this review article, we summarize the current knowledge of TGFBI corneal dystrophies including clinical manifestations, epidemiology, most common and recently reported associated mutations for each disease, and treatment modalities. We review our current understanding of the molecular mechanisms of granular corneal dystrophy type 2 (GCD2) and studies of other TGFBI corneal dystrophies. In GCD2 corneal fibroblasts, alterations of morphological characteristics of corneal fibroblasts, increased susceptibility to intracellular oxidative stress, dysfunctional and fragmented mitochondria, defective autophagy, and alterations of cell cycle were observed. Other studies of mutated TGFBIp show changes in conformational structure, stability and proteolytic properties in lattice and granular corneal dystrophies. Future research should be directed toward elucidation of the biochemical mechanism of deposit formation, the relationship between the mutated TGFBIp and the other materials in the extracellular matrix, and the development of gene therapy and pharmaceutical agents.
... A second possibility is that PN or βig-h3 secreted from mesenchymal stromal or adenocarcinoma cells undergo loss of N-or C-terminal basic residues as a result of proteolysis, thus lowering the pI. Such has been demonstrated for βig-h3 from cornea [40]. A final possibility is that PN or βig-h3 carry other acidic modifications or are phosphorylated. ...
Periostin (PN, gene name POSTN) is an extracellular matrix protein that is up-regulated in bronchial epithelial cells and lung fibroblasts by TH-2 cytokines. Its paralog, TGF-β-induced protein (βig-h3, gene name TGFBI), is also expressed in the lung and up-regulated in bronchial myofibroblasts by TGF-β. PN and βig-h3 contain fasciclin 1 modules that harbor putative recognition sequences for γ-glutamyl carboxylase and are annotated in UniProt as undergoing vitamin K-dependent γ-carboxylation of multiple glutamic acid residues. γ-carboxylation profoundly alters activities of other proteins subject to the modification, e.g., blood coagulation factors, and would be expected to alter the structure and function of PN and βig-h3. To analyze for the presence of γ-carboxylation, proteins extracted from fibrotic lung were reacted with monoclonal antibodies specific for PN, βig-h3, or modification with γ-carboxyglutamic acid (Gla). In Western blots of 1-dimensional gels, bands stained with anti-PN or -βig-h3 did not match those stained with anti-Gla. In 2-dimensional gels, anti-PN-positive spots had pIs of 7.0 to >8, as expected for the unmodified protein, and there was no overlap between anti-PN-positive and anti-Gla-positive spots. Recombinant PN and blood coagulation factor VII were produced in HEK293 cells that had been transfected with vitamin K 2, 3-epoxide reductase C1 to optimize γ-carboxylation. Recombinant PN secreted from these cells did not react with anti-Gla antibody and had pIs similar to that found in extracts of fibrotic lung whereas secreted factor VII reacted strongly with anti-Gla antibody. Over 67% coverage of recombinant PN was achieved by mass spectrometry, including peptides with 19 of the 24 glutamates considered targets of γ-carboxylation, but analysis revealed no modification. Over 86% sequence coverage and three modified glutamic acid residues were identified in recombinant fVII. These data indicate that PN and βig-h3 are not subject to vitamin K-dependent γ-carboxylation.
... It is particularly puzzling why peptides from PN and big-h3 could be generated from protein focusing at pIs well below those of the unmodified forms of these proteins (Coutu et al., 2008), whereas we detected no PN in this region of two-dimensional gels. Possibilities are that PN or big-h3 secreted from mesenchymal stem or adenocarcinoma cells are lacking N-or C-terminal basic residues as a result of proteolysis (Karring et al., 2010) or are phosphorylated or carry other acidic modifications. PhosphoSitePlus (www. ...
Periostin (PN) and TGF-β-induced protein (βig-h3) are paralogs that contain a single emilin and four fasciclin-1 modules and are secreted from cells. PN receives attention because of its up-regulation in cancer and degenerative and allergic diseases. βig-h3 is highly enriched in cornea and best known for harboring mutations in humans associated with corneal dystrophies. Both proteins are expressed widely, and many functions, some over-lapping, have been attributed to PN and βig-h3 based on biochemical, cell culture, and whole animal experiments. We attempt to organize this knowledge so as to facilitate research on these interesting and incompletely understood proteins. We focus particularly on whether PN and βig-h3 are modified by vitamin K-dependent γ-glutamyl carboxylation, a question of considerable importance given the profound effects of γ-carboxylation on structure and function of other proteins. We consider the roles of PN and βig-h3 in formation of extracellular matrix and as ligands for integrin receptors. We attempt to reconcile the contradictory results that have arisen concerning the role of PN, which has emerged as a marker of TH2 immunity, in murine models of allergic asthma. Finally, when possible we compare and contrast the structures and functions of the two proteins.
... The mutation of TGFB-I induces alterations in the processing and degradation patterns of TGFB-I p characterized by progressive deposition of proteinaceous material in the cornea. Karring at al. [2] demonstrated that the C-terminal of the mutant TGFB-I p (the last to be turned over in the normal human cornea) becomes more resistant to proteolysis and less degradable. Thus, increased accumulation of C-terminal mutant TGFB-I p isoforms might be the direct cause of the TGFB-I p aggregation (increased protein stability with reduced proteolysis) and crystalloid accumulation in GCD corneas. ...
Clinical, instrumental, and genetic findings are reported in Italian families with Type II Granular Corneal Dystrophies (GCD2) presenting an initial unusual presentation of a Granular Corneal Dystrophy Type I (GCD1) phenotypic spectrum in female descendants. Slit-lamp examinations showed the typical phenotypic features of GCD2 in both mothers and a phenotypic appearance of GCD1 in both daughters. Despite the different phenotypic onset, the genetic diagnostic testing revealed the presence of a mutation in the TGFB-I gene, typical of GCD2 in both cases, excluding GCD1. Patients who were clinically suspected of corneal dystrophy need a genetic confirmatory testing for certain diagnosis. Genetic test may help to find the specific mutation distinguishing between different phenotypic spectra with relative diagnostic and prognostic implications. The study demonstrates that the phenotypic spectrum of genetically confirmed granular corneal dystrophies in patients may change over time. Since the R124H mutation has also been described in clinically asymptomatic individuals prior to LASIK, who then develop dramatic deposition, suggesting that this particular mutation and phenotype may be sensitive to, precipitated, or modified by central cornea trauma, a careful familial anamnesis excluding cornel dystrophies and specific preoperative genetic test are recommended prior to LASIK.
... mg/mg of the wet tissue in child to about 0.7-0. 8 36 Rawe et al have shown that, in the rabbit corneal stromal extract, TGFBIp can be co-purified under non-reducing conditions along with type VI collagen, and the two can be separated by reducing agents. 37 TGFBI transcripts are primarily seen in the corneal epithelial cells, but increased levels have been shown in the stroma during development and wound healing. ...
TGFBI-associated corneal dystrophies are characterized by accumulation of insoluble deposits of the mutant protein transforming growth factor β−induced protein (TGFBIp) in the cornea. Depending on the nature of mutation, the lesions appear as granular (non-amyloid) or lattice lines (amyloid) in the Bowman’s layer or in the stroma. This review article emphasizes the structural biology aspects of TGFBIp. We discuss the tinctorial properties and ultrastructure of deposits observed in granular and lattice corneal dystrophic mutants with amyloid and non-amyloid forms of other human protein deposition diseases and review the biochemical and putative functional role of the protein. Using bioinformatics tools, we identify intrinsic aggregation propensity and discuss the possible protective role of gatekeepers close to the “aggregation-prone” regions of native TGFBIp. We describe the relative aggregation rates of lattice corneal dystrophy (LCD) and granular corneal dystrophy (GCD2) mutants using the three-parameter model, which is based on intrinsic properties of polypeptide chains. The predictive power of this model is compared with two other algorithms. We conclude that the model is able to predict the aggregation rate of mutants which do not alter overall net charge of the protein. The need to understand the mechanism of corneal dystrophies from the structural biology viewpoint is emphasized.
... of the proteolytic events revealed that 31 of the 38 TGFBIp cleavage sites (82%) from the amyloid deposits and eight of the 12 cleavage sites (67%) from the periamyloid tissue were not detected in the healthy cornea tissue, suggesting that altered proteolytic events occur in LCD type 1 (Appendix 5). Previous studies have shown that TGFBIp from normal human corneas is proteolytically processed [9,50], but except for the cleavage at the C-terminus of A657 in mature TGFBIp [51], the specific cleavage sites have remained unknown. The cleavage sites in normal TGFBIp from the healthy cornea were distributed throughout the TGFBIp sequence, while those from the amyloid deposits were more clustered (Appendix 5A). ...
Specific mutations in the transforming growth factor beta induced (TGFBI) gene are associated with lattice corneal dystrophy (LCD) type 1 and its variants. In this study, we performed an in-depth proteomic analysis of human corneal amyloid deposits associated with the heterozygous A546D mutation in TGFBI.
Corneal amyloid deposits and the surrounding corneal stroma were procured by laser capture microdissection from a patient with an A546D mutation in TGFBI. Proteins in the captured corneal samples and healthy corneal stroma were identified with liquid chromatography-tandem mass spectrometry and quantified by calculating exponentially modified Protein Abundance Index values. Mass spectrometry data were further compared for identifying enriched regions of transforming growth factor beta induced protein (TGFBIp/keratoepithelin/βig-h3) and detecting proteolytic cleavage sites in TGFBIp.
A C-terminal fragment of TGFBIp containing residues Y571-R588 derived from the fourth fasciclin 1 domain (FAS1-4), serum amyloid P-component, apolipoprotein A-IV, clusterin, and serine protease HtrA1 were significantly enriched in the amyloid deposits compared to the healthy cornea. The proteolytic cleavage sites in TGFBIp from the diseased cornea are in accordance with the activity of serine protease HtrA1. We also identified small amounts of the serine protease kallikrein-14 in the amyloid deposits.
Corneal amyloid caused by the A546D mutation in TGFBI involves several proteins associated with other varieties of amyloidosis. The proteomic data suggest that the sequence 571-YHIGDEILVSGGIGALVR-588 contains the amyloid core of the FAS1-4 domain of TGFBIp and point at serine protease HtrA1 as the most likely candidate responsible for the proteolytic processing of amyloidogenic and aggregated TGFBIp, which explains the accumulation of HtrA1 in the amyloid deposits. With relevance to identifying serine proteases, we also found glia-derived nexin (protease-nexin 1) in the amyloid deposits, making this serine protease inhibitor a good candidate for the physiologically relevant inhibitor of one of the amyloid-associated serine proteases in the cornea and probably in other tissues. Noteworthy, the present results are in accordance with our findings from a previous study of corneal amyloid deposits caused by the V624M mutation in TGFBI, suggesting a common mechanism for lattice corneal dystrophies (LCDs) associated with mutations in the TGFBIp FAS1-4 domain.
... A healthy human cornea was obtained post mortem at the Department of Forensic Medicine, Aarhus University Hospital, Denmark as described previously (32). Two pieces of 0.5 mg cornea were dried in a SpeedVac concentrator. ...
TGFBIp, also known as keratoepithelin and βig-h3, is among the most abundant proteins in the human cornea and approximately 60% is associated with the insoluble fraction following extraction in sodium dodecyl sulfate (SDS) sample buffer. TGFBIp is of particular interest because a wide range of mutations causes amyloid or fuchsinophilic crystalloid deposits in the cornea leading to visual impairment. In the present study, we show that the SDS-insoluble fraction of TGFBIp from porcine and human corneas is covalently linked to the NC3 domain of type XII collagen in a TGFBIp:type XII collagen stoichiometric ratio of 2:1 via a reducible bond. Since type XII collagen is anchored to striated collagen fibers of the extracellular matrix, its interaction with TGFBIp is likely to provide anchoring for cells to the extracellular matrix through the integrin-binding capability of TGFBIp. Furthermore, the TGFBIp-type XII collagen molecule will affect our understanding of the molecular pathogenesis of the TGFBI-linked corneal dystrophies.
... RGD is known to act as a ligand recognition sequence for integrins in modulating cell adhesion within the extracellular matrix (ECM). Ser658-His683 in the C-terminal is critical for function malignance of RGD because the lack of these residues is liable to expose the RGD sequence to interactions [15,16]. TGFBIp widely expresses in the extracellular matrix of many organs and interacts with other matrix proteins, such as fibronectin, different integrins, and collagens [17-19]. ...
To identify the gene mutation underlying Avellino corneal dystrophy in a four-generation Chinese pedigree.
Patients from the affected family underwent detailed clinical examination involving slit-lamp photography and confocal microscopy. Genomic DNA extracted from peripheral leukocytes was amplified using touch-down PCR for gene scanning. Two-point linkage analysis and haplotyping were performed to map the relevant chromosome region. The candidate gene in this region was sequenced to screen out the disease-causing mutation.
Patients in the pedigree were diagnosed with Avellino corneal dystrophy. Using linkage analysis, the responsible gene was mapped to chromosome 5q31.2 with a maximum LOD (log odds) score (Z(max)) of 3.23 at D5S479 (θ(max)=0.0). Haplotypes constructed from 11 microstallite markers identified the disease-linked chromosome region as being below D5S808. Sequencing of TGFBI (transforming growth factor-beta induced gene), a known gene in this region, revealed a heterozygous transition (c.418 G>A) in exon 4 resulting in Arg124His (R124H) being co-segregated with the disease in affected family members but not in the unaffected members or the 50 unrelated controls.
Our study demonstrated that a G>A transition in Arg124His of TGFBI was responsible for Avellino corneal dystrophy in a Chinese pedigree. This result further supports the importance of TGFBIp in maintaining transparency of the cornea.
... This view is further supported by the fact that the relative spectral count ratios of tryptic peptides from the FAS1-4 domain were generally higher for the LCD samples than for the GCD samples indicating that the amyloidogenic V624M mutation destabilises the FAS1-4 domain structure making it more susceptible to tryptic digestion ( Fig. 3A and Fig. S2A, Supplemental Data). Interestingly, the identified Y571-R588 peptide of TGFBIp accumulated in the amyloid deposits is included in the multiple C-terminal fragments of TGFBIp observed in the normal human cornea (Karring et al., 2010) suggesting that it is part of a structural motif resistant to proteolysis in vivo. Our results are in accordance with the observations by Stix et al. (Stix et al., 2005) showing that corneal amyloid caused by mutation P540S in the FAS1-4 domain of TGFBIp reacts strongly with antiserum raised against C-terminal residues 426-682 but not antiserum against the N-terminus of TGFBIp (residues 69-364) and that they contain proteolytic fragments of TGFBIp. ...
Different types of granular corneal dystrophy (GCD) and lattice corneal dystrophy (LCD) are associated with mutations in the transforming growth factor beta induced gene (TGFBI). These dystrophies are characterized by the formation of non-amyloid granular deposits (GCDs) and amyloid (LCD type 1 and its variants) in the cornea. Typical corneal non-amyloid deposits from GCD type 2 (R124H), amyloid from a variant of LCD type 1 (V624M) and disease-free tissue controls were procured by laser capture microdissection and analyzed by tandem mass spectrometry. Label-free quantitative comparisons of deposits and controls suggested that the non-amyloid sample (R124H) specifically accumulated transforming growth factor beta induced protein (TGFBIp/keratoepithelin/βig-h3), serum amyloid P-component, clusterin, type III collagen, keratin 3, and histone H3-like protein. The amyloid (V624M) similarly accumulated serum amyloid P-component and clusterin but also a C-terminal fragment of TGFBIp containing residues Y571-R588 derived from the fourth fasciclin-1 domain (FAS1-4), apolipoprotein E and apolipoprotein A-IV. Significantly, analyses of the amyloid sample also revealed the presence of the serine protease Htr (High-temperature requirement) A1 and a number of proteolytic cleavage sites in the FAS1-4 domain of TGFBIp. These cleavage sites were consistent with the ligand binding and proteolytic activity of HtrA1 suggesting that it plays a role in the proteolytic processing of the amyloidogenic FAS1-4 domain. Taken together, the data suggest that the amyloidogenic-prone region of the fourth FAS1 domain of TGFBIp encompasses the Y571-R588 peptide and that HtrA1 is involved in the proteolytic processing of TGFBIp-derived amyloid in vivo.
The accumulation and propagation of hyperphosphorylated tau (p-Tau) is a neuropathological hallmark occurring with neurodegeneration of Alzheimer’s disease (AD). Extracellular vesicles, exosomes, have been shown to initiate tau propagation in the brain. Notably, exosomes from human-induced pluripotent stem cell (iPSC) neurons expressing the AD familial A246E mutant form of presenilin 1 (mPS1) are capable of inducing tau deposits in the mouse brain after in vivo injection. To gain insights into the exosome proteome cargo that participates in propagating tau pathology, this study conducted proteomic analysis of exosomes produced by human iPSC neurons expressing A246E mPS1. Significantly, mPS1 altered the profile of exosome cargo proteins to result in (1) proteins present only in mPS1 exosomes and not in controls, (2) the absence of proteins in the mPS1 exosomes which were present only in controls, and (3) shared proteins which were upregulated or downregulated in the mPS1 exosomes compared to controls. These results show that mPS1 dysregulates the proteome cargo of exosomes to result in the acquisition of proteins involved in the extracellular matrix and protease functions, deletion of proteins involved in RNA and protein translation systems along with proteasome and related functions, combined with the upregulation and downregulation of shared proteins, including the upregulation of amyloid precursor protein. Notably, mPS1 neuron-derived exosomes displayed altered profiles of protein phosphatases and kinases involved in regulating the status of p-tau. The dysregulation of exosome cargo proteins by mPS1 may be associated with the ability of mPS1 neuron-derived exosomes to propagate tau pathology.
Corneal dystrophies are a group of genetically inherited disorders with mutations in the TGFBI gene affecting the Bowman’s membrane and the corneal stroma. The mutant TGFBIp is highly aggregation-prone and is deposited in the cornea. Depending on the type of mutation the protein deposits may vary (amyloid, amorphous powdery aggregate or a mixed form of both), making the cornea opaque and thereby decreases visual acuity. The aggregation of the mutant protein is found to be specific with a unique aggregation mechanism distinct to the cornea. The proteolytic processing of the mutant protein is reported to be different compared to the WT protein. The proteolytic processing of mutant protein gives rise to highly amyloidogenic peptide fragments. The current treatment option, available for patients, is tissue replacement surgery that is associated with high recurrence rates. The clinical need for a simple treatment option for corneal dystrophy patients has become highly essential either to prevent the protein aggregation or to dissolve the preformed aggregates. Here, we report the screening of 2500 compounds from the Maybridge RO3 fragment library using weak affinity chromatography (WAC). The primary hits from WAC were validated by ¹⁵N-HSQC NMR assays and specific regions of binding were identified. The recombinant mutant proteins (4th FAS-1 domain of R555W and H572R) were subjected to limited proteolysis by trypsin together with the lead compounds identified by NMR assays. The lead compounds (MO07617, RJF00203 and, BTB05094) were effective to delay/prevent the generation of amyloidogenic peptides in the R555W mutant and compounds (RJF00203 and BTB05094) were effective to delay/prevent the generation of amyloidogenic peptides in the H572R mutant. Thus the lead compounds reported here upon further validation and/or modification might be proposed as a potential treatment option to prevent/delay aggregation by inhibiting the formation of amyloidogenic peptides in TGFBI-corneal dystrophy.
Purpose: To produce an acellular small intestine submucosa (SIS) that would be a suitable scaffold for corneal epithelium tissue engineering.
Methods: The SIS was decellularized by immersion in 0.1% (wt/vol) sodium dodecyl sulfate (SDS). The efficacy of acellularization was confirmed by histological observation and DNA quantification. The mechanical properties were evaluated by uniaxial tensile testing. ELISA was performed to assess the growth factor contents. The cytotoxicity of SIS scaffolds and extracts to rabbit corneal epithelial cells was determined by CCK-8 assay. We also investigated the inflammatory reaction of SIS implanted subcutaneously in a rat. The biocompatibility was studied by rabbit interlamellar corneal transplantation and reseeding assay with cornea-derived cells. Immunofluorescent staining was used to detect the expression of CK3, ZO-1 and K13.
Results: Histological analyses showed that complete cell removal was achieved, and the DNA quantity, which reflects the presence of cellular materials, was significantly diminished in acellular SIS. Collagen fibers were properly preserved and appeared in an orderly fashion. The tissue structure, the mechanical properties and the growth factor contents within the acellular SIS were well retained. The CCK8 assay demonstrated that the acellular SIS scaffolds and extracts had no cytotoxicity to rabbit corneal epithelial cells. There was no sign that an immune reaction occurred with acellular SIS implanted subcutaneously in a rat. In fact, in vivo implantation to rabbit interlamellar stromal pockets showed good biocompatibility. We also observed that clusters of rabbit corneal epithelial cells were growing well on the surface of the SIS in vitro and the distinctive CK3, ZO-1 for corneal epithelial cells was detected.
Conclusions: The decellularized SIS retained the major structural components. The matrix is biocompatible with cornea-derived cells and might be a suitable scaffold for corneal epithelium tissue engineering.
The serine protease high-temperature requirement protein A1 (HtrA1) is associated with protein-misfolding disorders such as Alzheimer's disease and transforming growth factor b-induced protein (TGFBIp)-linked corneal dystrophy. In this study, using several biochemical and biophysical approaches, including recombinant protein expression, LC-MS/MS and 2D-PAGE analyses and thioflavin-T (ThT) fluorescence assays for amyloid fibril detection, and FTIR assays, we investigated the role of HtrA1 both in normal TGFBIp turnover and in corneal amyloid formation. We show that HtrA1 can cleave wild-type TGFBIp but prefers amyloidogenic variants. Corneal TGFBIp is extensively processed in healthy people, resulting in C-terminal degradation products spanning the FAS1-4 domain of TGFBIp. We show here that HtrA1 cleaves the wild-type FAS1-4 domain only inefficiently, whereas the amyloidogenic FAS1-4 mutations transform this domain into a considerably better HTRA1 substrate. Moreover, HtrA1 cleavage of the mutant FAS1-4 domains generated peptides capable of forming in vitro amyloid aggregates. Significantly, these peptides have been previously identified in amyloid deposits in vivo, supporting the idea that HtrA1 is a causative agent for TGFBIp-associated amyloidosis in corneal dystrophy. In summary, our results indicate that TGFBIp is an HtrA1 substrate and that some mutations in the gene encoding TGFBIp cause aberrant HtrA1-mediated processing that results in amyloidogenesis in corneal dystrophies.
Human Transforming Growth Factor β‐induced (TGFBI), is a gene responsible for various corneal dystrophies. TGFBI produces a protein called TGFBI which is involved in cell adhesion and serves as a recognition sequence for integrins. An alteration in cell surface interactions could be the underlying cause for the progressive accumulation of extracellular deposits in different layers of the cornea with the resulting changes of refractive index and transparency. To this date 69 different pathogenic or likely pathogenic variants in TGFBI have been identified in heterozygous or homozygous state in various corneal dystrophies, including a novel variant reported here. All disease‐associated variants were inherited as autosomal dominant traits but one; this latter was inherited as an autosomal recessive trait (Afshari et al., 2008). Most corneal dystrophy‐associated variants are located at amino acids Arg124 and Arg555. In order to keep the list of corneal dystrophy‐associated variant current, we generated a locus‐specific database for TGFBI (http://databases.lovd.nl/shared/variants/TGFBI) containing all pathogenic and likely pathogenic variants reported so far. Non disease associated variants are described in specific databases, like gnomAD and ExAC and are not listed here. This article presents the most recent up‐to‐date list of disease‐associated variants.
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Corneal Dystrophies (CD) constitute a group of genetically inherited protein aggregation disorders that affect different layers of the cornea. With accumulated protein deposition, the cornea becomes opaque with decreased visual acuity. CD affecting the stroma and Bowman's membrane is associated with mutations in transforming growth factor β‐induced (TGFBI) gene. The purpose of this study was to identify and visualize the spatial distribution of proteins present in amyloid corneal deposits of TGFBI‐CD patients using Mass Spectrometry Imaging (MSI) and compare it with healthy control cornea. MALDI‐MSI provided a relative abundance and two‐dimensional spatial protein signature of key proteins (TGFBIp, apolipoprotein A‐I, apolipoprotein A‐IV, apolipoprotein E, kaliocin‐1, pyruvate kinase and ras‐related protein Rab‐10) in the patient deposits compared to the control. This is the first report of the anatomical localization of key proteins on corneal tissue section from CD patients. This may provide insight in understanding the mechanism of amyloid fibril formation in TGFBI‐corneal dystrophy.
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TGFBIp is a constituent of the extracellular matrix in many human tissues including the cornea, where it is one of the most abundant proteins expressed. TGFBIp interacts with Type I, II, IV, VI and XII collagens as well as several members of the integrin family, suggesting it plays an important role in maintaining structural integrity and possibly corneal transparency as well. Significantly, more than 60 point mutations within the TGFBI gene have been reported to result in aberrant TGFBIp folding and aggregation in the cornea, resulting in severe visual impairment and blindness. Several studies have focused on targeting TGFBIp in the cornea as a therapeutic approach to treat TGFBI-linked corneal dystrophies, but the effect of this approach on corneal homeostasis and matrix integrity remained unknown. In the current study, we evaluated the histological and proteomic profiles of corneas from TGFBI-deficient mice as well as potential redundant functions of the paralogous protein POSTN. The absence of TGFBIp in mouse corneas did not grossly affect the collagen scaffold, and POSTN is unable to compensate for loss of TGFBIp. Proteomic comparison of wildtype and TGFBI(-/-) mice revealed 11 proteins were differentially regulated, including Type VI and XII collagens. However, as these alterations did not manifest at the macroscopic and behavioral levels, these data support partial or complete TGFBI knockdown as a potential therapy against TGFBI-linked corneal dystrophies. Lastly, in situ hybridization verified TGFBI mRNA in the epithelial cells but not in other cell types, supportive of a therapy directed specifically at this lineage. This article is protected by copyright. All rights reserved.
TGFBI-associated corneal dystrophies are inherited disorders caused by TGFBI gene variants that promote deposition of mutant protein (TGFBIp) as insoluble aggregates in the cornea. Depending on the type and position of amino acid substitution, the aggregates may be amyloid fibrillar, amorphous globular or both, but the molecular mechanisms that drive these different patterns of aggregation are not fully understood. In the current study, we report the protein composition of amyloid corneal aggregates from lattice corneal dystrophy patients of Asian origin with H626R and R124C mutation and compared it with healthy corneal tissues via LC-MS/MS. We identified several amyloidogenic, non-fibrillar amyloid associated proteins and TGFBIp as the major components of the deposits. Our data indicates that apolipoprotein A-IV, apolipoprotein E, and serine protease HTRA1 were significantly enriched in patient deposits compared to healthy controls. HTRA1 was also found to be seven fold enriched in the amyloid deposits of patients compared to the controls. Peptides sequences (G511DNRFSMLVAAIQSAGLTETLNR533 and Y571HIGDEILVSGGIGALVR588) derived from the 4th FAS-1 domain of TGFBIp were enriched in the corneal aggregates in a mutation-specific manner. Biophysical studies of these two enriched sequences revealed high propensity to form amyloid fibrils under physiological conditions. Our data suggests a possible proteolytic processing mechanism of mutant TGFBIp by HTRA1 and peptides generated by mutant protein may form the β-amyloid core of corneal aggregates in dystrophic patients.
Transforming growth factor beta-induced protein (TGFBIp) is an extracellular matrix protein composed of an NH2-terminal cysteine-rich domain (CRD) annotated as an emilin (EMI) domain, and four fasciclin-1 (FAS1-1 to FAS1-4) domains. Mutations in the gene cause corneal dystrophies, a group of debilitating protein misfolding diseases that lead to severe visual impairment. Previous studies have shown that TGFBIp in the cornea is cross-linked to type XII collagen through a reducible bond. TGFBIp contains 11 cysteine residues and is thus able to form five intra-molecule disulfide bonds leaving a single cysteine residue available for the collagen cross-link. The structures of TGFBIp and its homologs are unknown. We here present the disulfide bridge pattern of TGFBIp, which was solved by generating specific peptides. These were separated by ion exchange followed by reverse phase HPLC and analyzed by mass spectrometry and Edman degradation. The NH2-terminal CRD contains six cysteine residues, one of these (Cys65) was identified as the candidate for the reducible cross-link between TGFBIp and type XII collagen. In addition, the CRD contained two intra-domain disulfide bridges (Cys49-Cys85 and Cys84-Cys97) and one inter-domain disulfide bridge to FAS1-2 (Cys74-Cys339). Significantly, this arrangement violates the predicted disulfide bridge pattern of an EMI domain. The cysteine residues in FAS1-3 (Cys473 and Cys478) were shown to form an intra-domain disulfide bridge. Finally, an inter-domain disulfide bridge between FAS1-1 and FAS1-2 (Cys214-Cys317) was identified. The inter-domain disulfide bonds indicate that the NH2-terminal of TGFBIp (CRD, FAS1-1 and FAS1-2) adopts a compact globular fold leaving FAS1-3 and FAS1-4 exposed.
Several forms of the familial protein aggregation disease corneal dystrophy (CD) have been linked to mutations in transforming growth factor β-induced protein (TGFBIp). More than 30 point mutations in TGFBIp lead to CD, but the mutations induce many different aggregates in the cornea, ranging from granular to lattice and rod-like deposits. Biophysical methods have begun to help us elucidate how and why these mutations lead to polymorphic aggregates. Most CD-inducing mutations are found in the fourth fasciclin-1 domain of TGFBIp, and this domain also controls the stability of the entire TGFBIp molecule. Some mutations decrease TGFBIp stability, others increase it, and there is as yet no simple link between phenotype and stability. The mutations also affect surface electrostatics, proteolytic cleavage susceptibility, oligomerization propensities and interactions with other macromolecules. We highlight ways in which these changes can affect corneal aggregation. Future investigations will hopefully provide us with a clearer view of the links between biophysical properties and clinical manifestations.
Mutations in the transforming growth factor beta-induced (TGFBI) gene result in a group of hereditary diseases of the cornea that are collectively known as TGFBI corneal dystrophies. These mutations translate into amino acid substitutions mainly within the fourth fasciclin 1 domain (FAS1-4) of the transforming growth factor beta-induced protein (TGFBIp) and cause either amyloid or non-amyloid protein aggregates in the anterior and central parts of the cornea, depending on the mutation. The A546T substitution in TGFBIp causes lattice corneal dystrophy (LCD), which manifests as amyloid-type aggregates in the corneal stroma. We previously showed that the A546T substitution renders TGFBIp and the FAS1-4 domain thermodynamically less stable compared with the wild-type (WT) protein, and the mutant FAS1-4 is prone to amyloid formation in vitro. In the present study, we identified the core of A546T FAS1-4 amyloid fibrils. Significantly, we identified the Y571-R588 region of TGFBIp, which we previously found to be enriched in amyloid deposits in LCD patients. We further found that the Y571-R588 peptide seeded fibrillation of A546T FAS1-4 and, more importantly, we demonstrated that native TGFBIp aggregates in the presence of fibrils formed by the core peptide. Collectively, these data suggest an involvement of the Y571-R588 peptide in LCD pathophysiology.
To investigate whether mutations in TGFBI gene or CHST6 gene correlated with stromal corneal dystrophies (CD) in 8 Chinese probands.
Eight unrelated patients with stromal corneal dystrophies were recruited in this study; all affected members were assessed by completely ophthalmologic examinations. Genomic DNA was extracted from peripheral leukocytes, 17 exons of TGFBI gene and the exon of CHST6 gene were amplified by polymerase chain reaction (PCR), sequenced directly and compared with the reference database.
Three heterozygous mutations in TGFBI gene were identified in six patients: c. 370C>T (p.Arg124Cys) was found in exon 4 of TGFBI gene in three members, c. 371G>A (p.Arg124His) was found in one patient; c. 1663C>T (p.Arg555Trp) was found in exon 12 in other two members. In addition, four polymorphisms with the nucleotide changes rs1442, rs1054124, rs4669, and rs35151677 were found in TGFBI gene. Mutations were not identified in the rest of 2 affected individuals in TGFBI gene or CHST6 gene.
Within these patients, R124C, R124H and R555W mutations were co-segregated with the disease phenotypes and were specific mutations for lattice corneal dystrophy type I (LCD I), Avellino corneal dystrophy (ACD, GCD II), granular corneal dystrophy type I (GCD I), respectively. Our study highlights the prevalence of codon 124 and codon 555 mutations in the TGFBI gene among the Chinese stromal corneal dystrophies patients.
Transforming growth factor beta-induced protein (TGFBIp) has been linked to several corneal dystrophies as certain point mutations in the protein may give rise to a progressive accumulation of insoluble protein material in the human cornea. Little is known about the biological functions of this extracellular protein, which is expressed in various tissues throughout the human body. However, it has been found to interact with a number of extracellular matrix macromolecules such as collagens and proteoglycans. Structural information about TGFBIp might prove to be a valuable tool in the elucidation of its function and its role in corneal dystrophies caused by mutations in the TGFBI gene. A simple method for the purification of wild-type and mutant forms of recombinant human TGFBIp from human cells under native conditions is presented here. Moreover, the crystallization and preliminary X-ray analysis of TGFBIp are reported.
The term corneal dystrophy embraces a heterogenous group of bilateral genetically determined non-inflammatory corneal diseases that are restricted to the cornea. The designation is imprecise but remains in vogue because of its clinical value. Clinically, the corneal dystrophies can be divided into three groups based on the sole or predominant anatomical location of the abnormalities. Some affect primarily the corneal epithelium and its basement membrane or Bowman layer and the superficial corneal stroma (anterior corneal dystrophies), the corneal stroma (stromal corneal dystrophies), or Descemet membrane and the corneal endothelium (posterior corneal dystrophies). Most corneal dystrophies have no systemic manifestations and present with variable shaped corneal opacities in a clear or cloudy cornea and they affect visual acuity to different degrees. Corneal dystrophies may have a simple autosomal dominant, autosomal recessive or X-linked recessive Mendelian mode of inheritance. Different corneal dystrophies are caused by mutations in the CHST6, KRT3, KRT12, PIP5K3, SLC4A11, TACSTD2, TGFBI, and UBIAD1 genes. Knowledge about the responsible genetic mutations responsible for these disorders has led to a better understanding of their basic defect and to molecular tests for their precise diagnosis. Genes for other corneal dystrophies have been mapped to specific chromosomal loci, but have not yet been identified. As clinical manifestations widely vary with the different entities, corneal dystrophies should be suspected when corneal transparency is lost or corneal opacities occur spontaneously, particularly in both corneas, and especially in the presence of a positive family history or in the offspring of consanguineous parents. Main differential diagnoses include various causes of monoclonal gammopathy, lecithin-cholesterol-acyltransferase deficiency, Fabry disease, cystinosis, tyrosine transaminase deficiency, systemic lysosomal storage diseases (mucopolysaccharidoses, lipidoses, mucolipidoses), and several skin diseases (X-linked ichthyosis, keratosis follicularis spinolosa decalvans). The management of the corneal dystrophies varies with the specific disease. Some are treated medically or with methods that excise or ablate the abnormal corneal tissue, such as deep lamellar endothelial keratoplasty (DLEK) and phototherapeutic keratectomy (PTK). Other less debilitating or asymptomatic dystrophies do not warrant treatment. The prognosis varies from minimal effect on the vision to corneal blindness, with marked phenotypic variability.
To investigate the differential expression of TGFBIp in normal human and Fuchs endothelial corneal dystrophy (FECD) endothelial cell-Descemet's membrane (HCEC-DM) complex, and to asses the structural role of TGFBIp and clusterin (CLU) in guttae formation.
HCEC-DM complex was dissected from stroma in normal and FECD samples. Proteins were separated by 2-D gel electrophoresis and subjected to proteomic analysis. N-terminal processing of TGFBIp was detected by Western blot analysis with two separate antibodies against the N- and C-terminal regions of TGFBIp. Expression of TGFBI mRNA was compared by using real-time PCR. Subcellular localization of TGFBIp and CLU in corneal guttae was assessed by fluorescence confocal microscopy.
A major 68-kDa fragment and a minor 39-kDa fragment of TGFBIp were identified on 2-D gels. Western blot analysis revealed an age-dependent proteolytic processing of the TGFBIp N terminus resulting in the increased formation of 57-kDa (P = 0.04) and 39-kDa (P = 0.03) fragments in older donors. FECD HCEC-DM showed a significant increase in the 68-kDa (P = 0.04), 57-kDa (P = 0.01), and 39- kDa (P = 0.03) fragments of TGFBIp. Real-time PCR analysis revealed that TGFBI mRNA was significantly increased (P = 0.04) in FECD samples. TGFBIp formed aggregates at the lower portions of guttae, next to Descemet's membrane, whereas CLU localized mostly on top of the TGFBIp-stained areas at the level of the endothelial cell nuclear plane.
The overexpression of proaggregative protein CLU, and proadhesive protein TGFBIp, have been colocalized in the guttae. Such findings provide us with a better understanding of the major contributors involved in the aberrant cell-extracellular matrix interactions seen in the guttae of patients with FECD.
Small amounts (7-250 pmol) of myoglobin, beta-lactoglobulin, and other proteins and peptides can be spotted or electroblotted onto polyvinylidene difluoride (PVDF) membranes, stained with Coomassie Blue, and sequenced directly. The membranes are not chemically activated or pretreated with Polybrene before usage. The average repetitive yields and initial coupling of proteins spotted or blotted into PVDF membranes ranged between 84-98% and 30-108% respectively, and were comparable with the yields measured for proteins spotted onto Polybrene-coated glass fiber discs. The results suggest that PVDF membranes are superior supports for sequence analysis of picomole quantities of proteins purified by gel electrophoresis.
To confirm the mutation of the keratoepithelin gene in patients with a severe form of superficial juvenile granular corneal dystrophy (GCD).
Five Japanese probands in whom GCD was diagnosed after histopathological examination and who developed severe manifestations of GCD in their first decade of life were investigated. Other affected family members of two probands were also examined. All probands were the offspring of consanguineous parents. DNA was extracted from their peripheral blood leucocytes and mutational analysis of the gene was performed by the polymerase chain reaction and direct sequencing.
Four of the five probands underwent their first keratectomy or keratoplasty in their teens and subsequently underwent a second or third keratoplasty. Each proband had a homozygous G-->A transition at codon 124, replacing Arg-->His, of the keratoepithelin gene. Their moderately affected family members were heterozygous for the mutation.
This finding suggests that the severity of the corneal phenotype depends on the dose effect of the mutant gene.
Mutations in kerato-epithelin are responsible for a group of hereditary cornea-specific deposition diseases, 5q31-linked corneal dystrophies. These conditions are characterized by progressive accumulation of protein deposits of different ultrastructure. Herein, we studied the corneas with mutations at kerato-epithelin residue Arg-124 resulting in amyloid (R124C), non-amyloid (R124L), and a mixed pattern of deposition (R124H). We found that aggregated kerato-epithelin comprised all types of pathological deposits. Each mutation was associated with characteristic changes of protein turnover in corneal tissue. Amyloidogenesis in R124C corneas was accompanied by the accumulation of N-terminal kerato-epithelin fragments, whereby species of 44 kDa were the major constituents of amyloid fibrils. R124H corneas with prevailing non-amyloid inclusions showed accumulation of a new 66-kDa species altogether with the full-size 68-kDa form. Finally, in R124L cornea with non amyloid deposits, we found only the accumulation of the 68-kDa form. Two-dimensional gels revealed mutation-specific changes in the processing of the full-size protein in all affected corneas. It appears that substitutions at the same residue (Arg-124) result in cornea-specific deposition of kerato-epithelin via distinct aggregation pathways each involving altered turnover of the protein in corneal tissue.
betaig-h3 is a transforming growth factor-beta-inducible cell adhesion molecule that has four characteristic homologous repeated domains. We made recombinant betaig-h3 proteins, which were highly active in mediating human corneal epithelial (HCE) cell adhesion and spreading. The 2nd and the 4th repeated domains were sufficient to mediate HCE cell adhesion. A sequence analysis showed that aspartic acid (Asp) and isoleucine (Ile) of the 2nd and the 4th domains are highly conserved in many fasciclin 1 homologous (fas-1) domains. Substitution mutational study identified these two amino acids are essential for cell adhesion. Synthetic peptides containing Asp and Ile, NKDIL and EPDIM derived from the 2nd and the 4th domains, respectively, almost completely blocked cell adhesion mediated by not only wild type betaig-h3 but also each of the 2nd and the 4th domains. These peptides alone were fully active in mediating cell adhesion. In addition, we demonstrated the functional receptor for betaig-h3 is alpha(3)beta(1) integrin. These results, therefore, establish the essential motifs within the 2nd and the 4th domains of betaig-h3, which interact with alpha(3)beta(1) integrin to mediate HCE cell adhesion to betaig-h3 and suggest that other proteins containing Asp-Ile in their fas-1 domains could possibly function as cell adhesion molecules.
Proper growth and development require the orderly synthesis and deposition of individual components of the extracellular matrix
(ECM) into well ordered networks. Once formed, the ECM maintains tissue structure and houses resident cells. One ECM component,
βig-h3, is a highly conserved transforming growth factor-β-inducible protein that has been hypothesized to function as a bifunctional
linker between individual matrix components and resident cells. To gain insights into its physiological function, full-length
βig-h3 protein was produced using a baculovirus expression system and purified under native conditions. Human fibroblasts
attached and spread on βig-h3-coated plates and developed actin stress fibers. Purified βig-h3 binds fibronectin (FN) and
type I collagen (Col I) but does not bind gelatin. Using defined fragments of FN, we localized the βig-h3 recognition region
to the gelatin/collagen binding domain present in the N-terminal region of the FN molecule. Our results identify FN and Col
I as two ligands of βig-h3 in the ECM.
betaig-h3 is an extracellular matrix protein and its expression is highly induced by TGF-beta and it has also been suggested to play important roles in skin wound healing. In this paper, we demonstrate that betaig-h3 is present in the papillary layer of dermis and synthesized in the basal keratinocytes in vivo and its expression is induced by TGF-beta in normal human keratinocytes (NHEK) and HaCaT cells. betaig-h3 mediates not only adhesion and spreading of keratinocytes but also supports migration and proliferation. These activities are mediated through interacting with alpha3beta1 integrin. Previously identified two alpha3beta1 integrin-interacting motifs of betaig-h3, EPDIM, and NKDIL, are responsible for these activities. The results suggest that betaig-h3 may regulate keratinocyte functions in normal skin and potentially during wound-healing process.
Transforming growth factor-beta induced gene-h3 (betaig-h3) was found to co-purify with collagen VI microfibrils, extracted from developing fetal ligament, after equilibrium density gradient centrifugation under both nondenaturing and denaturing conditions. Analysis of the collagen VI fraction from the non-denaturing gradient by gel electrophoresis under non-reducing conditions revealed the present of a single high molecular weight band that immunostained for both collagen VI and betaig-h3. When the fraction was analyzed under reducing conditions, collagen VI alpha chains and betaig-h3 were the only species evident. The results indicated that betaig-h3 is associated with collagen VI in tissues by reducible covalent bonding, presumably disulfide bridges. Rotary shadowing and immunogold staining of the collagen VI microfibrils and isolated tetramers indicated that betaig-h3 was specifically and periodically associated with the double-beaded region of many of the microfibrils and that this covalent binding site was located in or near the amino-terminal globular domain of the collagen VI molecule. Using solid phase and co-immunoprecipitation assays, recombinant betaig-h3 was found to bind both native and pepsin-treated collagen VI but not individual pepsin-collagen VI alpha chains. Blocking experiments indicated that the major in vitro betaig-h3 binding site was located in the pepsin-resistant region of collagen VI. In contrast to the tissue situation, the in vitro interaction had the characteristics of a reversible non-covalent interaction, and the Kd was measured as 1.63 x 10(-8) m. Rotary shadowing of immunogold-labeled complexes of recombinant betaig-h3 and pepsin-collagen VI indicated that the in vitro betaig-h3 binding site was located close to the amino-terminal end of the collagen VI triple helix. The evidence indicates that collagen VI may contain distinct covalent and non-covalent binding sites for betaig-h3, although the possibility that both interactions use the same binding region is discussed. Overall the study supports the concept that betaig-h3 is extensively associated with collagen VI in some tissues and that it plays an important modulating role in collagen VI microfibril function.
To determine normal central and paracentral corneal thickness measurements in the pediatric population and to determine if these measurements are consistent across different pediatric age groups and different racial groups.
Prospective observational case series.
Pachymetry measurements were performed on 198 eyes of 108 children. The measurements were taken centrally as well as at four paracentral sites 3 mm from the corneal center at the 3, 6, 9, and 12 o'clock positions. The two-tailed t test was used for comparison of the continuous means for values of corneal thickness. Analysis of variance (ANOVA) was performed to determine differences among age and ethnic groups
The mean central corneal thickness (CCT) was 549 +/- 46 microm. Paracentral corneal thickness mean values, as measured 3 mm from the corneal center, were as follows: superior, 575 +/- 52 microm; nasal, 568 +/- 50 microm; inferior, 568 +/- 51 microm; and temporal, 574 +/- 47 microm. The mean CCT values were significantly thinner than at each of the mean paracentral points (P < .05 for each comparison, paired t test). Paracentral corneal thickness measurements demonstrated no significant differences between locations (P > .05, variance analysis). The mean CCT +/- SD for each age group was as follows: 6 to 23 months, 538 +/- 40 microm; 2 to 4 years, 546 +/- 41 microm; 5 to 9 years, 566 +/- 48 microm; and 10 to 18 years, 554 +/- 35 microm (ANOVA P = .012). ANOVA performed on central pachymetry values demonstrated no significant differences among racial subgroups.
Pediatric central and paracentral corneal thicknesses increase slowly over time and reach adult thicknesses at 5 to 9 years of age.
Diseases of the cornea are extremely common and cause severe visual impairment worldwide. To explore the basic molecular mechanisms involved in corneal health and disease, the present study characterizes the proteome of the normal human cornea. All proteins were extracted from the central 7-mm region of 12 normal human donor corneas containing all layers: epithelium, Bowman's layer, stroma, Descemet's membrane, and endothelium. Proteins were fractionated and identified using two different procedures: (i) two-dimensional gel electrophoresis and protein identification by MALDI-MS and (ii) strong cation exchange or one-dimensional SDS gel electrophoresis followed by LC-MS/MS. All together, 141 distinct proteins were identified of which 99 had not previously been identified in any mammalian corneas by direct protein identification methods. The characterized proteins are involved in many processes including antiangiogenesis, antimicrobial defense, protection from and transport of heme and iron, tissue protection against UV radiation and oxidative stress, cell metabolism, and maintenance of intracellular and extracellular structures and stability. This proteome study of the healthy human cornea provides a basis for further analysis of corneal diseases and the design of bioengineered corneas.
To determine genotypes in 2 Indian families with severe granular corneal dystrophy, to document clinical and histopathologic features, and to attempt a genotype-phenotype correlation.
Mutation analysis of exon 12 of the TGFBI gene was carried out in 9 individuals from 2 families.
A C-->T mutation at residue 1710 of TGFBI complementary DNA, corresponding to an Arg555Trp mutation in keratoepithelin, was found in affected members of both families. In 5 patients, this mutation was homozygous, and it was heterozygous in the other 4. Clinical examination revealed a severe form of granular corneal dystrophy with early onset and superficial lesions in the homozygous individuals and a milder phenotype in the heterozygous individuals. Histopathologic evaluation of corneal specimens from 2 homozygous patients confirmed the presence of superficial granular deposits.
To our knowledge, this is the first molecular and clinical characterization of severe granular corneal dystrophy in India. Genotype-phenotype correlation and comparison with earlier reports on this entity highlight the uniform expressivity of the Arg555Trp allele in homozygous individuals.
Homozygous granular corneal dystrophy has a severe phenotype and can be recognized based on clinical and histopathologic features, especially in association with consanguinity or inbreeding.
The lattice corneal dystrophies (LCD) and granular corneal dystrophies (GCD) are autosomal dominant disorders of the corneal stroma. They are bilateral, progressive conditions characterized by the formation of opacities arising due to the deposition of insoluble material in the corneal stroma leading to visual impairment. The LCDs and GCDs are distinguished from each other and are divided into subtypes on the basis of the clinical appearance of the opacities, clinical features of the disease, and on histopathological staining properties of the deposits. The GCDs and most types of LCD arise from mutations in the transforming growth factor beta-induced (TGFBI) gene on chromosome 5q31. Over 30 mutations causing LCD and GCD have been identified so far in the TGFBI. There are two mutation hotspots corresponding to arginine residues at positions 124 and 555 of the transforming growth factor beta induced protein (TGFBIp) and they are the most frequent sites of mutation in various populations. Mutations at either of these two hotspots result in specific types of LCD or GCD. The majority of identified mutations involve residues in the fourth fasciclin-like domain of TGFBIp.
Several inherited corneal disorders in humans result from mutations in the transforming growth factor beta induced gene (TGFBI), which encodes for the extracellular transforming growth factor beta induced protein (TGFBIp) that is one of the most abundant proteins in the cornea. We previously reported a significant amount of TGFBIp in plasma by immunoblotting using the only TGFBIp antiserum (anti-p68(beta ig-h3)) available at that time (anti-p68(beta ig-h3) was generated against residues Val210-His683 of TGFBIp). This observation raised the possibility that a fraction of corneal TGFBIp may originate from the plasma. However, recent experiments in our laboratory indicated that the anti-p68(beta ig-h3) antiserum cross-reacts with an environmental protein contaminant. Therefore, we investigated the specificity of the originally utilized anti-p68(beta ig-h3) antiserum and re-evaluated the amount of TGFBIp in human plasma by immunoblotting using a new specific antiserum.
The observed cross-reactivity of the previously utilized anti-p68(beta ig-h3) antiserum was tested by immunoblotting and the antigen identity was determined by mass spectrometry. A part of human TGFBI encoding an NH2-terminal 11.4 kDa fragment of TGFBIp (residues Gly134-Ile236) was amplified by polymerase chain reaction (PCR) and cloned in E. coli. The TGFBIp fragment was expressed in E. coli, purified by Ni2+-affinity chromatography, and used to immunize rabbits to produce a specific antiserum (anti-TGFBIp(134-236)). To enhance the detection of possible TGFBIp in plasma by allowing a higher sample load, albumin and immunoglobulin G (IgG) were specifically depleted from normal human plasma by affinity chromatography. The presence of TGFBIp in plasma was investigated by immunoblotting using the anti-TGFBIp(134-236) antiserum. Purified TGFBIp from porcine corneas was used for estimation of the TGFBIp detection limit.
The previously utilized TGFBIp antiserum, anti-p68(beta ig-h3), cross-reacted with human keratin-1, a common environmental protein contaminant. Thus, the anti-p68(beta ig-h3) antiserum recognizes both TGFBIp and keratin-1. In contrast, the anti-TGFBIp(134-236) antiserum reacted with TGFBIp but showed no indication of reactivity with other proteins in plasma. Using this antiserum, TGFBIp was not detected in crude or albumin/IgG-depleted human plasma and the detection limit of TGFBIp using immunoblotting was estimated to be 10 ng.
Our failure to detect TGFBIp in human plasma using a highly specific antiserum suggests that TGFBIp is not present in a physiologically relevant concentration in human plasma. The previous impression that normal human plasma contains a significant amount of TGFBIp by immunoblotting was due to the utilization of a less specific antiserum that recognizes both TGFBIp and human keratin-1. Together with other results, our observation makes it unlikely that TGFBIp is imported into the cornea from the circulation as reported for other abundant extracellular corneal proteins and suggests corneal origin of TGFBIp deposits in individuals with inherited corneal diseases caused by mutations in the TGFBI gene.
Adult human corneal epithelial basement membrane (EBM) and Descemet's membrane (DM) components exhibit heterogeneous distribution. The purpose of the study was to identify changes of these components during postnatal corneal development.
Thirty healthy adult corneas and 10 corneas from 12-day- to 3-year-old children were studied by immunofluorescence with antibodies against BM components.
Type IV collagen composition of infant corneal central EBM over Bowman's layer changed from alpha1-alpha2 to alpha3-alpha4 chains after 3 years of life; in the adult, alpha1-alpha2 chains were retained only in the limbal BM. Laminin alpha2 and beta2 chains were present in the adult limbal BM where epithelial stem cells are located. By 3 years of age, beta2 chain appeared in the limbal BM. In all corneas, limbal BM contained laminin gamma3 chain. In the infant DM, type IV collagen alpha1-alpha6 chains, perlecan, nidogen-1, nidogen-2, and netrin-4 were found on both faces, but they remained only on the endothelial face of the adult DM. The stromal face of the infant but not the adult DM was positive for tenascin-C, fibrillin-1, SPARC, and laminin-332. Type VIII collagen shifted from the endothelial face of infant DM to its stromal face in the adult. Matrilin-4 largely disappeared after the age of 3 years.
The distribution of laminin gamma3 chain, nidogen-2, netrin-4, matrilin-2, and matrilin-4 is described in the cornea for the first time. The observed differences between adult and infant corneal BMs may relate to changes in their mechanical strength, corneal cell adhesion and differentiation in the process of postnatal corneal maturation.
To report the clinical, ophthalmic, and genetic characteristics for lattice corneal dystrophy type I (LCDI) in a Chilean family.
Six affected family members were examined clinically including visual acuity, color cornea photography, applanation tonography, and fundoscopy. Genomic DNA was extracted from peripheral leukocytes from six affected and three unaffected members of a family with lattice corneal dystrophy type I. Exon 4 of the transforming growth factor-induced gene (TGFBI) was screened for the most frequent mutation, R124C, in the proband by sequencing. We also designed a rapid polymerase chain reaction-restriction fragment length polymorphism (PCR-RFLP) method to analyze the same mutation, amplifying exon 4 and digesting with PstI restriction enzyme. Using this strategy, we analyzed the mutation in six affected and three healthy family members.
Three generations of family members were positively diagnosed with lattice corneal dystrophy. Six participants demonstrated LCD1 in both eyes, most of whom were symmetric. Age at onset of symptoms was variable (3-42 years old). Moreover, in this family, the age of onset of the disease decreased in succeeding generations, which could be interpreted as anticipation. Visual acuity varied from 1.0 to 0.13. Two patients, ages 69 and 44 years old, demonstrated a degree of severity "Bad" according to best-corrected vision and corneal commitment. The exon 4 sequence of TGFBI of the proband exhibits the heterozygous single-nucleotide mutation, C417T, leading to amino acid substitution (R124C) in the encoded TGF-induced protein. Using PCR-RFLP, we confirmed the heterozygous mutation in six affected family members and excluded it in three healthy members.
The R124C mutation in TGFBI cosegregated with LCD type I in the investigated family. This is the first report of a molecular analysis of LCD type I in Chilean patients. The early onset affected persons in the fourth generation raises the possibility of anticipation.
Three buffer systems were compared for sodium dodecyl sulphate (SDS) electrophoresis of proteins and peptides in gels of 12.5% polyacrylamide. The system based on that of M. Wyckoff, A. Rodbard and A. Chrambach (Anal. Biochem., 78 (1977) 459) gave the best resolution, especially for polypeptides below 10,000 Mr, compared with the other two systems (based on those of D.M. Neville, J. Biol. Chem., 246 (1971) 6328 and U.K. Laemmli, Nature (London), 227 (1970) 680). The low concentration of SDS in the upper buffer (0.03%) suggested by Wyckoff et al. was found to be unsatisfactory for the resolution of some proteins and peptides and I have maintained this at 0.1%. The migration velocities of standard proteins and tracking dye in the systems varied in the order predicted by the multiphasic zone electrophoresis (MZE) theory, even in the presence of SDS. Differences of resolution in the systems were rationalized using MZE theory.
Transforming growth factor-beta (TGF-beta) is capable of affecting the proliferation of many cell types. To identify novel genes whose protein products may mediate cellular responses to this factor, a cDNA library was made from mRNA isolated from a human lung adenocarcinoma cell line (A549) that had been treated for 3 days with TGF-beta. The library was screened by differential hybridization and a cDNA clone, beta ig-h3, was isolated. This gene was induced up to 20-fold in A549 cells after 2 days of treatment with TGF-beta 1. It was also induced in several other cell lines, including PC-3 and H2981. DNA sequence analysis of beta ig-h3 indicated that it encoded a novel protein, beta IG-H3, of 683 amino acids, which contained an amino-terminal secretory sequence and a carboxy-terminal Arg-Gly-Asp (RGD) sequence that can serve as a ligand recognition site for several integrins. beta IG-H3 also contained short amino acid regions homologous to similar regions in Drosophila fasciclin-I and four homologous internal domains, which can be folded into a potential bivalent structure and could act as a bridge between cells expressing the appropriate ligand. beta ig-h3 RNA was detected in several cell lines and tissues. COS cells transfected with plasmids encoding beta IG-H3 secreted a major 68-kD protein that was detected by immunoblotting using antipeptide antibodies. Since beta ig-h3 is induced in several cell lines whose proliferation is affected by TGF-beta 1, it may be involved in mediating some of the signals of this multifunctional growth modulator.
βig-h3 is a TGF-β-induced matrix protein known to mediate the adhesion of several cell types. In this study, we found that
all four of the fas-1 domains in βig-h3 mediate MRC-5 fibroblast adhesion and that this was specifically inhibited by a function-blocking
monoclonal antibody specific for the αvβ5 integrin. Using deletion mutants of the fourth fas-1 domain revealed the MRC-5 cell
adhesion motif (denoted the YH motif) is located in amino acids 548–614. Experiments with substitution mutants showed that
tyrosine 571, histidine 572, and their flanking leucine and isoleucine amino acids, which are all highly conserved in many
fas-1 domains, are essential for mediating MRC-5 cell adhesion. A synthetic 18-amino acid peptide encompassing these conserved
amino acids could effectively block MRC-5 cell adhesion to βig-h3. Using HEK293 cells stably transfected with the β5 integrin
cDNA, we confirmed that the αvβ5 integrin is a functional receptor for the YH motif. In conclusion, we have identified a new
αvβ5 integrin-interacting motif that is highly conserved in the fas-1 domains of many proteins. This suggests that fas-1 domain-containing
proteins may perform their biological functions by interacting with integrins.
Central corneal thickness was measured optically in premature and full-term babies, and in children. The thickness was found to decrease from the values found in premature and full-term babies to those found in small children aged between 2–4 years. The thickness of the adult cornea is reached at the age of about 3 years.
These findings are discussed with respect to the possible role of corneal thickness as a biometric parameter, to intraocular pressure measurement, and to corneal thickness steady state regulation.
In previous studies, RGD-CAP (collagen-associated protein containing the RGD sequence) isolated from a collagen fiber-rich fraction of pig cartilage was found to be orthologous to human βig-h3, which is synthesized by lung adenocarcinoma cells in response to transforming growth factor-β. In the present study, we examined the effect of recombinant chick RGD-CAP on the spreading of chondrocytes and fibroblasts using RGD-CAP-coated dishes. When rabbit articular chondrocytes, chick embryonic sternal chondrocytes, rabbit peritoneal fibroblasts or human MRC5 fibroblasts were seeded on plastic dishes coated with RGD-CAP, cell spreading was enhanced compared with that on control dishes (bovine serum albumin- or β-galactosidase-coated dishes). The effect of RGD-CAP on the cell spreading required divalent cations (Mg2+ or Mn2+), and was reduced by EDTA. Monoclonal antibodies (mAbs) to the human integrin α1 or β1 subunit, but not to the α2, α3, α5 or β2 subunits, suppressed the RGD-CAP-induced spreading of human MRC5 fibroblasts. In a parallel experiment, the mAb to the α5 subunit, but not the mAb to the α1 subunit, suppressed fibronectin-induced spreading of these cells. These findings suggest that RGD-CAP is a novel ligand for integrin α1β1 that dose not bind to the RGD motif. Accordingly, an RGD-CAP fragment, which carries a deletion in the C-terminal region containing the RGD motif, was still capable of stimulating cell spreading.
Central corneal thickness was measured optically in premature and full-term babies and in children. The thickness was found to decrease from the values found in premature and full-term babies to those found in small children aged between 2-4 years. The thickness of the adult cornea is reached at the age of about 3 years. These findings are discussed with respect to the possible role of corneal thickness as a biometric parameter, to intraocular pressure measurement, and to corneal thickness steady state regulation.
Transforming growth factor-beta (TGF-beta) is capable of affecting the proliferation of many cell types. To identify novel genes whose protein products may mediate cellular responses to this factor, a cDNA library was made from mRNA isolated from a human lung adenocarcinoma cell line (A549) that had been treated for 3 days with TGF-beta. The library was screened by differential hybridization and a cDNA clone, beta ig-h3, was isolated. This gene was induced up to 20-fold in A549 cells after 2 days of treatment with TGF-beta 1. It was also induced in several other cell lines, including PC-3 and H2981. DNA sequence analysis of beta ig-h3 indicated that it encoded a novel protein, beta IG-H3, of 683 amino acids, which contained an amino-terminal secretory sequence and a carboxy-terminal Arg-Gly-Asp (RGD) sequence that can serve as a ligand recognition site for several integrins. beta IG-H3 also contained short amino acid regions homologous to similar regions in Drosophila fasciclin-I and four homologous internal domains, which can be folded into a potential bivalent structure and could act as a bridge between cells expressing the appropriate ligand. beta ig-h3 RNA was detected in several cell lines and tissues. COS cells transfected with plasmids encoding beta IG-H3 secreted a major 68-kD protein that was detected by immunoblotting using antipeptide antibodies. Since beta ig-h3 is induced in several cell lines whose proliferation is affected by TGF-beta 1, it may be involved in mediating some of the signals of this multifunctional growth modulator.
The fasciclin I, II, and III glycoproteins are expressed on different subsets of axon bundles (fascicles) in insect embryos and are thus candidates for surface recognition molecules involved in growth cone guidance. Here we present the sequence of grasshopper fasciclin I and the identification and sequence of the Drosophila fasciclin I homolog. In both species, fasciclin I appears to be an extrinsic membrane protein with a signal sequence but no transmembrane region; the protein comprises four homologous domains of approximately 150 amino acids each. Antibodies against Drosophila fasciclin I reveal that it is expressed on the surface of a subset of commissural axon pathways in the embryonic central nervous system and on all sensory axon pathways in the peripheral nervous system. This pattern of expression is similar to that in grasshopper.
The origin, growth in thickness, and differentiation of Descemet's membrane was studied by light, electron microscopic, morphometric, and statistical methods in 67 specimens from 12 weeks of gestation to 98 years. Descemet's membrane is formed by three major processes: growth in thickness during the prenatal period, prenatal differentiation into a striated basement membrane, and growth in thickness during the postnatal period. The initial step is the synthesis of an ordinary basement membrane, which is very thin and quite different in appearance from the adult Descemet's membrane. Growth of the prenatal Descemet's membrane then proceeds by deposition of a series of similar "membrane units," which are stacked to form a lamellar structure consisting of at least 30 layers by the end of gestation. Second, during prenatal life, differentiation of the membrane leads to the formation of a striated structure through the gradual addition of short and thin cross-linking bridges separated by 110-nm intervals that are disposed in a plane perpendicular to the lamellae. The third process occurs in postnatal life when the membrane continues to grow in thickness by deposition of a nonstriated, nonlamellar material posterior to the striated prenatal layer. Regression analysis suggests that prenatal growth proceeds at a rapid but variable rate best described by a "sigmoid-like" function of age. Postnatal growth, in contrast, proceeds in a predominantly exponential manner but at a slower pace than in the prenatal period. The low variability and large size of our set of measurements make these data especially useful for comparisons with pathologic specimens.
The non-pigmented ciliary epithelium is largely responsible for the formation of aqueous humor in the mammalian eye. To provide a basis for studies at the molecular level, a directional expression cDNA library was constructed in Uni-ZAP XR vector from poly A+ RNA of the human non-pigmented ciliary epithelial derived ODM-2 cell line. Fifty-three cDNA clones were isolated from the library and characterized by partial sequence analysis. Approximately 49% of the clones exhibited homology with known genes in the GenBank/EMBL databases. The putative identification of these clones may reflect the transcriptional activity of the ODM-2 cells in culture. One of the identified clones, ODM-42-I, was found to be specific and highly expressed in the corneal epithelium. This clone had an exact match with a recently discovered human gene, beta ig-h3 (Skonier et al., 1992, DNA Cell Biol., 11:511-522), which codes a surface recognition protein, inducible by transforming growth factor beta (TGF-beta), and containing a putative binding site (RDG) for integrins. The ODM-42-I cDNA clone displays a distinctive pattern of expression found in the human eye, expressed almost exclusively in the cornea. Further studies, using sera from a synthetic peptide to the carboxy-terminal region of ODM-42-I, reveal that the protein is heterogeneous in charge and is preferentially expressed on the extracellular surface of corneal epithelial cells, and might share immunologic properties with integrins beta 1.
A 66-kDa collagen fiber-associated protein (RGD-CAP) was isolated from a fiber-rich fraction of pig cartilage by ultrafiltration and collagen-affinity chromatography. Amino acid sequencing and cDNA cloning indicated that the RGD-CAP is identical or closely related to beta ig-h3 protein which is induced in human adenocarcinoma cells by transforming growth factor-beta (TGF-beta) (Skonier, J., Neubauer, M., Madisen, L., Bennett, K., Plowman, G.D., and Purchio, A.F. (1992) DNA Cell. Biol. 11, 511-522). The RGD-CAP, as well as beta ig-h3, has the RGD sequence in the C-terminal region. The native RGD-CAP bound to type I, II, and IV collagens even in the presence of 1 M NaCl. A recombinant preparation of RGD-CAP expressed in Escherichia coli cells also bound to collagen but not to gelatin. The RGD-CAP mRNA was expressed in chondrocytes throughout all stages, although the expression level was highest during the prehypertrophic stage. In addition, TGF-beta increased the RGD-CAP mRNA level in chondrocyte cultures. Since RGD-CAP transcripts were found in most tissues, this novel collagen-binding protein may play an important role in cell-collagen interactions in various tissues including developing cartilage.
To report a family with several members affected with granular corneal dystrophy Groenouw type 1. Three members of the family were affected with a severe placoid type of corneal dystrophy. To determine the relationship between gene mutations and phenotypic variations of the disease, we analyzed the kerato-epithelin gene.
The pedigree included a consanguineous marriage of two affected individuals. The three family members affected with a severe form of corneal dystrophy were offspring of these parents. However, the phenotype of other affected family members was typical granular corneal dystrophy. We isolated genomic DNA from leukocytes of the family members. Exons of the keratoepithelin gene were amplified by the polymerase chain reaction and were analyzed using the single-strand conformation polymorphism technique. Mutations were identified by direct sequencing method and restriction digestion analysis.
The three severely affected family members exhibited homozygous mutations at codon 555 (arginine to tryptophan) in the keratoepithelin gene, whereas those with typical granular corneal dystrophy had the heterozygous mutation at the same codon. Unaffected family members did not have the mutation.
We determined that the severe phenotype of granular corneal dystrophy is caused by homozygous mutations in the kerato-epithelin gene. Clinical manifestation of the severe phenotype is a placoid type of corneal dystrophy and early recurrence after surgery. Granular corneal dystrophy appears to be the first ophthalmic disease in which homozygosity for a dominant allele has been genetically identified.
Mutations in the betaIGH3 gene on chromosome 5q31 cause five distinct autosomal dominant corneal dystrophies: granular Groenouw type I, Reis-Bücklers', lattice type I and IIIA. and Avellino corneal dystrophies. We present here a new mutation of the betaIGH3 gene in patients with late-onset lattice corneal dystrophy manifest as a deep stromal opacity. To test the previously reported R124C, R124H, P501T, R555W, and R555Q mutations of the betaIGH3 gene, 30 patients and 11 normal relatives from 16 independently ascertained families with lattice corneal dystrophy, 49 patients and 12 normal relatives from 40 independently ascertained families with other corneal dystrophies, and 40 unrelated normal volunteers, were analyzed. A L527R (CTG/CGG) mutation of the betaIGH3 gene was found in 6 unrelated patients with lattice corneal dystrophy. A retrospective review of the patients' records showed that the opacities were deep in the stromal layer and of late onset. The mutation was a heterozygous single base-pair transversion from T to G of the second nucleotide position of codon 527. This caused the substitution of arginine for leucine. These six patients did not have mutations in codons 124, 501, or 555. The L527R mutation was not detected in the other corneal dystrophies or 40 normal volunteers. Although phenotypic variations in the size and shape of the deposits were found, all patients with the L527R mutation showed deposits deep in the stromal layer. We conclude that there are now at least six different mutations that have been detected in the betaIGH3 gene on chromosome 5q31 and that lead to corneal dystrophy.
In previous studies, RGD-CAP (collagen-associated protein containing the RGD sequence) isolated from a collagen fiber-rich fraction of pig cartilage was found to be orthologous to human (beta)ig-h3, which is synthesized by lung adenocarcinoma cells in response to transforming growth factor-beta. In the present study, we examined the effect of recombinant chick RGD-CAP on the spreading of chondrocytes and fibroblasts using RGD-CAP-coated dishes. When rabbit articular chondrocytes, chick embryonic sternal chondrocytes, rabbit peritoneal fibroblasts or human MRC5 fibroblasts were seeded on plastic dishes coated with RGD-CAP, cell spreading was enhanced compared with that on control dishes (bovine serum albumin- or beta-galactosidase-coated dishes). The effect of RGD-CAP on the cell spreading required divalent cations (Mg(2+) or Mn(2+)), and was reduced by EDTA. Monoclonal antibodies (mAbs) to the human integrin alpha(1) or beta(1) subunit, but not to the alpha(2), alpha(3), alpha(5) or beta(2) subunits, suppressed the RGD-CAP-induced spreading of human MRC5 fibroblasts. In a parallel experiment, the mAb to the alpha(5) subunit, but not the mAb to the alpha(1) subunit, suppressed fibronectin-induced spreading of these cells. These findings suggest that RGD-CAP is a novel ligand for integrin alpha(1)beta(1) that dose not bind to the RGD motif. Accordingly, an RGD-CAP fragment, which carries a deletion in the C-terminal region containing the RGD motif, was still capable of stimulating cell spreading.
To characterize the betaig-h3 gene defect in a French family affected with lattice corneal dystrophy type IIIA (LCDIIIA).
Histologic examination was performed from corneal buttons of two patients. Genomic DNA was extracted from leukocytes, and exons of the betaig-h3 gene were amplified by polymerase chain reaction to be directly sequenced.
Numerous deposits were evident in the stroma and beneath the Bowman membrane, which had all the features of amyloid deposits. Analysis of exon 12 revealed a heterozygous G to A transition on codon 546.
In contrast to Japanese patients, these French patients affected with LCDIIIA carry a distinct mutation of the betaig-h3 gene (A546T instead of P501T). Therefore, it is unclear whether different mutations could result in the same dystrophy or whether we are dealing with clinical heterogeneity of LCDIIIA.
The N-terminal cysteine-rich domain (EMI domain) of EMILIN-1 is a new protein domain that is shared with two proteins (multimerin and EMILIN-2) and with four additional database entries. The EMI domains are always located at the N-terminus, have a common gene organization, and belong to proteins that are forming or are compatible with multimer formation. The potential role of the EMI domain in the assembly of EMILIN-1 was investigated by the two-hybrid system. No reporter gene activity was detected when EMI-1 was co-transformed with the C-terminal gC1q-1 domain excluding a head-to-tail multimerization; conversely, a strong interaction was detected when the EMI-1 domain was co-transformed with the gC1q-2 domain of EMILIN-2.
General Protein/Mass Analysis for Windows (GPMAW) is a valuable piece of software for any molecular biologist, biochemist or mass spectrometrist wishing to analyze protein or peptide sequences. All steps from the acquisition of protein sequence from a built-in web interface, to proteolytic digests, theoretical peptide fragmentation, detailed annotation of sequences and secondary structure prediction, can be performed rapidly and intuitively without first having to spend days reading manuals.
βig-h3 is a TGF-β-induced matrix protein known to mediate the adhesion of several cell types. In this study, we found that
all four of the fas-1 domains in βig-h3 mediate MRC-5 fibroblast adhesion and that this was specifically inhibited by a function-blocking
monoclonal antibody specific for the αvβ5 integrin. Using deletion mutants of the fourth fas-1 domain revealed the MRC-5 cell
adhesion motif (denoted the YH motif) is located in amino acids 548–614. Experiments with substitution mutants showed that
tyrosine 571, histidine 572, and their flanking leucine and isoleucine amino acids, which are all highly conserved in many
fas-1 domains, are essential for mediating MRC-5 cell adhesion. A synthetic 18-amino acid peptide encompassing these conserved
amino acids could effectively block MRC-5 cell adhesion to βig-h3. Using HEK293 cells stably transfected with the β5 integrin
cDNA, we confirmed that the αvβ5 integrin is a functional receptor for the YH motif. In conclusion, we have identified a new
αvβ5 integrin-interacting motif that is highly conserved in the fas-1 domains of many proteins. This suggests that fas-1 domain-containing
proteins may perform their biological functions by interacting with integrins.
Beta(ig)-h3 is a secretory protein that is induced by transforming growth factor (TGF)-beta. We have recently found that beta(ig)-h3 expression is induced in cultured astrocytes by TGF-beta1 and in rat cerebral cortex by stab wound. The purpose of this study was to examine the effect of the secreted beta(ig)-h3 on cell adhesion of astrocytes and the underlying mechanisms. When U87 human astrocytoma cells were seeded on dishes coated with recombinant beta(ig)-h3, cell adhesion was significantly enhanced. Blocking experiments using various antibodies to the integrin subunit suggested alpha6beta4 integrin could be involved in the beta(ig)-h3-mediated astrocyte cell adhesion. Cell adhesion to beta(ig)-h3 substrate was substantially blocked by preincubation with the inhibitor to the src kinase. When cells were plated on beta(ig)-h3-coated dishes, tyrosine phosphorylation of focal adhesion kinase was prominently increased within 20 min in a beta4 integrin-dependent manner. The results suggest that alpha6beta4 integrin-mediated interactions of astrocytes with beta(ig)-h3 transduce intracellular signals through the focal adhesion proteins, which may regulate certain aspects of astrocyte response to brain injury.
The pertinent literature on inherited corneal diseases is reviewed in terms of the chromosomal localization and identification of the responsible genes. Disorders affecting the cornea have been mapped to human chromosome 1 (central crystalline corneal dystrophy, familial subepithelial corneal amyloidosis, early onset Fuchs dystrophy, posterior polymorphous corneal dystrophy), chromosome 4 (Bietti marginal crystalline dystrophy), chromosome 5 (lattice dystrophy types 1 and IIIA, granular corneal dystrophy types 1, 2 and 3, Thiel-Behnke corneal dystrophy), chromosome 9 (lattice dystrophy type II), chromosome 10 (Thiel-Behnke corneal dystrophy), chromosome 12 (Meesmann dystrophy), chromosome 16 (macular corneal dystrophy, fish eye disease, LCAT disease, tyrosinemia type II), chromosome 17 (Meesmann dystrophy, Stocker-Holt dystrophy), chromosome 20 (congenital hereditary endothelial corneal dystrophy types I and II, posterior polymorphous corneal dystrophy), chromosome 21 (autosomal dominant keratoconus) and the X chromosome (cornea verticillata, cornea farinata, deep filiform corneal dystrophy, keratosis follicularis spinulosa decalvans, Lisch corneal dystrophy). Mutations in nine genes (ARSC1, CHST6, COL8A2, GLA, GSN, KRT3, KRT12, M1S1and TGFBI [BIGH3]) account for some of the corneal diseases and three of them are associated with amyloid deposition in the cornea (GSN, M1S1, TGFBI) including most of the lattice corneal dystrophies (LCDs) [LCD types I, IA, II, IIIA, IIIB, IV, V, VI and VII] recognized by their lattice pattern of linear opacities. Genetic studies on inherited diseases affecting the cornea have provided insight into some of these disorders at a basic molecular level and it has become recognized that distinct clinicopathologic phenotypes can result from specific mutations in a particular gene, as well as some different mutations in the same gene. A molecular genetic understanding of inherited corneal diseases is leading to a better appreciation of the pathogenesis of these conditions and this knowledge has made it imperative to revise the classification of inherited corneal diseases.
Mutations in the TGFBI (BIGH3) gene that encodes for transforming growth factor beta induced protein (TGFBIp) are the cause of several phenotypically different corneal dystrophies. While the genetics of these protein misfolding diseases are well documented, relatively little is known about this extracellular matrix protein itself. In this study, we have purified TGFBIp from normal human and porcine corneas using nondenaturing conditions and standard chromatography techniques. The two homologues were shown to be monomers, and we did not find evidence for posttranslational additions. The C-terminal of both human and porcine TGFBIp is truncated predominantly after the integrin binding sequence Arg(642)-Gly(643)-Asp(644) (RGD). However, using an antibody against the C-terminal fragment (residues 648-683), we also detected a small amount of full-length TGFBIp in corneal extracts. Approximately 60% of TGFBIp was covalently associated with insoluble components of the extracellular matrix in both human and porcine corneas through a disulfide bridge.
To investigate the location and tissue-specificity of the pathologic keratoepithelin (KE) deposition in a patient with a keratoepithelinopathy (KEP), TGFBI/BIGH3-related corneal dystrophy.
An autopsy was performed in a patient with lattice type I corneal dystrophy (LCDI) after authorization was obtained from the family. Mutation screening in TGFBI/BIGH3 was done on the patient several years ago. Eighteen different tissues or organs, including brain, heart, lung, kidney, liver, lymph nodes, spleen, aorta, esophagus, bone marrow, urinary bladder (including a papillary urothelial carcinoma), samples of a metastatic squamous cell carcinoma, adrenal gland, parathyroid gland, muscle, prostate, and cornea were investigated, and sections from the tissues were labeled with KE2 rabbit TGFBI/BIGH3 antiserum.
The patient, diagnosed with LCDI and Alzheimer's disease, died at 79 years of age from a complicated chronic obstructive lung disease. Mutation analysis showed the classical Arg124Cys mutation in exon 4 of TGFBI/BIGH3, associated with LCDI. Except for the cornea, immunostaining with KE2 antisera did not reveal any deposits in any of the 17 other organs analyzed.
Pathologic deposits caused by KE accumulation were only observed in the cornea and in no other tissue or organ in this patient. These results suggest a cornea-specific mechanism in the aggregation of KE. Further studies need to be done to investigate whether the degradation of mutated KE generates cornea-specific fragments that aggregate or whether the clearing of normal fragments is different in affected corneas, which then leads to aggregation.
Increased biochemical knowledge of normal and diseased corneas is essential for the understanding of corneal homeostasis and pathophysiology. In a recent study, we characterized the proteome of the normal human cornea and identified 141 distinct proteins. This dataset represents the most comprehensive protein study of the cornea to date and provides a useful reference for further studies of normal and diseased human corneas. The list of identified proteins is available at the Cornea Protein Database. In the present paper, we review the utilized procedures for extraction and fractionation of corneal proteins and discuss the potential roles of the identified proteins in relation to homeostasis, diseases, and wound-healing of the cornea. In addition, we compare the list of identified proteins with high quality gene expression libraries (cDNA libraries) and Serial Analysis of Gene Expression (SAGE) data. Of the 141 proteins, 86 (61%) were recognized in cDNA libraries from the corneas of dogs and rabbits, or humans with keratoconus, and 98 (69.5%) were recognized in SAGE data of mouse and human corneas. However, the percentages of identified genes in each of the protein functional groups differed markedly. Thus, exceptionally few of the traditional blood/plasma proteins and immune defense proteins that were identified in the human cornea were recognized in the gene expression libraries of the cornea. This observation strongly indicates that these abundant corneal proteins are not expressed in the cornea but originate from the surrounding pericorneal tissue.
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Identification of motifs for cell adhesion within the repeated domains of transforming growth factor-beta-induced gene, betaig-h3
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Analysis of protein and peptide mixtures. Evaluation of three sodium dodecyl sulfate-polyacrylamide gel electrophoresis buffer systems
- Bury
The transforming growth factor-beta-inducible matrix protein (beta)ig-h3 interacts with fibronectin
- Billings
Discovery of a novel protein (big-h3) in normal human cornea
- Klintworth