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Identification and Localization of Type IV Collagen Chains in the Inner Ear Cochlea
Abstract
Mutations in the genes encoding the alpha3(IV), alpha4(IV) and alpha5(IV) chains of type IV collagen have been implicated in the pathogenesis of Alport's syndrome, a hereditary disorder characterized by progressive nephropathy and sensorineural deafness. The known expression of these chains in kidney basement membranes supports the contention that they play a crucial role in the ultrafiltration function. Whether they play a role in auditory signal transduction remains unknown as heretofore, they have not been identified in the inner ear. In the present study, the expression of type IV collagen in cochlea of the inner ear of guinea pigs was determined. All six alpha-chains of type IV collagen were identified by biochemical and immunological methods. By indirect immunofluorescence, alpha1(IV) and alpha2(IV) chains were localized to the spiral limbus, basilar membrane and tectorial membrane. The alpha3(IV), alpha4(IV), alpha5(IV) and alpha6(IV) chains localized exclusively to the tectorial membrane and basilar membrane. These results suggest a possible role of type IV collagen chains in the active tuning of the basilar and tectorial membrane, an essential step in frequency discrimination and amplification of auditory signals.
... BMs are intertwined networks of polymeric laminin and type IV collagen, bridged by noncovalent interactions with nidogens and proteoglycans (Kalluri, 2003). Most of the studies of collagen IV, laminin, and nidogen expression in cochlear BMs have been in rodent species: mouse (Cosgrove et al., 1996;Rodgers et al., 2001;Meyer zum Gottesberge and Felix, 2005), guinea pig (Weinberger et al., 1999;Kalluri et al., 1998;Takahashi and Hokunan, 1992), rat (Satoh et al., 1998) or chinchilla (Tsuprun and Santi, 2001). There are only a few such studies of the vestibular periphery in any mammalian model (Yamashita et al., 1991;Swartz and Santi, 1999), and there are less than a handful of studies that investigate the composition of human cochlear BMs (Zehnder et al., 2005;Yamashita et al., 1991;Kleppel et al., 1989). ...
... Kleppel et al. (1989) also studied collagen IVα1 distribution in normative human, and reported a similar distribution, including perivascular and perineural cochlear BMs, spiral limbus, spiral ligament, the pars pectinata of the basilar membrane, and the osseous spiral lamina. Colocalization of collagen IVα1 and α2 chains has been described in the mouse and guinea pig cochlear BMs (Cosgrove et al., 1996;Kalluri et al., 1998;Meyer zum Gottesberge and Felix, 2005). Mouse kidney glomerular BMs also coprecipitated α1 and α2 (Kalluri and Cosgrove, 2000), and it is now widely believed that the six genetically distinct collagen IVα chains assemble into only three heterotrimers: α1α1α2; α3α4α5; α5α5α6 (Khoshnoodi et al., 2008). ...
The immunolocalization of several basement membrane (BM) proteins was investigated in vestibular endorgans microdissected from temporal bones obtained from subjects with a documented normal auditory and vestibular function (n=5, average age=88 years old). Fluorescent immunostaining using antibodies directed at collagen IV alpha 2, nidogen-1, laminin-beta2, alpha-dystroglycan, and tenascin-C was applied to cryosections from human cochlea, cristae ampullares, utricular and saccular maculae. Collagen IV alpha 2, nidogen-1, and laminin-beta2 localized to all subepithelial cochlear BMs, Reissner's membrane, strial and spiral ligamental perineural and perivascular BMs, and the spiral limbus. Tenascin-C localized to the basilar membrane and the osseous spiral lamina. alpha-Dystroglycan localized to most cochlear BMs except those in the spiral ligament, basilar membrane and spiral limbus. Collagen IV, nidogen-1, and laminin-beta2 localized to the subepithelial BMs of the maculae and cristae ampullares, and the perineural and perivascular BMs within the underlying stroma. The BM underlying the transitional and dark cell region of the cristae ampullares also expressed collagen IV, nidogen-1, and laminin-beta2. Tenascin-C localized to the subepithelial BMs of the utricular maculae and cristae ampullares, and to calyx-like profiles throughout the vestibular epithelium, but not to the perineural and perivascular BMs. alpha-Dystroglycan colocalized with aquaporin-4 in the basal vestibular supporting cell, and was also expressed in the subepithelial BMs, as well as perivascular and perineural BMs. This study provides the first comprehensive immunolocalization of these ECM proteins in the human inner ear. The validity of the rodent models for inner ear disorders secondary to BM pathologies was confirmed as there is a high degree of conservation of expression of these proteins in the human inner ear. This information is critical to begin to unravel the role that BMs may play in human inner ear physiology and audiovestibular pathologies.
... 26 Interestingly type IV collagen fibres similar to those seen predominantly in the lens capsule have been isolated in both the tectorial and basilar membranes of an animal model. 27 Besides its primary mechanical function, the tectorial membrane appears to be involved in controlling the chemical microenvironment of the stereocilia. 7,8 Furthermore, the auditory hair cells appear to be actively involved in mechanically tuning the basilar membrane. ...
... Structural alteration of either (or both) the tectorial and basilar membranes by deposition of PXF fibrils could change the way in which vibratory energy is conducted to the sensory hair cells and alter their surrounding chemical environment resulting in sensorineural hearing loss. 26,27 Interestingly, the majority of ears ipsilateral to eyes without PXF also had elevated hearing thresholds. Examination of the laterality of hearing loss with regard to the presence of PXF in the ipsilateral eye may be artificial in view of the small numbers in this study. ...
There is increasing evidence that pseudoexfoliation (PXF) not only affects ocular anterior segment structures, but may also be a systemic disease. This study was undertaken to assess the relationship between PXF and sensorineural hearing loss.
Patients with PXF were identified from hospital records and underwent complete ocular examination. The sum of pure-tone hearing thresholds measured at 1, 2 and 3 kHz (HTL1,2,3) in each ear was compared with the ISO 7029 standard sex-matched, median age-associated hearing loss summed over the same frequencies (AAHL1,2,3). The proportion of ears with thresholds higher than the ISO 7029 median AAHL1,2,3 on the same side as eyes without PXF was compared with the proportion of ears ipsilateral to eyes with PXF but without glaucoma and similarly the proportion of ears on the same side as eyes with PXF and glaucoma.
In total, 69 patients were studied, of whom 39 were male (56.5%). The mean age of the male patients was 75.8 years, while that of the female group was 75.1 years. All patients had PXF affecting at least one eye. Overall 101 ears (73.7%) had a higher HTL1,2,3 than the ISO 7029 median AAHL1,2,3 which included 56 ears of 78 in the male group (71.8%) and 45 ears of 59 in the female group (76.3%). There was no significant difference between the proportion of ears with HTL1,2,3 higher than the ISO 7029 median AAHL1,2,3 on the same side as eyes without PXF, with PXF but not glaucoma and with PXF and glaucoma, in either the male or female groups.
A large proportion of patients with PXF have sensorineural hearing loss in comparison to age-matched controls, regardless of whether or not there is associated glaucoma. This finding supports the theory that PXF may be a systemic condition.
... As cadeias 3(IV), 4(IV), 5(IV) e 6(IV) foram localizadas, exclusivamente, nas membranas tectória e basilar, da cóclea de cobaias normais, por imunofluorescência indireta 17 . Estes resultados sugerem uma possível função das cadeias do colágeno tipo IV no ajuste ativo das membranas basilar e tectória, uma etapa essencial na discriminação da freqüência e na amplificação dos sinais auditivos 17 . ...
... As cadeias 3(IV), 4(IV), 5(IV) e 6(IV) foram localizadas, exclusivamente, nas membranas tectória e basilar, da cóclea de cobaias normais, por imunofluorescência indireta 17 . Estes resultados sugerem uma possível função das cadeias do colágeno tipo IV no ajuste ativo das membranas basilar e tectória, uma etapa essencial na discriminação da freqüência e na amplificação dos sinais auditivos 17 . ...
Alport Syndrome is a genetic disorder characterized by hematuria, which often leads to renal failure. It may also be accompanied by extra-renal alterations, such as: sensorineural hearing loss, and ocular abnormalities. Dominant forms related to the X chromosome and caused by mutations in the locus COL4A5 have been described, as well as an autossomic recessive form resulting from mutations in the locus COL4A3 or COL4A4. An autossomic dominant type of AS has also been reported. The disease is caused by changes in the collagen type IV chains, where symptoms reflect the damage to the basal membrane of several organs. The alpha3.alpha4.alpha5(IV) networks are found in the kidneys, cochlea and eyes. The objective was to characterize AS in this group of patients. In the current literature review it was found that: 1. AS is characterized by hematuria that may develop into renal failure and can also be accompanied by extra-renal manifestations. Hearing loss is a frequent extra-renal finding and one of the first symptoms of AS, therefore representing a relevant factor in the prognosis of the renal disease; 2. It is a genetic disorder resulting from abnormalities in the chains of collagen type IV in the basal membranes; 3. The hearing loss in AS is typically sensorineural with variable intensities, progressive and symmetrical, affecting middle and high frequencies; 4. Otolaryngologists should include a urine test in the SNHL work-up. It is essential to have an otologist involved in the treatment of these patients.
... Introduction membrane nephropathy, and focal segmental glomerulosclerosis [5,[28][29][30][31]. The α3α4α5 protomer is also noted in the basement membranes of many other tissues, including brain, alveoli, testes, cochlea, the synapses of neuromuscular junctions, and lung epithelia in mammals [32][33][34][35][36][37][38]. Genetic disruption of the α3α4α5 network composition in these basement membranes is associated with multiple organ impairment in Alport syndrome [39]. ...
Type IV collagen, the most abundant component of basement membranes, is essential for the formation of the extracellular scaffold that supports tissue architecture and function. Collagen IV is present in all multicellular species, with lower organisms typically possessing two type IV collagen genes, encoding α1 and α2 chains. The human genome encodes for six different type IV collagen genes, α1 to α6 chains. The α chains assemble into trimeric protomers, the building blocks of the type IV collagen network. The detailed evolutionary conservation of type IV collagen network organization remains to be studied. Here we report on the molecular evolution of type IV collagen genes. Specifically, we report on the zebrafish α4 NC1 domain, which, in contrast with its human ortholog, contains an additional cysteine residue and lacks the Met93 and Lys211 residues involved in sulfilimine bond formation between adjacent protomers. This may alter α4 chain interactions with other α chains, as supported by temporal and anatomic expression patterns of collagen IV chains during zebrafish development. Despite the divergence between zebrafish and human α3 NC1 domain (human endogenous inhibitor of angiogenesis, Tumstatin), the zebrafish α3 NC1 domain exhibits conserved anti-angiogenic activity in human endothelial cells. Our work supports that sequence identity, tissue expression, and function of type IV collagen have remained largely conserved between zebrafish and humans, with one possible difference involving the α4 NC1 domain.
... Most studies concerning collagen distribution in the inner ear concern the cochlea. Kalluri, Gattone 2nd, and Hudson (1998) determined the expression of collagen IV in the inner ear of guinea pigs: α1 and α2 chains were localized in the spiral limbus, basilar membrane and tectorial membrane; α3, α4, α5, and α6 chains were localized exclusively in the tectorial membrane and basilar membrane. ...
Alport syndrome (AS) is caused by mutations in collagen IV, which is widespread in the basement membranes of many organs, including the kidneys, eyes, and ears. Whereas the effects of collagen IV changes in the cochlea are well known, no changes have been described in the posterior labyrinth. The aim of this study was to investigate both the auditory and the vestibular function of a group of individuals with AS. Seventeen patients, aged 9–52, underwent audiological tests including pure‐tone and speech audiometry, immittance test and otoacoustic emissions and vestibular tests including video head impulse test, rotatory test, and vestibular evoked myogenic potentials. Hearing loss affected 25% of the males and 27.3% of the females with X‐linked AS. It was sensorineural with a cochlear localization and a variable severity. 50% of the males and 45.4% of the females had a hearing impairment in the high‐frequency range. Otoacoustic emissions were absent in about one‐third of the individuals. A peripheral vestibular dysfunction was present in 75% of the males and 45.4% of the females, with no complaints of vertigo or dizziness. The vestibular impairment was compensated and the vestibulo‐ocular reflex asymmetry was more evident in rotatory tests carried out at lower than higher speeds; a vestibular hypofunction was present in all hearing impaired ears although it was also found in subjects with normal hearing. A posterior labyrinth injury should be hypothesized in AS even when the patient does not manifest hearing disorders or evident signs of renal failure.
... Heterozygous COL4A3 or COL4A4 mutations can also cause autosomal dominant thin basement membrane nephropathy and benign familial hematuria (Kashtan, 1998Kashtan, , 2004 Tryggvason & Patrakka, 2006). The similarities and selectivity of the organs affected in Alport syndrome and Goodpasture disease are consistent with the tissue distributions of the collagen type IV alpha chains underlying these diseases (Kalluri, Gattone, & Hudson, 1998; Kruegel & Miosge, 2010; Ninomiya et al., 1995). During normal development, the a1a1a2 network in the GBM is gradually replaced by the a3a4a5 network (Hudson et al., 2003; Miner & Sanes, 1994). ...
Basement membranes are highly specialized extracellular matrices. Once considered inert scaffolds, basement membranes are now viewed as dynamic and versatile environments that modulate cellular behaviors to regulate tissue development, function, and repair. Increasing evidence suggests that, in addition to providing structural support to neighboring cells, basement membranes serve as reservoirs of growth factors that direct and fine-tune cellular functions. Type IV collagens are a major component of all basement membranes. They evolved along with the earliest multicellular organisms and have been integrated into diverse fundamental biological processes as time and evolution shaped the animal kingdom. The roles of basement membranes in humans are as complex and diverse as their distributions and molecular composition. As a result, basement membrane defects result in multisystem disorders with ambiguous and overlapping boundaries that likely reflect the simultaneous interplay and integration of multiple cellular pathways and processes. Consequently, there will be no single treatment for basement membrane disorders, and therapies are likely to be as varied as the phenotypes. Understanding tissue-specific pathology and the underlying molecular mechanism is the present challenge; personalized medicine will rely upon understanding how a given mutation impacts diverse cellular functions.
... Assembly of collagen IV networks is regulated developmentally. network occurs in the kidney (in glomerular basement membrane and some tubular basement membranes), lung, testis, cochlea, and eye (38,43,44) and the α5.α5.α6(IV) network is a feature of skin, smooth muscle, esophagus, and kidney (Bowman's capsule) (31,32,45,46). ...
... La sordera en el síndrome de Alport se debe a la pérdida de la red del colágeno α3(IV)-α4(IV)-α5(IV) en las membranas basales de la cóclea (26). ...
Presentamos el caso de una paciente de 4 años de edad, con hermano gemelo dicigoto asintomático, hija de padres no consanguíneos y sin antecedentes familiares de enfermedad renal. Inicia su cuadro clínico con edemas y proteinuria severa como manifestación de un síndrome nefrótico primario de cambios mínimos, que fue diagnosticado por biopsia renal y manejado inicialmente con esteroides. Su evolución no fue adecuada debido a múltiples recaídas que lo clasificaron como síndrome nefrótico corticorresistente, requiriendo cambio en su tratamiento y una segunda biopsia renal, cuyo resultado histológico sorprendió al grupo médico tratante porque los cambios en la membrana basal glomerular confirmaban que se trataba de un Síndrome de Alport.
... This network is also present in several ocular basement membranes including the lens capsule, the corneal epithelial and endothelial (Descemet's) basement membranes, the internal limiting membrane of the retina, and in Bruch's membrane specifically underlying retinal pigment epithelial cells (Kleppel et al., 1989a,b;Kleppel and Michael, 1990;Kelley et al., 2002;Chen et al., 2003;Ohkubo et al., 2003). The a3/a4/a5 network is also present in the inner ear, and its distribution among cochlear basement membranes has been studied in several species (Cosgrove et al.,22 S. J. Harvey and P. S. Thorner Kalluri et al., 1998;Harvey et al., 2001). This topic is reviewed by others in this series (Chapter X). ...
... We focused on the laminin and type IV collagen expression in BMs. Important contributions to characterize laminins and other extracellular matrix (ECM) molecules in the inner ear were performed by Rodgers et al. (2001), Takahashi and Hokunan (1992), Weinberger et al. (1999), Kalluri et al. (1998), Cosgrove et al. (1996, Swartz and Santi (1999), Santi (1999, 2001), Heaney and Schulte (2003). The first comprehensive immunolocalization of ECM proteins in the human inner ear was performed by Ishiyama et al. (2009). ...
Human auditory nerve afferents consist of two separate systems; one is represented by the large type I cells innervating the inner hair cells and the other one by the small type II cells innervating the outer hair cells. Type I spiral ganglion neurons (SGNs) constitute 96% of the afferent nerve population and, in contrast to other mammals, their soma and pre- and post-somatic segments are unmyelinated. Type II nerve soma and fibers are unmyelinated. Histopathology and clinical experience imply that human SGNs can persist electrically excitable without dendrites, thus lacking connection to the organ of Corti. The biological background to this phenomenon remains elusive. We analyzed the pre- and post-somatic segments of the type I human SGNs using immunohistochemistry and transmission electron microscopy (TEM) in normal and pathological conditions. These segments were found surrounded by non-myelinated Schwann cells (NMSC) showing strong intracellular expression of laminin-β2/collagen IV. These cells also bordered the perikaryal entry zone and disclosed surface rugosities outlined by a folded basement membrane (BM) expressing laminin-β2 and collagen IV. It is presumed that human large SGNs are demarcated by three cell categories: a) myelinated Schwann cells, b) non-myelinated Schwann cells (NMSC) and c) satellite glial cells (SGCs). Their BMs express laminin-β2/collagen IV and reaches the BM of the sensory epithelium at the habenula perforata. We speculate that the NMSC protect SGNs from further degeneration following dendrite loss. It may give further explanation why SGNs can persist as electrically excitable monopolar cells even after long time deafness, a blessing for the deaf treated with cochlear implantation.
... Normally, the α3(IV), α4(IV), and α5(IV) chains are highly expressed in basement membranes that are definitively, or in some cases possibly, involved in Alport syndrome: glomerular basement membrane (GBM), anterior lens capsule, Descemet's membrane, Bruch's membrane, and several basement membranes of the cochlea, including the basilar membrane and the basement membranes of the stria vascularis, spiral limbus, and spiral prominence [15,[23][24][25][26][27][28][29][30]. In 70%-80% of males with X-linked Alport syndrome (XLAS), the GBM, distal tubular basement membrane (TBM), and Bowman's capsules fail to stain for the α3(IV), α4(IV), and α5(IV) chains, but expression of the α1(IV) and α2(IV) chains is preserved and is increased [18,31]. ...
Alport syndrome is a primary genetic disease of basement membranes, manifested clinically as a progressive nephropathy variably associated with sensorineural deafness and a plethora of ocular abnormalities. The long-recognized phenotypic heterogeneity of Alport syndrome may be considered on several levels, including basement membrane biochemistry, basement membrane ultrastructure, the natural history of the nephropathy, and the occurrence of extrarenal abnormalities. This review discusses the possible molecular bases for the heterogeneity. The discussion draws upon recent insights into the molecular genetics of Alport syndrome, and the biochemistry of normal and Alport syndrome basement membranes, in order to provide a framework for understanding the variable renal and extrarenal manifestations of the disease.
... Additional coexpressed genes include hepsin, lysyl oxidase homolog precursor, and collagen type XV. Hepsin is a transmembrane serine protease involved in digestion of extracellular matrix (Tsuji et al., 1991); lysyl oxidase crosslinks extracellular matrix substrates such as collagen and elastin by catalyzing the oxidative deamination of peptidyl lysine (Jourdan-Le Saux et al., 1999); and collagen type XV has been implicated in the tectorial membrane (Kalluri et al., 1998). Some or all these additional genes may play roles in the formation of the tectorial membrane. ...
Understanding the development of the inner ear requires knowing the spatial and temporal pattern of gene expression, and the functions of those gene products. In the last decade, hearing research has benefited tremendously from the progress of the human and mouse genome projects, as amply illustrated by the identification of many deafness genes in both human and mouse. However, the sheer amount of information generated from the genome project has far outpaced the rate at which it is utilized. Microarray technology offers a means to quantify the expression level of transcripts at a whole-genome scale. Cross-tissue comparisons will identify genes unique to the inner ear, which will expedite the identification of new deafness genes. Microdissection and subtraction after ablation of cell types can reveal genes expressed in certain cells, such as hair cells. Expression profiling of both inner ear and other tissues, under a variety of conditions (such as during development, with drug treatment or in knock-out animals), can be used for cluster analysis to group genes of similar expression. Coexpression can suggest functional pathways and interactions between known genes, and can identify new genes in a structure or pathway. In this review we give examples for both transcription factors and cochlear structures.
Background:
Type IV collagen is an abundant component of basement membranes in all multicellular species and is essential for the extracellular scaffold supporting tissue architecture and function. Lower organisms typically have two type IV collagen genes, encoding α1 and α2 chains, in contrast with the six genes in humans, encoding α1 to α6 chains. The α chains assemble into trimeric protomers, the building blocks of the type IV collagen network. The detailed evolutionary conservation of type IV collagen network remains to be studied.
Results:
We report on the molecular evolution of type IV collagen genes. The zebrafish α4 non-collagenous (NC1) domain, in contrast with its human ortholog, contains an additional cysteine residue and lacks the M93 and K211 residues involved in sulfilimine bond formation between adjacent protomers. This may alter α4 chain interactions with other α chains, as supported by temporal and anatomic expression patterns of collagen IV chains during zebrafish development. Despite the divergence between zebrafish and human α3 NC1 domain (endogenous angiogenesis inhibitor, Tumstatin), the zebrafish α3 NC1 domain exhibits conserved anti-angiogenic activity in human endothelial cells.
Conclusions:
Our work supports type IV collagen is largely conserved between zebrafish and humans, with a possible difference involving the α4 chain. This article is protected by copyright. All rights reserved.
Basement membranes (BMs) are thin dense sheets of extracellular matrix that surround most tissues. When the BMs of neighboring tissues come into contact, they usually slide along one another and act to separate tissues and organs into distinct compartments. However, in certain specialized regions, the BMs of neighboring tissues link, helping to bring tissues together. These BM connections can be transient, such as during tissue fusion events in development, or long-term, as with adult tissues involved with filtration, including the blood brain barrier and kidney glomerulus. The transitory nature of these connections in development and the complexity of tissue filtration systems in adults have hindered the understanding of how juxtaposed BMs fasten together. The recent identification of a BM-BM adhesion system in C. elegans, termed B-LINK (BM linkage), however, is revealing cellular and extracellular matrix components of a nascent tissue adhesion system. We discuss insights gained from studying the B-LINK tissue adhesion system in C. elegans, compare this adhesion with other BM-BM connections in Drosophila and vertebrates, and outline important future directions towards elucidating this fascinating and poorly understood mode of adhesion that joins neighboring tissues.
Significance:
Basement membranes (BMs) are sheet-like structures of specialized extracellular matrix that underlie nearly all tissue cell layers including epithelial, endothelial, and muscle cells. BMs not only provide structural support, but are critical for the development, maintenance, and repair of organs. Animal heme peroxidases generate highly reactive hypohalous acids extracellularly and therefore target BMs for oxidative modification. Given the importance of BMs in tissue structure and function, hypohalous acid mediated oxidative modifications of BM proteins represent a key mechanism in normal development and pathogenesis of disease. Recent Advances: Peroxidasin, a BM associated animal heme peroxidase, generates hypobromous acid (HOBr) to form sulfilimine cross-links within the collagen IV network of BM. These cross-links stabilize BM and are critical for animal tissue development. These findings highlight a paradoxical anabolic role for HOBr, which typically damages protein structure leading to dysfunction.
Critical issues:
The molecular mechanism whereby peroxidasin uses HOBr as a reactive intermediate to cross-link collagen IV, yet avoid collateral damage to nearby BM proteins remains unclear.
Future directions:
The exact identification and functional impact of specific hypohalous acid mediated modifications of BM proteins needs to be addressed to connect these modifications to tissue development and pathogenesis of disease. As seen with the sulfilimine cross-link of collagen IV, hypohalous acid oxidative events may be beneficial in select situations rather than uniformly deleterious.
Type IV collagen is the main collagen component of the basement membrane. It is a network-forming collagen that underlies epithelial and endothelial cells and functions as a barrier between tissue compartments. Type IV collagen has many binding partners and forms the backbone of the basement membrane. It holds important signaling potential as subdomains such as tumstatin are released when the protein is degraded by special proteases. Consequently, type IV collagen is both the most important structural collagen of the basement membrane and it entails key signaling potential, which is important for various physiological and pathological functions. The most well-studied mutations in type IV collagen cause Alport syndrome, a chronic kidney disease. Several biomarkers of type IV collagen have been developed, both formation and degradation fragments as well as whole domains such as 7S, documenting the importance of type IV collagen turnover in most, if not all, connective tissue diseases.
Purpose: Determine hearing thresholds at sound frequencies important for speech comprehension in subject with ocular pseudoexfoliation (PXF) and to compare them with that of control without PXF. Method: 60 subjects with ocular PXF and 60 age and sex matched controls without PXF were enrolled in this case-control study. Pure tone audiometry (bone conduction) was performed at 1,2 and 3 kilohertz (KHz) in all subjects. Thresholds were compared to an age and sex stratified standard (ISO7029) and between study groups. Hearing loss was defined as sum of tested hearing thresholds (HTL-1,2,3) lower than the ISO7029 standard median. Results: The study included 35 males and 25 females subject in each group. Hearing loss was present in 97 of 120 (80.83%) of examined ears in the case group Vs 52 of 120 (43.3%) in the control group [P<0.001; odds ratio(OR) = 5.51; 95% confidence interval (CI)]. overall 54 subjects (90%) in the case group Vs 37 subjects 61.6% in the control group had hearing loss in one or both ears (P<0.001; odds ratio(OR) = 5.5; 95% confidence interval. Hearing thresholds at each of the examined frequencies and the HTL- 1,2,3 were also significantly higher in individuals with PXF. Conclusions: Hearing thresholds at frequencies which are important for speech comprehension are significantly worse in individuals with ocular PXF as compared to matched controls. This finding may support the multi-organ nature of PXF syndrome.
Renal diseases presenting in the Pediatric Intensive Care Unit are diverse. Amongst the most severe lesions are the thrombotic microangiopathies (TMA) and the glomerulonephritides. Both clinical scenarios necessitate nephrology consultation to facilitate diagnosis and timely treatment initiation in order to limit renal mobidity.
TMAs are represented by intravascular platelet aggregation, red blood cell shearing and thrombus formation. The resultant microangiopathic hemolytic anemia and thrombocytopenia are the clinical markers that suggest a risk for the ischemic loss of renal function. The broad differential for TMA includes typical hemolytic uremic syndrome (HUS), atypical hemolytic uremic syndrome (aHUS), and thrombotic thrombocytopenic purpura (TTP). Though histologically similar, typical HUS, aHUS, and TTP have different causal mechanisms and diverse treatments may be warrented. In contrast to the TMAs, glomerulonephritis is an inflammatory process leading to direct glomerular injury and loss of renal filtering function. The differential for glomerulonephritis can be broad with etiologies ranging from collagen abnormalities (e.g., Alport Syndrome) to antibody-mediated disease (e.g., anti-glomerular basement membrane disease). Acute renal replacement therapy and/or plasmapheresis may be required for successful treatment of disease due to TMA as well as glomerulonephritis.
The diagnosis of Alport syndrome, a type IV collagen hereditary disorder, should be considered in any child with unexplained, persistent microscopic hematuria of glomerular origin. Characteristic ultrastructural change in the glomerular basement membrane has been the primary pathologic criterion for diagnosis. New insights about the genes responsible for collagen type IV and its complex protein structure have led to alternative diagnostic avenues. Familiarity with the pediatric kidney is necessary to accurately interpret these exciting new data.
This chapter sheds light on clinical features, pathology, and diagnosis of Alport?s disease (AD) and thin basement membrane nephropathy (TBMN), one of the most common disorders of the kidney, affecting at least 1% of the total population. Alport's disease (AD) is an inherited kidney disease manifesting with hematuria and sensorineural deafness. AD is caused by mutations in the genes coding for type IV collagen a-chains, i.e., the COL4A3, COL4A4, and COL4A5 genes. In a great majority (about 85%) of patients with AD, there is X-linked inheritance of mutations in the COL4A5 gene on chromosome X and the remaining cases are explained by being compound heterozygotes or homozygous mutations in COL4A3 or COL4A4 genes on chromosome 2q35-37. There are two canine models and a transgenic mouse model for X-linked AD. All three X-linked AD models are caused by mutations in the gene coding for the type IV collagen a5 chains. Thin basement membrane nephropathy (TBMN) is the most common cause of persistent hematuria in children and adults and it is a major diagnostic problem. The characteristic clinical manifestation of TBMN is persistent microscopic hematuria. Most patients with TBMN present only with hematuria, without additional symptoms or progression to renal impairment, and the condition is usually incidentally detected during health control. TBMN mainly manifests as an inherited disorder with dominant transmission affecting approximately one-half of successive generations. TBMN is a disorder of the trimeric a 3: a 4: a 5 isoform of type IV collagen. Therefore, it could be anticipated that a single mutation in any of the alleles for these three genes would lead to a similar phenotype, as they all affect the same trimeric protein.
Metabolic syndrome (MS) is a cluster of metabolic abnormalities and serves as a precursor of cardiovascular disorders (CVD). The objective of the study was to determine the prevalence of MS and its association with Diabetes mellitus (DM) in an urban setup of Raipur city of Chattishgarh State. A cross sectional study was conducted on 400 randomly selected subjects (men 186, women 214 of age group 10-80 years) in the study region. Anthropometric variables, blood pressure, and lipid profile were monitored besides blood glucose in the study population. The incidence of MS and DM were worked out as per the criteria of National Cholesterol Education Programme-ATPIII (NCEPATPIII) and World Health Organization.The incidence of MS was found to be 15% and 5% by ATPIII and WHO criteria respectively. According to NCEP-ATPIII criterion, women recorded significantly high incidence of MS (65%) as compared to males (35%). About 28% of subjects with MS exhibited type II DM (males 16.6% and females 11.6%). On the other hand, the incidence of type II DM was found to be 6.7% in non-metabolic syndrome group of subjects as per NCEP-ATPIII criterion. By WHO criteria, 65% of the subjects with MS exhibited type II DM (males 35% and females 30%) while the incidence is 6.9% in non-metabolic syndrome group of subjects. Subjects in age group of 41-65 years showed the highest incidence of MS irrespective of the criteria employed. The incidence of MS differed significantly by the two criteria employed and it was more by NCEP-ATP-III criteria. The prevalence of type II DM was significantly high in subjects with metabolic syndrome and it was more in males of age group of 41-65 yrs.
Background
Since their establishment, the Primary Health Centers (PHCs) are being criticized for their inability to perform up to the expectations because of various reasons among which inadequate infrastructure and manpower is one reason.
Objective: To assess the infrastructure facilities and manpower among the selected PHCs of Chittoor
District.
Materials and Method
This cross-sectional study was conducted among 22 stratified randomly selected PHCs of Chittoor District, Andhra Pradesh which had 88 PHCs i.e. 25.0% of the total existing PHCs of the District. PHCs established within the last 5 years were excluded from the study. Assessment of the existing infrastructure and equipment and the manpower and health services being provided by the PHCs with respect to the Indian Public Health Standards was done. The data has been analyzed by Microsoft excel using proportions.
Results:
Each PHC on an average had 10 sub-centres, covering the population of 49,728. 63.6% of PHCs were providing in-patient services and 63.6%, the emergency services. 43.9% and 36.3% of the PHCs failed to meet the IPH Standards with respect to having Medical Officer and Lady Medical Officer. Only 59.0% of PHCs were conducting deliveries despite the presence of labour room. Sufficient quantity of drugs was present in only 71.9% of PHCs.
Conclusions:
Not all the PHCs were providing the in-patient as well as emergency services including MTP and round the clock services. Most PHCs were not staffed as per the IPHS norms and were also without sufficient quantity of drugs
Deafness affects more than 28 million people in European countries and a similar number in the USA. At present, a cochlear implant is the only option to treat deafness but it is expensive and not all kinds of deafness benefit from a cochlear implant. Gene therapy promises to be a clinically useful method to cure deafness, in particular genetic deafness. Thus, the present review will focus on the mechanisms underlying genetic deafness and possible treatment measures using gene therapy in the near future.Section editor:Richard Smith – University of Iowa, Iowa City, USA
Our conceptual thinking about hearing is confined, in part, by the “deafness” genes that have and have not identified. The
proper development of the auditory system and its associated electromechanical processes requires the orchestrated temporal
and spatial expression of numerous different genes. Further characterization of the genes for hearing loss will provide a
clearer vision of the structure and function of the auditory system in health and disease. It is hoped that these discoveries
will establish a conceptual basis for the rational therapy of hearing loss and deafness.
To determine the possible relation between pseudoexfoliation (PSX) and sensorineural hearing loss.
This study was carried out in Afyon Kocatepe University, Afyon, Turkey between July 2002 and June 2005. Sixty-three patients who were found to have ocular PSX on routine biomicroscopic examination, and 38 age-matched control subjects were evaluated for evidence of audiometric abnormality. The sum of pure-tone hearing threshold measured at 250-2000 Hz, 2000-6000 Hz, and 250-6000 Hz in each ear was compared with controls for the same frequencies.
The mean age of the patients was 68.4+/-10.3 years. All patients had PSX affecting at least one eye. Fifty (79.4%) patients with PSX, and 10 (26.3%) control subjects were found to have hearing loss (p=0.00, chi-square). From the 50 patients with PSX who had hearing loss, 34 patients had bilateral PSX, and 16 patients had unilateral PSX. Twenty-nine patients had high frequency hearing loss, while 20 patients had hearing loss in all frequencies. Forty-eight patients with PSX and 7 controls had bilateral sensorineural hearing loss (p=0.030).
Sensorineural hearing loss was seen more frequently in patients with PSX in comparison with age-matched control subjects.
Alport syndrome (AS) is a progressive renal disease that is characterised by hematuria and progressive renal failure, and often accompanied by progressive high-tone sensorineural hearing loss and ocular changes in form of macular flecks and lenticonus. AS is a genetic heterogenous disease, and X-linked dominant in about 85% of the families. The autosomal recessive and dominant forms constitute about 15% of the cases. In the first part of the study is a multipoint linkage analysis of 12 families suspected of X-linked AS. The aim of that part of the study was to map a number of X-chromosomal polymorphic markers in relation to the locus for AS, in order to be able to perform carrier detection and prenatal diagnosis in the families. In addition, a more precise map of the region could form the basis for positional cloning of the gene for X-linked AS. In 1990 it was found that the X-linked form of AS is caused by mutation in the COL4A5 gene located at Xq22, and encoding the alpha 5-chain of type IV-collagen. The COL4A5 gene is a very large gene spanning 257 kb with a transcript of 6.5 kb distributed on 51 exons. In addition, two alternatively transcribed exons have been identified. In the second part of the study methods were set up for detection and characterisation of mutations in the COL4A5 gene in 135 patients suspected of AS. The aims of that part of the study were to develop an efficient and reliable approach for mutation detection, and to implement the results of the mutation analysis in clinical practice for carrier detection and prenatal diagnosis, in order to be able to offer a better genetic counselling to the families. Knowledge of a possible correlation between genotype and phenotype can be of help in predicting the prognosis. Samples from 135 probands suspected of AS and 359 of their relatives were collected, together with available clinical information. Southern blotting analysis and multiplex ligation-dependent probe amplification (MLPA) were used to screen for larger structural rearrangements (deletions and duplications). cDNA probes covering the entire coding region of the COL4A5 gene were hybridised on restriction enzyme digested genomic DNA on Southern blots. Three different rearrangements were found by Southern blotting, two of which were caused by single base substitutions, and also detected by the PCR-SSCP analysis. One larger rearrangement was found, an inversion of 21 Mb with a proximal breakpoint in COL4A5 intron 8 at Xq22.3, and a distal breakpoint in the RAB33A gene at Xq26.1. This rearrangement was exclusively ascertained by the Southern blotting analysis. Three deletions of >or= 2 exons were detected by MLPA. One of these was detected in a female proband. A deletion in heterozygous form will not be detected by PCR-SSCP or direct sequencing. A method based on the PCR-SSCP technique was set up for screening of the COL4A5 gene exon-by-exon for mutation. All 51 COL4A5 exons with flanking intronic sequences were screened by this technique. The two alternatively transcribed exons 41A and 41B were directly sequenced. The PCR-SSCP method was compared to direct sequencing in 15 of the cases. No difference in mutation detection rates were found. Finally, a method based on RT-PCR analysis of mRNA extracted from cultured skin fibroblasts was established. A mutation in a patient previously screened by PCR-SSCP analysis with normal result, was detected. Another advantage of analysing a skin biopsy is that it is also possible to perform immunostaining for the alpha 5(IV)-chain in the epidermal basement membrane on sections from the biopsy. Absence of the alpha 5(IV)-chain support a diagnosis of X-linked AS. A total of 64 different and putative disease causing mutations were found in 72 of the families. Half of the mutations identified were missense mutations. The most frequent mutations in AS were glycine substitutions in the Gly-Xaa-Yaa repeat sequence in the collagenous domain of the alpha 5(IV)-chain, accounting for 47% of all mutations and 89% if the missense mutations. Frame-shift mutations accounted for 17% of the mutations, splice site mutations for 13%, nonsense mutations for 11%, in-frame deletions for 4%, and larger structural rearrangements for 6%. In addition, 5 different non-pathogenic sequence variations, polymorphisms and mutations of unknown effect on the phenotype, were found. Nineteen of the mutations are new and have not previously been published, and 55 of the mutations have exclusively been detected in this material. Two of the mutations (3%) are de novo mutations, and it has been possible to trace the mutation back in six of the families, and to determine the parental origin of the mutation in these six families. The origin of the mutation was found to be paternal in 4 of the families (67%), and maternal in 2 of the families (33%). We have demonstrated a highly efficient and sensitive molecular diagnostic approach for analysing the COL4A5 gene in putative AS cases. Based on the present results and the litterature, an algorithm for molecular genetic analysis of the COL4A5 gene is suggested. The overall mutation detection rate was found to be 53%. The mutation detection rate was 72% in patients fulfilling >or= 3 of the clinical criteria for AS, and 82% in families clearly demonstrating X-linked inheritance. No COL4A5 mutation could be detected in 63 (47%) of the families. X-linked inheritance could be excluded in seven of these families solely based on a pedigree analysis, and a diagnosis of Epstein syndrome was established in one of the patients by MYH9 mutation analysis. We found that the underlying COL4A5 mutation, truncating or non-truncating, can significantly predict the age at ESRD in male patients. Truncating mutations, comprising nonsense mutations, frame-shifts, and larger structural rearrangements, were found to cause a juvenile form of the disease with a mean age at ESRD of 21.6 years, compared to 33.1 years in patients with a non-truncating mutation. The effect of non-truncating mutations is, however, less clear-cut. Glycine substitutions in the collagenous domain of the alpha 5(IV)-chain will result in an adult form of AS with a mean age at ESRD of 35.8 years. Missense mutations in the NC1-domain and in-frame deletions result in a juvenile form of the disease with a mean age at ESRD of 23.3 and 20.3 years, respectively, but the number of patients in each group is limited. We found no significant differences in the presence of hearing defects or ocular manifestations between patients with the different types of mutations. The lack of distinct genotype-phenotype correlations implies that the usefulness of the result of the COL4A5 mutation analysis to predict the prognosis is limited. Future technological improvements e.g. automated sequencing strategies and implementation of microarray technology , may increase the mutation detection rates, and lower the time and costs of the analyses. Functional studies are hampered by the restricted expression of the type IV collagen chains. The many different animal models for AS are obvious and promising targets for functional studies, and an important resource for gene therapy studies. This makes AS a reliable candidate for future gene therapy in humans.
Alport syndrome is an inherited disorder of type IV collagen with progressive nephropathy, ocular abnormalities, and high-tone sensorineural deafness. In X-linked Alport syndrome, mutations in the COL4A5 gene encoding the α5 chain of type IV collagen lead to loss of the α3/α4/α5 network and increased susceptibility of the glomerular basement membrane to long-term damage. The molecular defects that underlie the otopathology in this disease remain poorly understood. We used a canine model of X-linked Alport syndrome to determine the expression of type IV collagen α-chains in the inner ear. By 1 month in normal adult dogs, the α3, α4, and α5 chains were co-expressed in a thin continuous line extending along the basilar membrane and the internal and external sulci, with the strongest expression along the lateral aspect of the spiral ligament in the basal turn of the cochlea. Affected dogs showed complete absence of the α3/α4/α5 network. The lateral aspect of the spiral ligament is populated by tension fibroblasts that express α-smooth muscle actin and nonmuscle myosin and are postulated to generate radial tension on the basilar membrane via the extracellular matrix for reception of high frequency sound. We propose that in Alport syndrome, the loss of the α3/α4/α5 network eventually weakens the interaction of these cells with their extracellular matrix, resulting in reduced tension on the basilar membrane and the inability to respond to high frequency sounds.
Alport syndrome (AS) is a genetically heterogeneous disease arising from mutations in genes coding for basement membrane type IV collagen. About 80% of AS is X-linked, due to mutations in COL4A5, the gene encoding the alpha 5 chain of type IV collagen (alpha 5[IV]). A subtype of X-linked Alport syndrome (XLAS) in which diffuse leiomyomatosis is an associated feature reflects deletion mutations involving the adjacent COL4A5 and COL4A6 genes. Most other patients have autosomal recessive Alport syndrome (ARAS) due to mutations in COL4A3 or COL4A4, which encode the alpha 3(IV) and alpha 4(IV) chains, respectively. Autosomal dominant AS has been mapped to chromosome 2 in the region of COL4A3 and COL4A4. The features of AS reflect derangements of basement membrane structure and function resulting from changes in type IV collagen expression. The primary pathologic event appears to be the loss from basement membranes of a type IV collagen network composed of alpha 3, alpha 4, and alpha 5(IV) chains. While this network is not critical for normal glomerulogenesis, its absence appears to provoke the overexpression of other extracellular matrix proteins, such as the alpha 1 and alpha 2(IV) chains, in glomerular basement membranes, leading to glomerulosclerosis. The diagnosis of AS still relies heavily on histologic studies, although routine application of molecular genetic diagnosis will probably be available in the future. Absence of epidermal basement membrane expression of alpha 5(IV) is diagnostic of XLAS, so in some cases kidney biopsy may not be necessary for diagnosis. Analysis of renal expression of alpha 3(IV)-alpha 5(IV) chains may be a useful adjunct to routine renal biopsy studies, especially when ultrastructural changes in the GBM are ambiguous. There are no specific therapies for AS. Spontaneous and engineered animal models are being used to study genetic and pharmacologic therapies. Renal transplantation for AS is usually very successful. Occasional patients develop anti-GBM nephritis of the allograft, almost always resulting in graft loss.
The family of type IV collagens continues to provide an important source of new information about basement-membrane molecules in epithelial tissues. Given the additional knowledge available today, we propose renaming the α3.α4.α5(IV) protomer the "Goodpasture protomer." This change honors the cornerstone role of the Goodpasture antigen in enlarging our knowledge of collagen IV biochemistry and relates the molecular understanding of protomer assembly to the pathogenesis of Goodpasture's and Alport's syndromes. The insights provided by the work completed to date suggest that a number of therapeutic advances may be forthcoming. Recognition that basement membranes in patients with Alport's syndrome are particularly susceptible to proteolysis may eventually lead to the prophylactic use of specific protease inhibitors or even gene-replacement therapy. Work with experimental models of anti-glomerular basement membrane disease already predicts a role for costimulatory blockade of T-cell activation, immune modulation with interleukin-4 and interleukin-10, or inhibition of macrophage migration. If nothing else, the future will be interesting, and work in this area will undoubtedly provide a new understanding of collagen-related diseases. Supported in part by grants (DK-46282 and DK-55926, to Dr. Neilson; DK-18381 and DK-53763, to Dr. Hudson; and DK-62524, to Dr. Sundaramoorthy) from the National Institutes of Health and a grant (to Dr. Tryggvason) from the Swedish Medical Research Council. We are indebted to Larry Howell for assistance in the preparation of the figures.
Ocular pseudoexfoliation (PXF) has been recently considered as a systemic disease affecting other organs as well as the eye. This prospective study is to assess the relationship between PXF and sensorineural hearing loss.
Patients attending a general ophthalmic clinic at King Hussein Medical Center, Amman, Jordan from (March 2002 through to March 2003) who were found to have ocular PXF on routine ophthalmic examination were referred to the Audiometric Department. Pure tone hearing threshold was measured at 1, 2, 3 kHz for each ear and was compared with International Standard (ISO 7029) median age associated hearing loss at 1, 2, 3 kHz (AAHL).
Forty-one patients were studied of whom 24 were males (58.5%); the mean age of the male patients was 78-years while that of the female group was 72-years. All patients had PXF affecting at least one eye, 16 patients (39%) had bilateral PXF. Overall (72-years) of 36 patients (87%) had a higher hearing threshold level (HTL) at 1, 2, 3 kHz (HTL (1, 2, 3) than the ISO 7029 median AAHL 1, 2, 3 which included (44 ears) of 22 patients in the male group (87%) and (28 ears) of 14 patients in the female group 82%. Approximately 26.8% of patients had glaucoma, however; there was no correlation between glaucoma and sensorineural hearing threshold level.
The majority of patients with ocular PXF had sensorineural hearing loss compared to age-matched controls. Thus, there is increasing evidence that PXF is a systemic disease.
The NC1 domain of alpha3 chain of type IV collagen, namely tumstatin, has been shown to display specific anti-angiogenic properties by inhibiting endothelial cells' proliferation and inducing their apoptosis via an interaction with alphavbeta3 integrin. Until now, the tumstatin anti-angiogenic effect has only been shown by in vitro studies or mouse xenograft experiments. In the present study, we examined the expression of tumstatin in relationship with tumor vascularization in 34 bronchopulmonary human carcinomas. We observed a clear association between tumstatin expression and tumor vascularization. Indeed, a strong expression of tumstatin in the tumor environment correlated with a mildly developed vascular network. In contrast, tumstatin was absent or poorly detected in highly vascularized tumors. Moreover, alphavbeta3 integrin and tumstatin colocalized in capillary endothelial cells, suggesting a potential interaction between these 2 molecules. Thus, our results plead in favor of an in vivo anti-angiogenic effect of tumstatin. This factor, largely expressed in well-differentiated lung carcinomas, could indeed reduce tumor vascularization and thereby limit tumor progression.
Outer hair cells (OHCs) are capable of altering their cell length in response to changes in membrane potential. Due to this electromotility, OHCs probably subject the basilar membrane to force, resulting in cochlear amplification. To understand the mechanism of such amplification, knowledge of the mechanical properties of OHCs is required since the force produced by OHC electromotility is thought to depend on such properties. Various studies have been conducted to investigate the mechanical properties of guinea pig OHCs. With regard to mice, however, although various kinds of transgenic and knockout mice possess great potential as research models, the mechanical properties of mouse OHCs have not as yet been reported since the cells and/or tissues in the mouse hearing organ are relatively small and vulnerable to external stimuli, rendering sample preparation difficult. In this study, therefore, to establish indicators of the mechanical properties of OHCs in mice, such properties were measured by atomic force microscopy (AFM).
CBA/JNCrj strain male mice aged 10-12 weeks (25-30 g) were used. Cochleae were dissected out from the animal and both the basilar membrane and the organ of Corti were simultaneously unwrapped from the modiolus with forceps. Dissected coiled tissue was then incubated with an enzymatic digestion medium for 15 min. After digestion, OHCs were isolated by gently triturating the coiled tissue. Local mechanical properties of the OHCs were then measured by an indentation test using an AFM.
Young's modulus and stiffness of the OHC in the apical turn of the mouse cochlea were 2.1+/-0.5 kPa and 4.4+/-1.2 mN/m, respectively.
Young's modulus of the OHC in the apical turn of the cochlea in mice was roughly the same as that in the apical turn of the cochlea in guinea pigs; however, the stiffness of the former was about two times greater than that of the latter because the cell length of the former was shorter than that of the latter.
We report a 9-year-old Icelandic male with Alport syndrome and nephrotic-range proteinuria who responded well to cyclosporine therapy. He presented at the age of 2 years with gross hematuria and proteinuria during an episode of upper respiratory tract infection. Three years later he had developed persistent proteinuria; kidney function was normal. A renal biopsy revealed marked irregularities in the glomerular basement membrane consistent with Alport syndrome. Mutation analysis revealed a single base insertion in COL4A5 which was predicted to cause a major structural defect in the collagen IV alpha5 chain. Despite angiotensin-converting enzyme inhibitor therapy his proteinuria progressed to the nephrotic range associated with edema. At the age of 7 years, cyclosporine therapy was instituted, which promptly resulted in almost complete resolution of proteinuria. Three years later his urinary protein excretion was close to the normal range and serum creatinine remained within normal limits. We conclude that closely monitored cyclosporine therapy may be a safe and effective treatment in patients with severe proteinuria and Alport syndrome.
Mutations in the COL4A5 collagen gene have been implicated as the primary defect in Alport syndrome, a heritable disorder characterized by sensorineural deafness and glomerulonephritis that progresses to end-stage renal failure. In the present study, the molecular nature of the defect in Alport glomerular basement membrane (GBM) was explored using anti-GBM alloantibodies (tissue-bound and circulating) produced in three Alport patients subsequent to renal transplantation. The alloantibodies bound to the alpha 3(IV)NC1 domain of type IV collagen and not to any other basement membrane component. In tissue sections, the alloantibodies bound specifically to peripheral GBM in normal kidney and the affected renal transplant but not to that of Alport kidney. These results establish that: the alpha 3 chain in type IV collagen molecules, the Goodpasture autoantigen, is the target alloantigen in post-transplant anti-GBM nephritis in patients with Alport syndrome, and that a molecular commonality exists in the pathogenesis of anti-GBM nephritis causing loss of renal allografts in patients with Alport syndrome and renal failure in patients with Goodpasture syndrome. These findings implicate: (1) defective assembly of type IV collagen molecules containing the alpha 3(IV) chain in Alport GBM; and (2) the existence of a mechanism linking the assembly of molecules containing the alpha 3(IV) chain with those containing the alpha 5(IV) chain.
The autoantibodies of patients with Goodpasture syndrome are primarily targeted to the noncollagenous (NC1) domain of the alpha 3(IV) chain of basement membrane collagen (Saus, J., Wieslander, J., Langeveld, J. P. M., Quinones, S., and Hudson, B. G. (1988) J. Biol. Chem. 263, 13374-13380). In the present study, the location of the Goodpasture epitope in human alpha 3NC1 was determined, and its structure was partially characterized. This was achieved by identification of regions of alpha 3NC1 which are candidates for the epitope and which are structurally unique among the five known homologous NC1 domains (alpha 1-alpha 5); amino acids that are critical for Goodpasture antibody binding, by selective chemical modifications; and regions that are critical for Goodpasture antibody binding, by synthesis of 12 alpha 3NC1 peptides and measurement of their antibody binding capacity. The carboxyl-terminal region, residues 198-233, was identified as the most likely region for the epitope. By experiment, lysine and cysteine were identified as critical amino acids for antibody binding. Three synthetic peptides were found to inhibit Goodpasture antibody binding to alpha 3NC1 markedly: a 36-mer (residues 198-233), a 12-mer (residues 222-233), and a 5-mer (residues 229-233). Together, these results strongly indicate that the Goodpasture epitope is localized to the carboxyl-terminal region of alpha 3NC1, encompassing residues 198-233 as the primary antibody interaction site and that its structure is discontinuous. These findings provide a conceptual framework for future studies to elucidate a more complete epitope structure by sequential replacement of residues encompassing the epitope using cDNA expression products and peptides synthesized chemically.
A third chain, alpha 3(IV), of basement membrane collagen was recently discovered and was identified as the primary target for the autoantibodies of patients with Goodpasture syndrome (Saus, J., Wieslander, J., Langeveld, J. P. M., Quinones, S., and Hudson, B. G. (1988) J. Biol. Chem. 263, 13374-13380). In the present study, this chain was excised in the form of a truncated promoter by cleavage of basement membrane with Pseudomonas aeruginosa elastase and characterized. The triple helical structure and NC1 domain were retained. Elastase selectively cleaved at a site within the triple helical domain of the alpha 3 chain that is distinct from the cleavage site of the alpha 1 and alpha 2 chains. The truncated alpha 3 chain was found to contain 1460 residues, of which 1225 comprise the collagenous domain, and is cross-linked within this domain by disulfide bonds, forming a high Mr complex (greater than 300,000). Truncated protomers with a length of 340 nm corresponding to the theoretical length for the truncated alpha 3 chain were observed by electron microscopy as suprastructures in which the triple helical domains of three protomers were interwined. These protomers were also connected to each other and to the 140-nm protomers that appear to be comprised of the alpha 1 and alpha 2 chains. These results extended the known length of the alpha 3 chain by about 1000 residues and suggested that protomers of this chain self-associate through interactions between their triple helical domains and between their NC1 domains.
The noncollagenous (NC1) domain hexamer of glomerular basement membrane (GBM) collagen is composed of a multiplicity of monomeric and dimeric subunits, and specific subunits are the targets for anti-GBM autoantibodies of patients with Goodpasture (GP) syndrome. The identity of GBM monomers has been established and the alpha 3(IV)NC1 monomer identified as the one that binds GP antibodies (Gunwar, S., Saus, J., Noelken, M. E., and Hudson, B. G. (1990) J. Biol. Chem. 265, 5466-5469). In the present study, the chain origin of 25 dimeric components and the identity of those that bound the anti-GBM antibodies from two GP patients were determined. This was accomplished by NH2-terminal sequence analysis and immunoblotting analysis of dimeric components that were resolved by two-dimensional electrophoresis in combination with high pressure liquid chromatography. The results revealed that (a) the components are mainly homodimers of the NC1 domains of alpha 1, alpha 2, alpha 3, alpha 4, and probably alpha 5 chains of collagen IV, reflecting a specificity of promoter-promoter association and (b) each homodimer had several size and charge isoforms. The GP antibodies bound exclusively to both alpha 3(IV)NC1 monomers and dimers and not to other basement membrane constituents. These findings provided new insights about the structure of GBM collagen and together with our previous findings firmly established the alpha 3(IV) chain as the target for the anti-GBM antibodies that mediate glomerulonephritis and pulmonary hemorrhage in patients with Goodpasture syndrome.
The noncollagenous domain hexamer of collagen IV from bovine glomerular basement membrane was further investigated to determine the types of collagen chain from which subunits M2*b and M3 are derived. M2*b was shown to be a shorter form, containing 9 fewer residues, of M2*a which was previously established as the noncollagenous domain of a third chain, alpha 3, of collagen IV (Saus, J., Wieslander, J., Langeveld, J.P.M., Quinones, S., and Hudson, B.G. (1988) J. Biol. Chem. 263, 13374-13380). M3 was identified as the noncollagenous domain of a fourth chain, alpha 4, of type IV collagen, on the basis of additional sequence data together with previous findings. A comparison of the collagenous-noncollagenous junction regions of alpha 3(IV) and alpha 4(IV) chains with those of classical alpha 1(IV) and alpha 2(IV) chains reveals structural information which provides a potential strategy for molecular cloning of these novel chains. The results further reveal the complexity of electrophoresis patterns of the hexamer and potential ambiguities in using one-dimensional patterns to determine whether molecular defects of collagen IV occur in pathological processes affecting basement membranes.
The authors have defined the specificity of monoclonal antibodies to collagen fragments of basement membrane (BM) and have used these highly specific antibodies to study the human tissue distribution of two novel 28 kd noncollagenous (NC1) peptides (M28 , M28+) compared with those derived from type IV collagen (alpha 1[26 kd] and alpha 2[24 kd] NC1). A limited distribution of the 28 kd peptides was observed in specialized BM of the kidney, eye, cochlea, lung, and brain, whereas type IV collagen is found in all human BM. These novel peptides, which colocalize with each other, are found in BM that also contain type IV collagen but do not, in all cases, colocalize with type IV collagen. The presence of the 28 kd peptides in the BM of the kidney, cochlea, and eye is in keeping with abnormalities involving these components in BM of patients with Alport familial nephritis (FN), who frequently have hearing loss, anterior lenticonus and retinal flecks in addition to renal disease. These 28 kd peptides are distinct, biochemically and immunochemically, from the alpha 1 and alpha 2 chain NC1 peptides of type IV collagen, and represent either peptide fragments of genetically distinct BM collagen molecules or additional molecules originating from the same gene family as type IV collagen.
The glomerular basement membranes (GBM) of Alport familial nephritis (FN) are laminated and split and fail to bind Goodpasture autoantibodies by indirect immunofluorescence. The Goodpasture antigen has been localized to multiple peptides of the noncollagenous C terminal (NC1) domain of type IV collagen. The principal target antigen is a 28-kDa peptide (M28) that coisolates with type IV collagen NC1 and which is derived from a larger collagenous molecule. We have shown that two novel 28-kDa peptides found in normal GBM (M28M28+) are absent from collagenase digests of X-linked dominant Alport FN GBM and that monoclonal antibodies specific for these collagen chains fail to bind to Alport GBM. In normal tissue these chains have a distribution restricted to specific basement membranes of kidney, eye, inner ear, lung, and brain, the former three of which are affected in Alport FN. Epitopes on a 26-kDa NC1 peptide identified by an antibody from a transplanted Alport patient (FN antibody) colocalized with the 28-kDa components in these tissues. The FN antibody did not bind to the GBM of homozygous Alport males. Antibodies to the 28-kDa peptides and the FN antibody colocalized in a segmental pattern in heterozygous Alport GBM by indirect immunofluorescence and were unrelated to the normal distribution of type IV collagen. Three of eight homozygous Alport FN tissues showed the presence of the 28-kDa components in Bowman's capsule in a focal distribution, and in four of eight tissues reactive antigen was present in the cytoplasm of some parietal and visceral epithelial cells. These observations support the hypothesis that the genetic abnormality in Alport FN is a defective parent chain of the 26-kDa peptide, which results in failure of normal 28-kDa collagen chain integration.
The noncollagenous domain of collagen from three different basement membranes of bovine origin (glomerular, lens capsule, and placental) was excised with bacterial collagenase, purified under nondenaturing conditions, and characterized. In each case the domain existed as a hexamer comprised of four distinct subunits (alpha 1 (IV) NC1, alpha 2 (IV) NC1, M2*, and M3). Each subunit exists in both monomeric and dimeric (disulfide-cross-linked) forms. Certain dimers also exist which contain nonreducible cross-links. The hexamers from the three membranes differ with respect to stoichiometry of subunits and subunit isoforms and to the degree of cross-linking of monomers into dimers. The minor subunits, M2* and M3, vary in quantity over a 20-fold range relative to the major ones among the three hexamers. The results indicate that: 1) at least two populations of triple-helical collagen molecules, differing in chain composition, exist in each membrane and that their relative proportions are tissue-specific; and 2) the chemical nature of the noncollagenous domain of these populations is tissue-specific with regard to subunit isoforms and relative proportion of reducible and nonreducible cross-links in dimers. A novel structural feature of the noncollagenous domain of basement membrane collagen was also evinced from these studies. Namely, that each of the four monomeric subunits exists in charge isoforms.
The X-linked Alport syndrome is associated with mutations and deletions in COL4A5 gene, one of six genes which constitute the alpha-chains of type IV collagen in basement membranes. The autosomal recessive form of Alport syndrome is characterized by mutations and deletions in the COL4A3 and COL4A4 genes. A fraction of Alport patients who undergo renal transplantation develop anti-glomerular basement membrane (GBM) nephritis, which results in loss of the renal allograft function. Recently, the target for alloantibodies from an X-linked Alport patient with complete COL4A5 gene deletion was determined to be the alpha 3 chain of type IV collagen. The present study characterized the post-transplant alloantibodies from an autosomal recessive Alport patient with anti-GBM glomerulonephritis and a COL4A3 gene mutation which predicted a loss of 85% of the alpha 3(IV) NC1 domain. The specificity of these new antibodies were studied using glomerular basement membrane constituents and recombinant type IV collagen domains. The results establish the target for the alloantibodies from an autosomal recessive Alport patient with COL4A3 deletion as principally the alpha 3(IV) collagen chain, similar to the post-transplant alloantibodies from X-linked Alport patients with COL4A5 gene deletions. The absence of alpha 3(IV) chain in the GBM of patients with both these forms of Alport syndrome, due either to a failure of synthesis or a failure of assembly, presumably leads to a loss of immunologic tolerance for the alpha 3(IV) NC1 domain in transplanted allografts.
Human Goodpasture syndrome is a lethal form of autoimmune disease that is characterized by pulmonary hemorrhage and glomerulonephritis. The tissue injury is mediated by autoantibodies that bind to glomerular and alveolar basement membrane. The target autoantigen is alpha 3(IV) collagen, one of six genetically distinct chains that comprise type IV collagen, and the epitope is sublocalized to the noncollagenous domain (NC1) of the alpha 3 chain. The present study reports the unique capacity of alpha 3(IV)NC1 dimer from bovine kidney to aberrantly engage the immune system of rabbits to respond to self, mimicking the organ-specific form of the human disease, whereas the other chains of type IV collagen are nonpathogenic. However, alpha 3(IV)NC1 hexamer was nonpathogenic, suggesting the exposure of a pathogenic epitope upon dissociation of hexamer into dimers. Exposure of the pathogenic epitope by infection or organic solvents, events which are thought to precede Goodpasture syndrome, may be the principal factor in the etiology of the disease. The pathogenicity of alpha 3(IV) collagen brings full circle a decade of research that has identified four novel chains (alpha 3-alpha 6) of type IV collagen.
Mutations in the COL4A5 gene encoding the alpha 5(IV) chain of type IV collagen have been implicated as the primary defect in X-linked Alport syndrome. Several kinds of mutations have been reported so far, spanning point mutations to complete gene deletions. About 5% of Alport patients, who undergo renal transplantation, develop anti-glomerular basement membrane (GBM) nephritis, causing loss of allograft function. In one such patient, COL4A5 gene deletion was recently identified. In the present study, the GBM constituent, targeted by the anti-GBM alloantibodies from the patient who had complete COL4A5 gene deletion was identified. Its identity was determined on the basis of circulating antibody binding to various GBM constituents, domains of bovine type IV collagen and recombinant NC1 domain of human type IV collagen. These results establish, for the first time, the absence of the alpha 5(IV) chain in Alport GBM and, in the same patient, the production of an alloantibody that is targeted to a different chain of type IV collagen, the alpha 3(IV) chain. These findings provide further support for the hypothesis that: (1) anti-alpha 3(IV) collagen alloantibodies mediate the allograft glomerulonephritis; and (2) COL4A5 gene mutations cause defective assembly of the alpha 3(IV) collagen alloantibodies mediate the allograft glomerulonephritis; and (2) COL4A5 gene mutations cause defective assembly of the alpha 3(IV) chain in Alport GBM, as reflected by the production of anti-alpha 3(IV) alloantibodies.
Antiglomerular basement membrane (GBM) antibodies can cause glomerulonephritis or pulmonary hemorrhage by themselves or Goodpasture syndrome when they occur together. It is unknown if variations in antibody reactivity contribute to the different patterns of organ involvement seen in this disease. This study examines the reactivity of the alpha 1-alpha 6 NC1 domains of Type IV collagen, the putative autoantigen, in sera from patients with anti-GBM antibodies after various clinical presentations of lung hemorrhage and renal injury. Serum or plasma containing anti-GBM antibodies from 35 patients with combined glomerulonephritis and pulmonary hemorrhage, 19 with glomerulonephritis alone, and 4 with pulmonary hemorrhage alone were compared with samples from 19 normal controls and 32 patients with other kidney diseases. Four different immunologic assays were performed with bovine alpha 1-alpha 6(IV) and recombinant human type alpha 1-alpha 5(IV) collagen NC1 domains. The study found that the anti-GBM antibodies from all patients reacted with the alpha 3(IV) NC1 (85% exclusively). Additional limited reactivity with the alpha 1(IV) NC1 and alpha 4(IV) NC1 was found in 15 and 3%, respectively. This non-alpha 3(IV) NC1 reactivity was most frequent in the patients with anti-GBM antibodies and glomerulonephritis alone. None of the patients had reactivity to other basement membrane components like laminin, fibronectin, heparan sulfate proteoglycan, entactin, or the 7S and triple helical fragments of Type IV collagen. The observed alpha-chain NC1 reactivity was confined to patients with anti-GBM antibodies with no additional reactivities detected among a large number of other kidney diseases controls. The correlation of alpha 1-alpha 6(IV) NC1 reactivity in a large number of patients with anti-GBM antibodies defined by classic assays definitively establishes that reactivity to alpha 3(IV) NC1 domains is both sufficient and necessary for the expression of autoimmune disease directed to the NC1 domain of Type IV collagen. On the basis of the evidence, the classification of antibasement membrane disease and Goodpasture syndrome as anti-Type IV collagen disease is proposed.
The family of type IV collagen comprises six chains numbered alpha1 through alpha6. The alpha3(IV) NC1 domain is the primary target antigen for autoantibodies from patients with anti-basement membrane disease and Goodpasture syndrome. Earlier peptide studies suggested that the last 36 amino acids of the alpha3 NC1 domain probably contains one recognition site for Goodpasture autoantibodies, and an algorithm analysis of secondary structure from a later study predicted a second possible upstream epitope near the triple helix junction. We have used several analytic approaches to evaluate the likelihood of two immunologic epitopes for the Goodpasture antigen. In our first set of studies, peptide antibodies directed against these two putative regions co-inhibited Goodpasture autoantibodies binding to denatured human alpha3(IV) NC1 monomer by nearly 80%, with the helix-junction region of the alpha3 NC1 domain contributing 26% of the binding sites and the C-terminal region contributing the remaining 50%. Second, both of these candidate regions are normally sequestered within the associated alpha3(IV) NC1 hexamer but become more visible for binding by anti-peptide antibodies upon their dissociation, a property that is shared by the Goodpasture autoantibodies. Third, segment deletions of recombinant alpha3 NC1 domain further confirmed the presence of two serologic binding sites. Finally, we looked more closely at the C-terminal binding region of the alpha3(IV) NC1 domain. Since the lysines in that region have been previously advanced as possible contact sites, we created several substitutions within the C-terminal epitope of the alpha3 NC1 domain. Substitution of lysines to alanines revealed lysines 219 and 229 as essential for antibody binding to this distal site; no lysines were present in the NC1 part of the helix-NC1 junction region. Substitutions involving arginine and cysteines to alanines in the same C-terminal region did not produce significant reductions in antibody binding. In summary, our findings characterize two Goodpasture epitopes confined to each end of the alpha3 NC1 domain; one is lysine-dependent, and the other is not. We propose, as a hypothetical model, that these two immunologically privileged regions fold to form an optimal pathogenic structure within the NC1 domain of the alpha3 chain. These sites are subsequently concealed by NC1 hexamer assembly of type IV collagen.
Normal glomerular capillaries filter plasma through a basement membrane (GBM) rich in alpha3(IV), alpha4(IV), and alpha5(IV) chains of type IV collagen. We now show that these latter isoforms are absent biochemically from the glomeruli in patients with X-linked Alport syndrome (XAS). Their GBM instead retain a fetal distribution of alpha1(IV) and alpha2(IV) isoforms because they fail to developmentally switch their alpha-chain use. The anomalous persistence of these fetal isoforms of type IV collagen in the GBM in XAS also confers an unexpected increase in susceptibility to proteolytic attack by collagenases and cathepsins. The incorporation of cysteine-rich alpha3(IV), alpha4(IV), and alpha5(IV) chains into specialized basement membranes like the GBM may have normally evolved to protectively enhance their resistance to proteolytic degradation at the site of glomerular filtration. The relative absence of these potentially protective collagen IV isoforms in GBM from XAS may explain the progressive basement membrane splitting and increased damage as these kidneys deteriorate.
A model for cochlear mechanics is proposed to take account of its two systems, one passive and one active. The classical passive system stimulates the inner hair cells directly at levels above about 40 dB SL. At intensities below about 60 dB an active process, the ‘cochlear amplifier’ (CA), somehow provides additional energy that enhances the vibration of a narrow segment of the basilar membrane near the apical foot of the familiar, traveling wave envelope. The outer hair cells are essential for CA. The active system acts like a high-Q acoustic resonator, and it accounts for the great sensitivity and sharp tuning expressed by the ‘tips’ of neural tuning curves. The tips are selectively vulnerable to anoxia, noise exposure and other trauma. The CA model explains the detection of small differences in time as well as in frequency, the dual character of the electrocochleogram, recruitment of loudness in cochlear hearing impairment, the long latency of normal neural responses near threshold, acoustic emissions (both stimulated and spontaneous) and the locus of TTS in the frequency range above the exposure tone. Both the classical high-intensity system and the active low-level CA system are highly nonlinear and they combine to compress the great dynamic range of hearing into a much narrower range of mechanical movement of the cilia of the inner hair cells. The mechanism of CA is unknown, and the problem remains of how its action can be triggered by submolecular movements near threshold.
The Alport antigen, a component of normal glomerular basement membranes (GBM) which is absent in Alport familial nephritis, is characterized as a 26 kD non-collagenous (NC1) peptide identified by a monoclonal antibody (Mab A7) and an Alport alloantibody. Both antibodies discriminate X-linkage of the Alport defect using indirect immunofluorescence of hemizygous and heterozygous Alport GBM and epidermal basement membrane (EBM). Immunoblotting of SDS-PAGE gels of collagenase-digested Alport renal BM shows absence of monomeric and dimeric components of the Alport antigen, alpha 3(IV) NC1, and alpha 4(IV) NC1. By immunoprecipitation experiments with specific antibodies, the Alport antigen is distinct from the 26 kD NC1 peptide derived from alpha 1(IV). The monoclonal antibody to the Alport antigen and rabbit antiserum to a non-consensus sequence of alpha 5(IV) NC1 react similarly by immunofluorescence with normal kidney and both fail to bind to Alport renal BM. Two dimension Western blots of collagenase-digested BM show that the anti-Alport antigen and the ant-alpha 5(IV) NC1 react similarly with monomeric and dimeric components of BM collagen. These studies are consistent with the likelihood that the Alport antigen and alpha 5(IV) NC1 are the same or are highly homologous molecules. The precise relationship will require characterization of alpha 5(IV) NC1 protein and determination of the nucleotide sequence of the Alport antigen. The associated absence of alpha 3(IV) NC1 and alpha 4(IV) (NC1) from Alport BM is consistent with other observations for a molecular association of these chains in a novel collagen network.
The noncollagenous domain hexamer of collagen IV from bovine alveolar basement membrane was excised with bacterial collagenase, purified under nondenaturing conditions, and characterized. The hexamer is comprised of four distinct subunits [alpha 1(IV)NC1, alpha 2(IV)NC1, alpha 3(IV)NC1, and alpha 4(IV)NC1]. Each subunit exists in both monomeric and dimeric (disulfide-crosslinked) form, and both monomers and dimers have charge isoforms. Certain dimers also contain nonreducible crosslinks. The alpha 3(IV)NC1 subunit, in both the monomeric and dimeric form, reacts with Goodpasture (GP) antibodies. The GP epitope is sequestered within the hexamer and becomes reactive with antibody upon exposure with protein denaturants. These results reveal that the alveolar basement membrane hexamer is identical to the hexamer from glomerular basement membrane with respect to subunit composition, identity of subunits reacting with GP antibodies, and sequestration of the GP epitope but differs greatly in the relative amount of the GP-reactive subunit and the degree of disulfide and nondisulfide crosslinking of subunits. This study leads to the conclusion that pulmonary hemorrhage associated with GP syndrome is mediated by the same autoantibody that mediates the glomerulonephritis, namely anti-collagen [alpha 3(IV)] antibody.
Collagen type IV, laminin, heparan sulfate proteoglycans, nidogen (entactin) and BM-40 (osteonectin, SPARC) represent major structural proteins of basement membranes. They are well-characterized in their domain structures, amino acid sequences and potentials for molecular interactions. Such interactions include self-assembly processes and heterotypic binding between individual constituents, as well as binding of calcium (laminin, BM-40) and are likely to be used for basement membrane assembly. Laminin, collagen IV and nidogen also possess several cell-binding sites which interact with distinct cellular receptors. Some evidence exists that those interactions are involved in the control of cell behaviour. These observations have provided a more defined understanding of basement membrane function and the definition of new research goals in the future.
In studies of victims from the influenza pandemic of 1918–1919, Ernest Goodpasture described the coexistence of fatal pulmonary hemorrhage and proliferative glomerulonephritis in a young man (25). The term Good- pasture syndrome was coined by Stanton and Tange in 1958 to describe cases characterized by the coexistence of these manifestations (73). The syndrome is now defined as an autoimmune disorder consisting of the triad of glomerulonephritis, lung hemorrhage, and antiglomerular basement membrane antibody formation, and it includes a broad spectrum of clinical features, ranging from massive pulmonary hemorrhage with little overt evidence of renal disease to fulminant crescentic glomerulonephritis and little overt evidence of pulmonary hemorrhage (24). Although the etiology of this syndrome remains unknown, profound advances have been made during the last 25 years in delineating the pathogenesis of the glomerulonephritis.
The mammalian auditory organs have a dual sensory system (inner vs. outer hair cells) with distinctly different cellular organizations and innervation patterns. However, the inner (IHCs) and outer (OHCs) hair cells are mechanoreceptors sharing similar general characteristics such as organization of stereocilia (including linkage system) and a gradation of stereociliary height along the length of the cochlea. This gradation of stereociliary height may be the single most important anatomic feature in the tuning capability of the sensory cell. Several lines of evidence suggest that the stereociliary stiffness may be modulated by the sensory cells themselves, most likely via the cuticular plate-rootlet complex.
The effects of the enzymes collagenase, pepsin, chondroitinase ABC and keratanase on the polypeptide composition of the mammalian tectorial membrane have been analysed using one dimensional SDS-polyacrylamide gel electrophoresis (SDS-PAGE). After reduction at least ten polypeptides can be consistently and clearly recognized in SDS gels with molecular weights relative to globular protein standards of 245, 235, 190, 165, 155, 145, 100, 93, 60-73 and 35-49 kDa. With the exception of the 60-73 and 35-49 kDa bands all these polypeptides are sensitive to digestion with bacterial collagenase. The 235, 165, 155, 145 and 93 kDa bands also resist degradation by cold, acidic pepsin. Amino acid analysis of whole tectorial membranes demonstrates that glycine accounts for nearly 25% of the total amino acid content, that proline, hydroxyproline and hydroxylysine are present and that amine sugars can be detected in fairly high concentrations. Estimates based on hydroxyproline content suggest that collagens account for 25-50% of the total tectorial membrane protein. Immunoblotting techniques demonstrate the presence of polypeptides cross reacting with antisera to Type II collagen, Type IX collagen and Type V collagen. Results from immunohistochemical studies confirm that these polypeptides are present in the tectorial membrane and are not contaminants of the isolation procedure. Collagenase treatment of tectorial membranes reveals the presence of an additional non-collagenous polypeptide with an apparent molecular weight of 173 kDa on 7.5% polyacrylamide gels, and polydisperse high molecular weight material spreading over a broad range at the top of the gels. This high molecular weight material and the 173, 60-73 and 35-49 kDa non-collagenous polypeptides are pepsin sensitive and all bind wheat germ agglutinin (WGA) suggesting that they contain N-acetyl glucosamine. The 173 kDa band also binds soybean agglutinin (SBA) suggesting the presence of N-acetyl galactosamine. In the absence of reducing agent the 173 and 60-73 kDa bands are no longer observed and high molecular weight material forming a broad band at the top of the separating gel is seen. The electrophoretic behaviour of this non-collagenous, glycosylated, disulphide bonded, high molecular weight material is altered by treatment with keratanase but not by chondroitinase ABC. The results of this study indicate the tectorial membrane contains at least three different collagen types and, in addition to these collagenous proteins, several non-collagenous, glycosylated polypeptides that may account for as much as 50% of the total tectorial membrane protein.
Goodpasture syndrome is an autoimmune disease causing rapidly progressive glomerulonephritis and pulmonary hemorrhage. The clinical manifestations are caused by autoantibodies that bind to a constituent, termed the Goodpasture autoantigen, of alveolar and glomerular basement membranes. Searches for the identity of this constituent have recently culminated in the discovery of two new chains (alpha 3 and alpha 4) of type IV collagen and the identification of the alpha 3 chain as the Goodpasture autoantigen. The gene, COL4A3, encoding this autoantigen was recently cloned and localized to the q35-37 region of chromosome 2. The major protomeric form of the alpha 3 chain is a homotrimer. The alpha 3-protomers associate through NC1-to-NC1 interactions mainly with each other to form a suprastructure, although some associate with protomers containing the alpha 1(IV) and alpha 2(IV) chains. The alpha 3-protomers also form suprastructures involving triple helical interactions of three or more protomers. The Goodpasture epitope is localized to the carboxylterminal region of the alpha 3(IV) chain, encompassing the last 36 residues of the chain, as the primary interaction site, and its structure is discontinuous.
It was previously demonstrated that about 40% of the protein of the tectorial membrane of the guinea pig consists of collagen type II, with lesser amounts of type IX and XI. In this paper we extend these studies on the tectorial membrane to the basilar membrane and to other accessory structures, the spiral ligament and spiral limbus. Earlier immunohistochemical data indicated that no collagen type II is present in the basilar membrane of the newborn guinea pig, but that it is present in the area of the basilar membrane in the embryo. However, by means of stringent extraction procedures we have determined biochemically that collagen type II and lesser amounts of type XI are present in the basilar membrane of the adult guinea pig, at similar levels (on the basis of total protein) to the tectorial membrane. Levels of collagen type II are much lower in the spiral ligament and spiral limbus. The presented studies demonstrate that classical techniques of collagen chemistry can be applied at the microscale on minute tissue elements. The significance of the presence of collagen in the tectorial membrane and basilar membrane is discussed in the light of known mechanical properties of these structures.
To determine the chain composition of type IV collagen of bovine thoracic aorta, we analyzed collagenase-solubilized carboxyl-terminal noncollagenous (NC1)-domains by high-pressure liquid chromatography, two-dimensional electrophoresis, immunoblotting and enzyme-linked immunoassay. In addition to the classical alpha 1- and alpha 2-chains, we found small amounts of the recently discovered alpha 3-, alpha 4- and alpha 5-chains. The alpha 3- and alpha 4-chains were, collectively, 7-13% of the total, and the alpha 5-chain was present in a low amount. Seventy-nine percent of the NC1-domains were dimerized. Immunolocalization studies on sections of aorta showed that the alpha 3- and alpha 5-chains were present, along with alpha 1- and alpha 2-chains, in the subendothelium and media. In capillaries of the media, the alpha 3-chain was found at relatively high levels and was co-localized with alpha 1- and alpha 2-chains. Digestion of aorta with Pseudomonas aeruginosa elastase yielded soluble multimolecular assemblies of type IV collagen. Electron microscopy results provided a direct demonstration of the supramolecular structure, in which the collagen molecules were tetramerized at the amino-terminal end and dimerized at the carboxyl-terminal end.
Goodpasture syndrome is an often fatal autoimmune disease associated with glomerulonephritis and/or pulmonary hemorrhage. The clinical manifestations of this disease correlate well with the presence of circulating antiglomerular basement membrane (GBM) autoantibodies. The primary target antigen in glomerular and alveolar basement membranes is thought to be the alpha 3 chain of type IV collagen. Nearly all that is known about anti-GBM antibodies in humans comes from work on unbound circulating antibody. We recently had the unique and rare opportunity to obtain early postmortem antibody and tissues from a patient who died with catastrophic Goodpasture syndrome. The specificity of circulating, kidney-bound and lung-bound autoantibodies from this patient was evaluated against a variety of purified basement membrane constituents. The results indicate that the primary target for the circulating and tissue-bound autoantibodies is the NC1 domain of the alpha 3(IV) chain of type IV collagen. Additionally, all the antibodies recognize a cryptic epitope/s on the alpha 3(IV)NC1 hexamer. Furthermore, tissue-bound and circulating antibodies compete with one another for overlapping epitopes on the antigen. These findings demonstrate that circulating autoantibodies in Goodpasture syndrome are highly representative of those bound to organ tissues, strengthening the notion that pathogenic autoantibodies are targeted to the alpha 3(IV)NC1 collagen, and that previous reports of findings in the circulation may be applicable to tissue injury.
Immunohistochemistry using antibodies specific for each of the basement membrane collagen chains was used to assess the location and composition of basement membranes in the mouse cochlea. The classical chains (COL4A1, 4A2) localized primarily in the osseous spiral lamina and in the capillaries of the spiral ligament. In contrast, the novel collagen chains (4A3, 4A4, and 4A5) localized to the interdental cells of the sulcus, the inner sulcus, the basilar membrane, and the region of type II fibrocytes in the spiral ligament. Antibodies against type 4A5 collagen also heavily stained the stria vascularis. Weak staining in the stria was observed with antibodies against 4A3. Basement membrane-associated proteins were also assessed. The basement membrane in the perineurium of the osseous spiral lamina immunostained using antibodies against laminin, heparan sulfate proteoglycan, and entactin. The basilar membrane contained only fibronectin in association with the novel collagen chains. The capillaries of the spiral ligament and the stria vascularis stained heavily for heparin sulfate proteoglycan and laminin. Generalized staining for laminin was observed in the spiral ligament. These results indicate that an abundance of basement membrane collagen containing extracellular matrix exists in the murine cochlea and that the composition of these matrices are surprisingly varied and tissue specific.
An immunofluorescence study was performed to examine the temporal and spatial patterns of expression for the different type IV collagen chains during postnatal cochlear development. At birth, the classical chains (4A1 and 4A2) were widely expressed, while the novel chains (4A3, 4A4, and 4A5) were completely absent. Activation of the novel chains was observed at 4 days of age, with intense, widely distributed immunostaining suggesting that most of the cells in the cochlea express the novel chains at this developmental stage. From day 8 through day 14, developmental inactivation of the novel chains results in a reduction of generalized immunoreactivity with a concomitant elevation of specific staining in the membranous structures bounding the interdental cells of the spiral limbus, the inner sulcus, the basilar membrane, and in a fibrous bed of staining radiating from the spiral prominence into the region of the spiral ligament which corresponds to the location of the root cell processes. This pattern of intense immunostaining for the novel chains persists through adulthood. The classical chains are expressed in these same anatomical regions only transiently (from day 6 to day 10), after which a gradual developmental inactivation leads to the adult expression pattern where classical collagen chains are found primarily in the perineurium, in the membranes surrounding the spiral ganglion cell bodies, and in the vascular basement membranes of the spiral ligament and the stria vascularis. The complex developmental pattern of expression for the type IV collagen chains in the murine cochlea is similar to that observed in the murine kidney, which is the other major site for basement membrane pathology in Alport syndrome.
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Immunological susceptibility to anti-glomerular basement disease is linked to MHC Class II genes and the emergence of a cell-mediated repertoire bearing a Th1 phenotype
- R Kalluri
- T M Danoff
- H Okada
- E G Neilson
Kalluri, R., Danoff, T. M., Okada, H. and Neilson, E. G. (1997). Immunological susceptibility to anti-glomerular base-ment disease is linked to MHC Class I1 genes and the emer-gence of a cell-mediated repertoire bearing a Thl phenotype. In Press. 1231
Alveolar basement membrane: molecular properties of the noncollagenous domain (hexamer) of collagen IV and its reactivity with Goodpasture autoantibodies (see comments)
- S Gunwar
- P A Bejarano
- R Kalluri
- J P Langeveld
- B J J Wisdom
- M E Noelken
- B G Hudson
An active process in cochlear mechanics
- H Davis
Immunologic Renal Diseases
- C M Meyers
- R Kalluri
- E Neilson
Immuno-localization of basement collagens and associated proteins in murine cochlea
- D Cosgrove
- G Sameulson
- J Pinnt