Publications

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    Saumya Prajapati · Jinhui Tao · Qichao Ruan · James J. De Yoreo · Janet Moradian-Oldak
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    ABSTRACT: Reconstruction of enamel-like materials is a central topic of research in dentistry and material sciences. The importance of precise proteolytic mechanisms in amelogenesis to form a hard tissue with more than 95% mineral content has already been reported. A mutation in the Matrix Metalloproteinase-20 (MMP-20) gene results in hypomineralized enamel that is thin, disorganized and breaks from the underlying dentin. We hypothesized that the absence of MMP-20 during amelogenesis results in the occlusion of amelogenin in the enamel hydroxyapatite crystals. We used spectroscopy and electron microscopy techniques to qualitatively and quantitatively analyze occluded proteins within the isolated enamel crystals from MMP-20 null and Wild type (WT) mice. Our results showed that the isolated enamel crystals of MMP-20 null mice had more organic macromolecules occluded inside them than enamel crystals from the WT. The crystal lattice arrangements of MMP-20 null enamel crystals analyzed by High Resolution Transmission Electron Microscopy (HRTEM) were found to be significantly different from those of the WT. Raman studies indicated that the crystallinity of the MMP-20 null enamel crystals was lower than that of the WT. In conclusion, we present a novel functional mechanism of MMP-20, specifically prevention of unwanted organic material entrapped in the forming enamel crystals, which occurs as the result of precise amelogenin cleavage. MMP-20 action guides the growth morphology of the forming hydroxyapatite crystals and enhances their crystallinity. Elucidating such molecular mechanisms can be applied in the design of novel biomaterials for future clinical applications in dental restoration or repair.
    Full-text · Article · Jan 2016 · Biomaterials
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    ABSTRACT: Amelogenin-chitosan (CS-AMEL) hydrogel has shown great potential for the prevention, restoration, and treatment of defective enamel. As a step prior to clinical trials, this study aimed to examine the efficacy of CS-AMEL hydrogel in biomimetic repair of human enamel with erosive or caries-like lesions in pH-cycling systems. Two models for enamel defects, erosion and early caries, were addressed in this study. Two pH-cycling systems were designed to simulate the daily cariogenic challenge as well as the nocturnal pH conditions in the oral cavity. After pH cycling and treatment with CS-AMEL hydrogel, a synthetic layer composed of oriented apatite crystals was formed on the eroded enamel surface. CS-AMEL repaired the artificial incipient caries by re-growing oriented crystals and reducing the depth of the lesions by up to 70% in the pH-cycling systems. The results clearly demonstrate that the CS-AMEL hydrogel is effective at the restoration of erosive and carious lesions under pH-cycling conditions.
    Full-text · Article · Jan 2016
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    ABSTRACT: Dry eye is a common disorder caused by inadequate hydration of the ocular surface that results in disruption of barrier function. The homeostatic protein clusterin (CLU) is prominent at fluid-tissue interfaces throughout the body. CLU levels are reduced at the ocular surface in human inflammatory disorders that manifest as severe dry eye, as well as in a preclinical mouse model for desiccating stress that mimics dry eye. Using this mouse model, we show here that CLU prevents and ameliorates ocular surface barrier disruption by a remarkable sealing mechanism dependent on attainment of a critical all-or-none concentration. When the CLU level drops below the critical all-or-none threshold, the barrier becomes vulnerable to desiccating stress. CLU binds selectively to the ocular surface subjected to desiccating stress in vivo, and in vitro to the galectin LGALS3, a key barrier component. Positioned in this way, CLU not only physically seals the ocular surface barrier, but it also protects the barrier cells and prevents further damage to barrier structure. These findings define a fundamentally new mechanism for ocular surface protection and suggest CLU as a biotherapeutic for dry eye.
    Full-text · Article · Sep 2015 · PLoS ONE
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    ABSTRACT: We have recently reported that the extracellular enamel protein amelogenin has affinity to interact with phospholipids and proposed that such interactions may play key roles in enamel biomineralization as well as reported amelogenin signaling activities. Here, in order to identify the liposome-interacting domains of amelogenin we designed four different amelogenin mutants containing only a single tryptophan at positions 25, 45, 112 and 161. Circular dichroism studies of the mutants confirmed that they are structurally similar to the wild-type amelogenin. Utilizing the intrinsic fluorescence of single tryptophan residues and fluorescence resonance energy transfer FRET, we analyzed the accessibility and strength of their binding with an ameloblast cell membrane-mimicking model membrane (ACML) and a negatively charged liposome used as a membrane model. We found that amelogenin has membrane-binding ability mainly via its N-terminal, close to residues W25 and W45. Significant blue shift was also observed in the fluorescence of a N-terminal peptide following addition of liposomes. We suggest that, among other mechanisms, enamel malformation in cases of Amelogenesis Imperfecta (AI) with mutations at the N-terminal may be the result of defective amelogenin-cell interactions. Copyright © 2015. Published by Elsevier Inc.
    No preview · Article · Jul 2015 · Biochemical and Biophysical Research Communications
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    Qichao Ruan · Janet Moradian-Oldak
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    ABSTRACT: Mature tooth enamel is acellular and does not regenerate itself. Developing technologies that rebuild tooth enamel and preserve tooth structure is therefore of great interest. Considering the importance of amelogenin protein in dental enamel formation, its ability to control apatite mineralization in vitro, and its potential to be applied in fabrication of future bio-inspired dental material this review focuses on two major subjects: amelogenin and enamel biomimetics. We review the most recent findings on amelogenin secondary and tertiary structural properties with a focus on its interactions with different targets including other enamel proteins, apatite mineral, and phospholipids. Following a brief overview of enamel hierarchical structure and its mechanical properties we will present the state-of-the-art strategies in the biomimetic reconstruction of human enamel.
    Full-text · Article · Mar 2015
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    ABSTRACT: The host immune system is known to influence mesenchymal stem cell (MSC)-mediated bone tissue regeneration. However, the therapeutic capacity of hydrogel biomaterial to modulate the interplay between MSCs and T-lymphocytes is unknown. Here it is shown that encapsulating hydrogel affects this interplay when used to encapsulate MSCs for implantation by hindering the penetration of pro-inflammatory cells and/or cytokines, leading to improved viability of the encapsulated MSCs. This combats the effects of the host pro-inflammatory T-lymphocyte-induced nuclear factor kappaB pathway, which can reduce MSC viability through the CASPASE-3 and CASPASE-8 associated proapoptotic cascade, resulting in the apoptosis of MSCs. To corroborate rescue of engrafted MSCs from the insult of the host immune system, the incorporation of the anti-inflammatory drug indomethacin into the encapsulating alginate hydrogel further regulates the local microenvironment and prevents pro-inflammatory cytokine-induced apoptosis. These findings suggest that the encapsulating hydrogel can regulate the MSC-host immune cell interplay and direct the fate of the implanted MSCs, leading to enhanced tissue regeneration.
    Full-text · Article · Mar 2015 · Advanced Functional Materials
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    ABSTRACT: Amelogenin protein has the potential to interact with other enamel matrix proteins, mineral and cell surfaces. We investigated the interactions of recombinant amelogenin rP172 with small unilamellar vesicles as model membranes, towards the goal of understanding the mechanisms of amelogenin-cell interactions during amelogenesis. Dynamic light scattering (DLS), fluorescence spectroscopy, circular dichroism (CD) and nuclear magnetic resonance (NMR) were used. In the presence of phospholipid vesicles, a blue shift in the Trp fluorescence emission maxima of rP172 was observed (~334 nm) and the Trp residues of rP172 were inaccessible to the aqueous quencher acrylamide. Though in DLS studies we cannot exclude the possibility of fusion of liposomes as the result of amelogenin addition, NMR and CD studies revealed a disorder-order transition of rP172 in a model membrane environment. Strong FRET from Trp in rP172 to DNS-bound-phospholipid was observed, and fluorescence polarization studies indicated that rP172 interacted with the hydrophobic core region of model membranes. Our data suggest that amelogenin has ability to interact with phospholipids and that such interactions may play key roles in enamel biomineralization as well as reported amelogenin signaling activities.
    Full-text · Article · Feb 2015 · Biopolymers

  • No preview · Conference Paper · Jan 2015
  • Saumya Prajapati · Qichao Ruan · Jinhui Tao · James DeYoreo · Janet Moradian-Oldak

    No preview · Conference Paper · Jan 2015
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    Qichao Ruan · Nadia Siddiqah · Xiaochen Li · Steven Nutt · Janet Moradian-Oldak
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    ABSTRACT: Abstract We recently reported an amelogenin-chitosan (CS-AMEL) hydrogel as a promising biomimetic material for future in situ human enamel regrowth. To further optimize the necessary conditions for clinical applicability of CS-AMEL hydrogel, herein we studied the effects of viscosity and supersaturation degree on the size and orientation of synthetic crystals by means of scanning electron microscopy (SEM) and X-ray diffraction (XRD). Raising the hydrogel viscosity by increasing chitosan concentration from 1% to 2% (w/v) improved the orientation of the crystals, while a higher supersaturation (σ(HAp) >10.06, [Ca(2+)] >5 mM) resulted in the formation of random crystals with larger sizes and irregular structures. We conclude that optimal conditions to produce organized enamel-like crystals in a CS-AMEL hydrogel are: 2% (w/v) chitosan, 2.5 mM calcium, and 1.5 mM phosphate (degree of supersaturation = 8.23) and 200 µg/ml of amelogenin.
    Full-text · Article · Aug 2014 · Connective Tissue Research
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    ABSTRACT: Epithelially-derived ameloblasts secrete extracellular matrix proteins including amelogenin, enamelin, and ameloblastin. Complex intermolecular interactions among these proteins are believed to be important in controlling enamel formation. Here we provide in vitro and in vivo evidence of co-assembly and co-localization of ameloblastin with amelogenin using both biophysical and immunohistochemical methods. We performed co-localization studies using immunofluorescence confocal microscopy with paraffin-embedded tissue sections from mandibular molars of mice at 1, 5, and 8 days of age. Commercially-available ameloblastin antibody (M300) against mouse ameloblastin residues 107-407 and an antibody against full-length recombinant mouse (rM179) amelogenin were used. Ameloblastin-M300 clearly reacted along the secretory face of ameloblasts from days 1-8. Quantitative co-localization was analyzed (QCA) in several configurations by choosing appropriate regions of interest (ROIs). Analysis of ROIs along the secretory face of ameloblasts revealed that at day 1, very high percentages of both the ameloblastin and amelogenin co-localized. At day 8 along the ameloblast cells the percentage of co-localization remained high for the ameloblastin whereas co-localization percentage was reduced for amelogenin. Analysis of the entire thickness on day 8 revealed no significant co-localization of amelogenin and ameloblastin. With the progress of amelogenesis and ameloblastin degradation, there was a segregation of ameloblastin and co-localization with the C-terminal region decreased. CD spectra indicated that structural changes in ameloblastin occurred upon addition of amelogenin. Our data suggest that amelogenin-ameloblastin complexes may be the functional entities at the early stage of enamel mineralization.
    Full-text · Article · Jul 2014 · Frontiers in Physiology
  • Qichao Ruan · Janet Moradian-Oldak
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    ABSTRACT: Biomimetic enamel reconstruction is a significant topic in material science and dentistry as a novel approach for the treatment of dental caries or erosion. Amelogenin has been proven to be a critical protein for controlling the organized growth of apatite crystals. In this paper, we present a detailed protocol for superficial enamel reconstruction by using a novel amelogenin-chitosan hydrogel. Compared to other conventional treatments, such as topical fluoride and mouthwash, this method not only has the potential to prevent the development of dental caries but also promotes significant and durable enamel restoration. The organized enamel-like microstructure regulated by amelogenin assemblies can significantly improve the mechanical properties of etched enamel, while the dense enamel-restoration interface formed by an in situ regrowth of apatite crystals can improve the effectiveness and durability of restorations. Furthermore, chitosan hydrogel is easy to use and can suppress bacterial infection, which is the major risk factor for the occurrence of dental caries. Therefore, this biocompatible and biodegradable amelogenin-chitosan hydrogel shows promise as a biomaterial for the prevention, restoration, and treatment of defective enamel.
    No preview · Article · Jul 2014 · Journal of Visualized Experiments
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    ABSTRACT: Objectives We recently demonstrated a significant correlation between enamel delamination and tooth-level radiation dose in oral cancer patients. Since radiation can induce the synthesis and activation of matrix metalloproteinases, we hypothesized that irradiated teeth may contain active matrix metalloproteinases. Materials and Methods: Extracted teeth from oral cancer patients treated with radiotherapy and from healthy subjects were compared. Extracted mature third molars from healthy subjects were irradiated in vitro and/or incubated for 0 to 6 months at 37 °C. All teeth were then pulverized, extracted, and extracts subjected to proteomic and enzymatic analyses. Results: Screening of irradiated crown extracts using mass spectrometry identified MMP-20 (enamelysin) which is expressed developmentally in dentin and enamel but believed to be removed prior to tooth eruption. MMP-20 was composed of catalytically active forms at Mr = 43, 41, 24 and 22 kDa and was immunolocalized predominantly to the morphological dentin enamel junction. The proportion of different sized MMP-20 forms changed with incubation and irradiation. While the pattern was not altered directly by irradiation of healthy teeth with 70 G, subsequent incubation at 37 °C for 3-6 months with or without prior irradiation caused the proportion of Mr = 24-22 kDa MMP-20 bands to increase dramatically. Extracts of teeth from oral cancer patients who received >70 Gy radiation also contained relatively more 24 and 22 kDa MMP-20 than those of healthy age-related teeth. Conclusion: MMP-20 is a radiation-resistant component of mature tooth crowns enriched in the dentin-enamel. We speculate that MMP-20 catalyzed degradation of organic matrix at this site could lead to enamel delamination associated with oral cancer radiotherapy.
    No preview · Article · May 2014 · Journal of dentistry
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    ABSTRACT: Amelogenin, the major extracellular matrix protein of developing tooth enamel is intrinsically disordered. Through its interaction with other proteins and mineral, amelogenin assists enamel biomineralization by controlling the formation of highly organized enamel crystal arrays. We used circular dichroism (CD), dynamic light scattering (DLS), fluorescence and NMR spectroscopy to investigate the folding propensity of recombinant porcine amelogenin rP172 following its interaction with SDS, at levels above critical micelle concentration. The rP172-SDS complex formation was confirmed by DLS, while an increase in the structure moiety of rP172 was noted through CD and fluorescence experiments. Fluorescence quenching analyses performed on several rP172 mutants where all but one Trp was replaced by Tyr at different sequence regions confirmed that the interaction of amelogenin with SDS micelles occurs via the N-terminal region close to Trp25 where helical segments can be detected by NMR. NMR spectroscopy and structural refinement calculations using CS-Rosetta modelling confirm that the highly conserved N-terminal domain is prone to form helical structure when bound to SDS micelles. Our findings reported here reveal interactions leading to significant changes in the secondary structure of rP172 upon treatment with SDS. These interactions may reflect the physiological relevance of the flexible nature of amelogenin and its sequence specific helical propensity that might enable it to structurally adapt with charged and potential targets such as cell surface, mineral, and other proteins during enamel biomineralization.
    Full-text · Article · May 2014 · Biopolymers
  • P. MAZUMDER · V. GALLON · L. CHEN · S. PRAJAPATI · J. MORADIAN-OLDAK
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    ABSTRACT: We recently reported interactions between amelogenin and enamelin using both in vitro and in vivo strategies [J Struct Biol. 183, 239-49]. Objectives: To quantitatively analyze co-localization of ameloblastin and amelogenin providing evidence for their interactions during enamel formation using immunofluorescence confocal microscopy. Methods: Fluorescent-IHC staining has been performed on paraffin-embedded tissue sections from 1 to 8 days mouse mandibular molars. Commercially available antipeptide antibodies against N-terminal (N18) and C-terminal (M300) of ameloblastin, and an antibody against the full-length recombinant mouse (rM179) amelogenin were used. Co-localization patterns were analyzed using two different regions of interests (ROIs): a large (40 µm height) and a small (7 µm diameter). Results: Ameloblastin-M300 clearly reacted along the secretary face of ameloblasts from day 2-8, while significant reaction against ameloblastin-N18 was observed throughout the entire thickness of enamel from day 5-8. Co-localization analysis by small ROIs along the secretary face of ameloblasts reveled that at day 2, equal amounts (~80%) of both the ameloblastin C-terminal and amelogenin co-localized. Co-localization decreased at day 8 when only ~66% of amelogenin and ~57% of ameloblastin-C-terminal co-localized. Analysis of the entire thickness from day 1 to 5 using the N-terminal antibody revealed lower and different values of co-localization for amelogenin and ameloblastin (48% and 30% respectively) while on day 8 co-localization was low and the same for both (~40%). Conclusions: Amelogenin and ameloblastin co-localize at the secretary face of ameloblasts at the early stage of amelogenesis, suggesting that they are secreted simultaneously. With the progress of amelogenesis and ameloblastin degradation co-localization pattern changes as following: 1) there is a segregation of ameloblastin C- and N-terminal, 2) co-localization with C-terminal decreases while co-localization with the N-terminal is low and does not change. Our data suggest that amelogenin-ameloblastin complexes may be the functional entities at the early stage of enamel mineralization.
    No preview · Conference Paper · Mar 2014
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    Qichao Ruan · Yuzheng Zhang · Xiudong Yang · Steven Nutt · Janet Moradian-Oldak
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    ABSTRACT: Biomimetic reconstruction of tooth enamel is a significant topic of study in material science and dentistry as a novel approach for prevention, restoration, and treatment of defective enamel. We developed a new amelogenin-containing chitosan hydrogel for enamel reconstruction that works through amelogenin supramolecular assembly, stabilizing Ca-P clusters and guiding their arrangement into linear chains. These amelogenin Ca-P composite chains further fuse with enamel crystals and eventually evolve into enamel-like co-aligned crystals, anchoring to the natural enamel substrate through a cluster growth process. A dense interface between the newly-grown layer and natural enamel was formed and the enamel-like layer had improved hardness and elastic modulus compared to etched enamel. We anticipate that chitosan hydrogel will provide effective protection against secondary caries because of its pH-responsive and antimicrobial properties. Our studies introduce amelogenin-containing chitosan hydrogel as a promising biomaterial for enamel repair and demonstrate the potential of applying protein-directed assembly to biomimetic reconstruction of complex biomaterials.
    Full-text · Article · Apr 2013 · Acta biomaterialia
  • Victoria Gallon · Lisha Chen · Xiudong Yang · Janet Moradian-Oldak
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    ABSTRACT: Enamelin and amelogenin are vital proteins in enamel formation. The cooperative function of these two proteins controls crystal nucleation and morphology in vitro. We quantitatively analyzed the co-localization between enamelin and amelogenin by confocal microscopy and using two antibodies, one raised against a sequence in the porcine 32 kDa enamelin region and the other raised against full-length recombinant mouse amelogenin. We further investigated the interaction of the porcine 32 kDa enamelin and recombinant amelogenin using immuno-gold labeling. This study reports the quantitative co-localization results for postnatal days 1, 2, 3, 4, 5, 6, 7 and 8 mandibular mouse molars. We show that amelogenin and enamelin are secreted into the extracellular matrix on the cuspal slopes of the molars at day 1 and that secretion continues to at least day 8. Quantitative co-localization analysis (QCA) was performed in several different configurations using large (45 μm height, 33 μm width) and small (7 μm diameter) regions of interest to elucidate any patterns. Co-localization patterns in day 8 samples revealed that enamelin and amelogenin co-localize near the secretory face of the ameloblasts and appear to be secreted approximately in a 1:1 ratio. The degree of co-localization decreases as the enamel matures, both along the secretory face of ameloblasts and throughout the entire thickness of the enamel. Immuno-reactivity against enamelin is concentrated along the secretory face of ameloblasts, supporting the theory that this protein together with amelogenin is intimately involved in mineral induction at the beginning of enamel formation.
    No preview · Article · Apr 2013 · Journal of Structural Biology
  • S.B. LOKAPPA · K.B. CHANDRABABU · J. MORADIAN-OLDAK
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    ABSTRACT: Amelogenin, an intrinsically disordered protein, is the major component in the dental enamel extracellular matrix (ECM). ECM also constitutes other phosphorylated, glycosylated and sulfated proteins, proteinases and lipids. Due to this heterogeneity, complex protein-protein, protein-mineral and protein-lipid interactions can be envisaged during amelogenesis. Lipids constitute 0.2% of the developing enamel matrix. As amelogenin is synthesized by the ameloblast cells and secreted via matrix secretory vesicles, the study of its structure in the presence of cell membrane or membrane mimicking models can give more insight into its function during amelogenesis. Objectives: To investigate interactions of amelogenin with liposomes as artificial models for ameloblast cell membrane and matrix vesicles, to analyze the structural changes involved in amelogenin upon its binding to liposomes, and address the transformation between its disordered and ordered conformation. Methods: We used recombinant porcine full-length amelogenin (rP172) and a mixture of commercially available lipids and phospholipids. Binding between amelogenin and liposomes was analyzed by fluorescence spectroscopy, dynamic light scattering and transmission electron microscopy. Conformational changes in amelogenin were analyzed by circular dichroism. Results: Fluorescence studies revealed that monomeric rP172 is peripherally bound to the zwitterionic 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine (POPC) liposomes but associated strongly with the anionic 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphoglycerol (POPG) liposomes, as well as with the ameloblast cell membrane mimicking liposomes (ACML). Further, circular dichroism studies revealed that rP172 coils to form alpha helix following its interaction with the anionic POPG and the ACML liposomes. Fluorescence and CD studies indicated weak interactions of rP172 nanospheres with POPG and ACML liposomes. TEM studies suggests that amelogenin interactions are highly dependent on the lipid composition. Conclusions: Amelogenin molecules interact weakly with neutral liposomes, but strongly with negatively charged liposomes and the ameloblast mimicking liposomes, changing their conformation. These findings have important implications for understanding the interactions of amelogenin with ameloblasts and matrix secretory vesicles.
    No preview · Conference Paper · Mar 2013
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    ABSTRACT: Objectives: To identify MMP-20 proteinase and to detect its activity in extracts of mature human teeth. Methods: Individual third molar crowns, without caries or restorations, were pulverized and extracted with a guanidine chloride/ethylenediaminetetraacetic acid cocktail containing protease inhibitors, exchanged into 5% acetic acid and extracts were lyophilized. Western blotting for MMP-20 was done using either antibodies specific for the C-terminus, where the hemopexin domain is present, or for the N-terminus, where the catalytic domain is present. Immunoprecipitation of MMP-20 was performed using the N-terminus antibody. Enzymatic activity in dental extracts was analyzed with zymography using casein, gelatin, or porcine recombinant amelogenin (rP172) gels. Results: Western blotting with the N-terminal antibody routinely detected six bands at 53, 43, 41, 22, 19 and 11 kDa; the first five bands are identical with the sizes of MMP-20 precursor and four activated forms. The C-terminal antibody only recognized the 53 kDa precursor. Casein zymography with crown extracts detected three major bands at 43 kDa, 41 kDa and 22 kDa and two minor bands at 31 and 14 kDa. After immunoprecipitation, only the three major bands at 43, 41 and 22 kDa were observed identifying these forms as catalytically active MMP-20. The relative activity of each band was 33%, 33% and 34%, respectively. Consistent with this assignment, none of the MMP-20 forms were detected in gelatin gels and activity was only detected in the presence of calcium and zinc. Finally, when the dental extracts were analyzed for enzymatic activity using an amelogenin containing gel, three active bands were also observed at 43, 41 and 22 kDa. Conclusions: Our data show that active MMP-20 proteinase is readily detectable in extracts of mature human teeth. This assignment was confirmed by immunoprecipitation and cleavage of amelogenin. Active MMP-20 could play a degradative role in tooth damage associated with caries and oral cancer radiotherapy.
    No preview · Conference Paper · Mar 2013
  • Qichao Ruan · Yuzheng Zhang · Xiudong Yang · Steven Nutt · Janet Moradian-Oldak

    No preview · Conference Paper · Jan 2013

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