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.
Highlights
Tooth enamel, the hardest substance in the body, is formed by an evolutionarily highly conserved biomineralization process that is controlled by extracellular matrix proteins
DEGRADATION AND DISTRIBUTION OF MOUSE AMELOBLASTIN We analyzed the degradation of mouse ameloblastin immunochemically using Western blot analysis (Figure 2, lane 3) with proteins extracted from 9 day old mouse enamel
To observe the distribution of ameloblastion and its proteolytic products in enamel we have incubate tissue sections from day 1 (Figure 3A), 5 (Figure 3B) and 8 (Figure 3C) with antiameloblastin M300 antibody followed by immunofluorescence labeling with Texas red conjugated anti-rabbit secondary antibody
Summary
The hardest substance in the body, is formed by an evolutionarily highly conserved biomineralization process that is controlled by extracellular matrix proteins. Proper mineralization of enamel depends upon the secretion of these proteins, as well as their being processed into smaller functional components and eventually degraded by proteinases such as enamelysin (matrix metalloproteinase 20, Mmp-20) and kallikrein 4 (Klk4) (Bartlett and Simmer, 1999). It was suggested that those proteins of enamel must have specific protein–protein interactions to assemble an organic matrix that is capable of undergoing mineral replacement and forming the highly ordered three-dimensional structure of the hydroxyapatite crystallites (Hu et al, 2001a; Bouropoulos and Moradian-Oldak, 2004; Bartlett et al, 2006; Fan et al, 2008; Iijima et al, 2010; Yang et al, 2011; Gallon et al, 2013). We have recently reported cooperative function of amelogenin and enamelin as well as their interactions using both in vitro and in vivo strategies (Bouropoulos and Moradian-Oldak, 2004; Fan et al, 2011; Yang et al, 2011; Gallon et al, 2013)
Sign-in/Register to view highlights & summary Talk to us
Join us for a 30 min session where you can share your feedback and ask us any queries you have
Schedule a callDisclaimer: All third-party content on this website/platform is and will remain the property of their respective owners and is provided on "as is" basis without any warranties, express or implied. Use of third-party content does not indicate any affiliation, sponsorship with or endorsement by them. Any references to third-party content is to identify the corresponding services and shall be considered fair use under The CopyrightLaw.
