Cleavage Site Specificity of MMP-20 for Secretory-stage Ameloblastin

Abstract

Ameloblastin is a secreted phosphorylated glycoprotein that is processed by protease(s) during enamel formation. We test the hypothesis that Mmp-20 catalyzes the cleavages that generate the Ambn cleavage products that accumulate in developing enamel. We isolated a 23-kDa Ambn cleavage product from developing enamel and determined its N-terminus sequence started at Tyr223. Ameloblastin was stably expressed and secreted from HEK293-H cells, purified and digested with Mmp-20 or Klk4. The digests were analysed by SDS-PAGE and Western blotting, and the cleavage products were characterized by N-terminal sequencing. Six fluorescent peptides were digested with Mmp-20 and Klk4 and analyzed by RP-HPLC and by mass spectrometry. Mmp-20 cleaved each peptide exactly at the sites correspsonding to Ambn cleavages catalyzed in vivo: on the N-terminal sides of Met32, Gln131, Leu171, Tyr223, Leu301, and Tyr343. Klk4 cleaved Ambn and the fluorescent peptides at many sites not observed in vivo, and was only able to cleave at a single correct site: before Leu171. We conclude that Mmp-20 is the enzyme that processes Ambn during the secretory stage of amelogenesis.

El papel de la enamelisina (MMP-20) en el desarrollo dentario. Revisión sistemática

“…Therefore, in the development of pig tooth, MMP-20 is the only that presents proteolytic activity in the extracellular space during the secretory phase. This is confirmed by Chun et al, 15 who concluded that MMP-20 -and not Klk4-catalyzes enamel proteins that are processed during the amelogenesis secretory phase. Other authors, such as Sun et al, 16 studied MMP-20 and Klk4.…”

“…Por lo tanto, en el diente en desarrollo del cerdo, la MMP-20 es la única que presenta actividad proteolítica en el espacio extracelular durante la fase secretora. Esto es confirmado por Chun y colaboradores, 15 donde concluyen que MMP-20 pero no Klk4 cataliza las proteínas de esmalte procesadas durante la fase secretora de amelogénesis. También otros autores, como Sun y colaboradores, 16 estudiaron MMP-20 y Klk4.…”

“…Esto lo corroboraron Bartlett y colaboradores 26 y Shin y colaboradores 27 donde, con un modelo de ratón knockout para MMP-20, demostraron la importancia de MMP-20 como mediador necesario para el mantenimiento de la superficie de esmalte, una unión amelo-dentinal fuerte y para el establecimiento del patrón de varillas de esmalte. Estos resultados validan lo encontrado en modelos in vitro por Nagano y colaboradores 14 y Chun y colaboradores 15 .…”

Protein Interaction between Ameloblastin and Proteasome Subunit α Type 3 Can Facilitate Redistribution of Ameloblastin Domains within Forming Enamel

“…In this study, we generated the ameloblastin N-terminal domain and C-terminal domain by separating the full-length protein between Arg 222 and Leu 223 according to the previous experimental evidence (42,54). We found that Psma3 coimmunoprecipitated with both full-length and C-terminal ameloblastin protein, indicating that the binding site for Psma3 maps to the C-terminal domain of ameloblastin.…”

“…Ameloblastin protein is highly expressed by the secretory-stage ameloblasts and diminishes in abundance during the maturation stage (38 -40). Ameloblastin is processed by matrix metalloproteinase 20 (also known as enamelysin or MMP20) (41,42) immediately upon being secreted into the extracellular space. Remarkably, the ameloblastin cleavage products redistribute into different areas within the enamel rod, producing a pattern.…”