Dataset Information

Cleavage site specificity of MMP-20 for secretory-stage ameloblastin.

ABSTRACT: Ameloblastin is processed by protease(s) during enamel formation. We tested the hypothesis that MMP-20 (enamelysin) catalyzes the cleavages that generate secretory-stage ameloblastin cleavage products. We isolated a 23-kDa ameloblastin cleavage product from developing enamel and determined its N-terminus sequence. Ameloblastin was stably expressed and secreted from HEK293-H cells, purified, and digested with MMP-20 or Klk4 (kallikrein 4). The digests were analyzed by SDS-PAGE and Western blotting, and 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 corresponding to ameloblastin cleavages catalyzed in vivo. Klk4 cleaved ameloblastin and the fluorescent peptides at sites not observed in vivo, and cleaved at only a single correct site: before Leu(171). We conclude that MMP-20 is the enzyme that processes ameloblastin during the secretory stage of amelogenesis, and we present a hypothesis about the sequence of ameloblastin cleavages.

SUBMITTER: Chun YH

PROVIDER: S-EPMC2909333 | biostudies-literature | 2010 Aug

REPOSITORIES: biostudies-literature

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Cleavage site specificity of MMP-20 for secretory-stage ameloblastin.

Chun Y-H P YH Yamakoshi Y Y Yamakoshi F F Fukae M M Hu J C-C JC Bartlett J D JD Simmer J P JP

Journal of dental research 20100416 8

Ameloblastin is processed by protease(s) during enamel formation. We tested the hypothesis that MMP-20 (enamelysin) catalyzes the cleavages that generate secretory-stage ameloblastin cleavage products. We isolated a 23-kDa ameloblastin cleavage product from developing enamel and determined its N-terminus sequence. Ameloblastin was stably expressed and secreted from HEK293-H cells, purified, and digested with MMP-20 or Klk4 (kallikrein 4). The digests were analyzed by SDS-PAGE and Western blottin ...[more]

PMID: 20400724

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Dataset Information

Cleavage site specificity of MMP-20 for secretory-stage ameloblastin.

Publications

Cleavage site specificity of MMP-20 for secretory-stage ameloblastin.

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