| 0804 Processing of Ameloblastin by MMP-20 | ||
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T. IWATA1, Y. YAMAKOSHI1, I. ISHIKAWA2, J.C. HU1, and J.P. SIMMER1, 1University of Michigan, Ann Arbor, USA, 2Tokyo Medical & Dental University, Japan Porcine ameloblastin (AMBN) is a secreted O-glycoprotein having 395 amino acids and an apparent Mr on SDS-PAGE of ~65 kDa. AMBN cleavage products are the most abundant nonamelogenin proteins in secretory stage enamel. AMBN N-terminal cleavage products are relatively stable and accumulate in the sheath space between enamel rods. They migrate as three bands on SDS-PAGE: 17 kDa, Val1-Arg170; 15 kDa, Val1-Gln130; and 13 kDa, Met32-Gln130. In contrast, C-terminal AMBN peptides localize to the enamel rod and do not accumulate in the matrix. MMP-20 (enamelysin) is the predominant protease in secretory stage enamel. Objective: to test the hypothesis that MMP-20 is the protease that cleaves AMBN. Methods: For recombinant expression, amplified porcine AMBN cDNA was ligated into pEF6/V5-His-TOPO (Invitrogen), which fused histo-V5-epitope tags to the AMBN C-terminus. 293-F cells were transfected with the expression construct and grown in serum-free FreeStyle 293 expression medium. Recombinant porcine enamelysin was expressed in bacteria. Recombinant AMBN and recombinant MMP-20 were both isolated using a TALON affinity column (Clontech) followed by RP-HPLC. AMBN and MMP-20 were incubated together at an enzyme: substrate ratio of 1:100. The digestion products were fractionated by RP-HPLC, analyzed by SDS-PAGE and densitometry, transblotted to a PVDF membrane and analyzed by Edman sequencing and MALDI mass spectrometry. Results: Recombinant AMBN migrates as a wide band between 65 and 75 kDa that is completely digested by enamelysin in 24 h. After 10 min, the wide AMBN band is diminished, while a broad smear band centered at 50 kDa appears, along with a triplet of lower molecular weight bands. Conclusions: Enamelysin cleaves AMBN in vitro in a pattern that closely matches the AMBN cleavage products that are observed in vivo, and is likely process AMBN during amelogenesis. This study is supported by NIDCR grant 12769 and DE15846. | ||
| Seq #70 - Mechanisms of Odontogenesis 11:00 AM-12:00 PM, Thursday, 29 June 2006 Brisbane Convention & Exhibition Centre Exhibit Hall 1 | ||
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