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Processing of Ameloblastin by MMP-20
T. Iwata1,3,
Y. Yamakoshi1,
J.C.-C. Hu2,
I. Ishikawa3,
J.D. Bartlett4,
P.H. Krebsbach1 and
J.P. Simmer1,*
1 Department of Biologic and Materials Sciences and
2 Department of Orthodontics and Pediatric Dentistry, Dental Research Lab, University of Michigan School of Dentistry, 1210 Eisenhower Place, Ann Arbor, MI 48108, USA;
3 Department of Hard Tissue Engineering, Tokyo Medical and Dental University, Tokyo, Japan; and
4 Department of Cytokine Biology, The Forsyth Institute Boston, MA, USA
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Figure 1. Purified rpAMBN expressed in 293F mammalian cells and activated rpMMP-20. (A) The rpAMBN migrated as a smeared band at 65 kDa on 4–12% NuPage gels. The rAMBN reacted with Coomassie brilliant blue (CBB), stains-all (SA), and a glycoprotein gel stain (Pro-Q). The rpAMBN reacted with 3 antibodies: 2 AMBN anti-peptide antibodies (AMBN-63 and AMBN-381) and a monoclonal antibody specific for the V5 epitope (V5) fused to the AMBN C-terminus. (B) The 25-kDa rpMMP-20 catalytic domain used to digest rpAMBN is indicated by an arrowhead on a casein zymogram (Zym).
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Figure 2. Time-course for the digestion of rpAMBN by rpMMP-20. (A) 4–12% NuPage gels stained with CBB (left) and silver (right). (B) Western blots of a similar time-course immunostained with the AMBN N-terminal (AMBN-63) and C-terminal (AMBN-381) anti-peptide antibodies. The lanes are marked according to the times (in hrs) of the digestions. An asterisk marks negative controls with no rpMMP-20 added. The rpAMBN incubated without rpMMP-20 was stable and was undigested for the duration of the time-course, indicating that rpMMP-20 was solely responsible for the AMBN cleavages in the experimental conditions. Western blot analyses with region-specific anti-peptide antibodies showed that the smeared band at 48 kDa did not contain the AMBN N-terminus, while the smaller bands at 16, 18, and 19 kDa did.
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Figure 3. N-terminal sequence characterization of rpAMBN polypeptides generated by rpMMP-20 digestion. The rpAMBN digestions after 0.5 and 4 hrs were separated on 4–12% NuPage gels, transblotted to a membrane, and lightly stained with CBB; 7 strips of membrane were analyzed by N-terminal sequencing. CBB-stained replica gels of the ones used for this analysis are shown here. Arrowheads indicate the positions of the bands excised for analysis. The band labels (i.e., S65, etc.) indicate the apparent molecular weight of the excised protein. The numbers at the start of the protein sequences indicate the number of the first amino acid in the 365-amino-acid porcine AMBN sequence. When more than one sequence was obtained for a single strip of membrane, the separate sequences are indicated by a, b, or c.
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Figure 4. Ameloblastin cleavage sites in vivo and in vitro. Analysis of porcine ameloblastin cleavage products isolated from developing teeth show that ameloblastin is cleaved in vivo after Asn31 (a), Gln130 (b), Arg170 (c), Ala222 (d), Gly300 (e), Arg319 (f), and Ala342 (g). Three AMBN cleavage sites targeted by MMP-20 in vitro are identical to known in vivo cleavage sites (after Gln130, Arg170, and A222). Two sites that were cleaved in vitro have not been identified in vivo: Pro2 (h*) and Gln139 (i*). The context of the Gln139 site (Q:QV) is identical to the Gln130 site, suggesting that this site is likely to be cleaved in vivo, but in vivo cleavage products starting with this cleavage have not yet been described. Key: The in vivo and in vitro AMBN cleavage sites are marked by arrows. The arrows are boxed for the sites cleaved in vitro.
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Journal of Dental Research, Vol. 86, No. 2,
153-157 (2007)
DOI: 10.1177/154405910708600209
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