Activation of recombinant bovine matrix metalloproteinase-20 and its hydrolysis of two amelogenin oligopeptides.
Enamelysin is a matrix metalloproteinase (MMP-20) secreted by ameloblasts, previously shown to hydrolyze recombinant amelogenin. The purpose of this study was to use recombinant MMP-20 to further investigate the specific hydrolysis of peptide fragments containing cleavage sites for tyrosine-rich amelogenin peptide (TRAP) and leucine-rich amelogenin peptide (LRAP). MMP-20 cDNA was isolated from a subtracted bovine cDNA library, reconstructed into pRSET A vector, and overexpressed in BL21 Escherichia coli. The recombinant MMP-20 was purified using Mono-S ion exchange and nickel affinity chromatography. The proteinase was renatured by dialysis against buffer containing 50 microM zinc and 5 mM calcium and autolysed to form several active fragments. The varying sizes and activities of the activated enzyme fragments appeared to be due to sequential autolysis at different location of the carboxyl terminus of the intact enzyme. Two synthetic peptides corresponding to amelogenin amino acid sequences 36-49 and 181-188 were hydrolyzed by the activated rMMP-20. Mass spectrometry and amino acid composition analysis showed that the cleavage sites were between the tryptophan and leucine (45 and 46) for TRAP and between proline and alanine (186-187) for LRAP. These results indicate that MMP-20 can be autoactivated, and activated MMP-20 has a functional role in the initial cleavage of amelogenin.
Li
W
W
Growth and Development Department, School of Dentistry, University of California at San Francisco, USA.
Machule
D
D
Gao
C
C
DenBesten
P K
PK
D000409:Alanine / Q000032:analysis; D000565:Ameloblasts / Q000201:enzymology; D053523:Amelogenin; D000596:Amino Acids / Q000032:analysis; D000818:Animals; D002118:Calcium; D002417:Cattle; D002846:Chromatography, Affinity; D002852:Chromatography, Ion Exchange; D003746:Dental Enamel Proteins / Q000032:analysis / Q000187:drug effects*; D003956:Dialysis; D004789:Enzyme Activation; D004926:Escherichia coli / Q000235:genetics; D015971:Gene Expression Regulation, Enzymologic; D005822:Genetic Vectors; D006868:Hydrolysis; D007930:Leucine / Q000032:analysis; D013058:Mass Spectrometry; D053519:Matrix Metalloproteinase 20; D020782:Matrix Metalloproteinases / Q000494:pharmacology*; D011392:Proline / Q000032:analysis; D011994:Recombinant Proteins; D014364:Tryptophan / Q000032:analysis; D015032:Zinc
D053523:Amelogenin; D000596:Amino Acids; D003746:Dental Enamel Proteins; D011994:Recombinant Proteins; C067432:leucine-rich amelogenin peptide; C084495:tyrosine-rich amelogenin polypeptide; D014364:Tryptophan; D011392:Proline; D053519:Matrix Metalloproteinase 20; D020782:Matrix Metalloproteinases; D007930:Leucine; D015032:Zinc; D000409:Alanine; D002118:Calcium