Characterization of recombinant pig enamelysin activity and cleavage of recombinant pig and mouse amelogenins.
Enamelysin (MMP-20) is a tooth-specific matrix metalloproteinase that is initially expressed by ameloblasts and odontoblasts immediately prior to the onset of dentin mineralization, and continues to be expressed throughout the secretory stage of amelogenesis. During the secretory stage, enamel proteins are secreted and rapidly cleaved into a large number of relatively stable cleavage products. Multiple proteinases are present in the developing enamel matrix, and the precise role of enamelysin in the processing of enamel proteins is unknown. We have expressed, activated, and purified the catalytic domain of recombinant pig enamelysin, and expressed a recombinant form of the major secreted pig amelogenin rP172. These proteins were incubated together, and the digestion products were analyzed by SDS-PAGE and mass spectrometric analyses. We assigned amelogenin cleavage products by selecting among the possible polypeptides having a mass within 2 Daltons of the measured values. The polypeptides identified included the intact protein (amino acids 2-173), as well as 2-148, 2-136, 2-107, 2-105, 2-63, 2-45, 46-148, 46-147, 46-107, 46-105, 64-148, 64-147, and 64-136. These fragments of rP172 include virtually all of the major amelogenin cleavage products observed in vivo. We propose that enamelysin is the predominant proteinase that processes enamel proteins during the secretory phase of amelogenesis.
Ryu
O H
OH
University of Texas Health Science Center at San Antonio, School of Dentistry, Department of Pediatric Dentistry, 78284-7888, USA.
Fincham
A G
AG
Hu
C C
CC
Zhang
C
C
Qian
Q
Q
Bartlett
J D
JD
Simmer
J P
JP
D000566:Amelogenesis; D053523:Amelogenin; D000595:Amino Acid Sequence; D000818:Animals; D003746:Dental Enamel Proteins / Q000737:chemistry* / Q000378:metabolism*; D004591:Electrophoresis, Polyacrylamide Gel; D004658:Enamel Organ / Q000201:enzymology*; D013058:Mass Spectrometry; D053519:Matrix Metalloproteinase 20; D020782:Matrix Metalloproteinases; D008666:Metalloendopeptidases / Q000378:metabolism*; D051379:Mice; D008970:Molecular Weight; D010446:Peptide Fragments / Q000737:chemistry; D011480:Protease Inhibitors / Q000378:metabolism; D011499:Protein Processing, Post-Translational; D011994:Recombinant Proteins / Q000378:metabolism; D013552:Swine; D019716:Tissue Inhibitor of Metalloproteinase-2 / Q000378:metabolism
D053523:Amelogenin; C507200:Amelx protein, mouse; D003746:Dental Enamel Proteins; D010446:Peptide Fragments; D011480:Protease Inhibitors; D011994:Recombinant Proteins; D019716:Tissue Inhibitor of Metalloproteinase-2; D053519:Matrix Metalloproteinase 20; D020782:Matrix Metalloproteinases; D008666:Metalloendopeptidases; C507113:Mmp20 protein, mouse