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Characterization of Recombinant Pig Enamelysin Activity and Cleavage of Recombinant Pig and Mouse Amelogenins
O.H. Ryu
University of Texas Health Science Center at San Antonio, School of Dentistry, Department of Pediatric Dentistry, 7703 Floyd Curl Drive, San Antonio, Texas 78284-7888
A.G. Fincham
Center for Craniofacial Molecular Biology, University of Southern California, School of Dentistry, 2250 Alcazar Street, CSA 1st Floor, Los Angeles, California 90033
C.-C. Hu
University of Texas Health Science Center at San Antonio, School of Dentistry, Department of Pediatric Dentistry, 7703 Floyd Curl Drive, San Antonio, Texas 78284-7888
C. Zhang
University of Texas Health Science Center at San Antonio, School of Dentistry, Department of Pediatric Dentistry, 7703 Floyd Curl Drive, San Antonio, Texas 78284-7888
Q. Qian
University of Texas Health Science Center at San Antonio, School of Dentistry, Department of Pediatric Dentistry, 7703 Floyd Curl Drive, San Antonio, Texas 78284-7888
J.D. Bartlett
Department of Biomineralization, Forsyth Dental Center, 140 Fenway, Boston, Massachusetts 02115
J.P. Simmer
University of Texas Health Science Center at San Antonio, School of Dentistry, Department of Pediatric Dentistry, 7703 Floyd Curl Drive, San Antonio, Texas 78284-7888
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.
Key Words: enamel • matrix • metalloproteinase • enamelysin • amelogenin • MMP-20
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Journal of Dental Research, Vol. 78, No. 3,
743-750 (1999)
DOI: 10.1177/00220345990780030601
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