PURIFICATION, CHARACTERIZATION, AND CLONING OF ENAMEL MATRIX SERINE PROTEINASE-1

The maturation of dental enamel succeeds the degradation of organic ma trix. inhibition studies have shown that this degradation is accomplis hed by a serine-type proteinase. To isolate and characterize cDNA clon es encoding this proteinase, we used two degenerate primer approaches to amplify part of the coding region using polymerase chain-reaction ( PCR). First, we purified the proteinase from porcine transition-stage enamel matrix and characterized it by partial protein sequencing. The enzyme was isolated from the neutral soluble enamel extract by success ive ammonium sulfate precipitations, hydroxyapatite HPLC, reverse-phas e HPLC, DEAE ion exchange, and affinity chromatography with a Benzamid ine Sepharose 6B column. The intact protein and lysylendopeptidase-gen erated cleavage products were characterized by amino acid sequence ana lyses. Degenerate oligonucleotide primers encoding two of the polypept ide sequences were synthesized. In a complementary strategy, degenerat e oligonucleotide primers were designed against highly conserved activ e-site regions of chymotrypsin-like proteinases. Both approaches yield ed PCR amplification products that served as probes for screening a po rcine enamel organ epithelia-specific cDNA library. The longest full-l ength clone is 1133 nucleotides and encodes a preproprotein of 254 ami no acids. We designate this protein enamel matrix serine proteinase 1 or EMSP1. The active protein has 224 amino acids, an isotope-averaged molecular mass of 24.1 kDa, and an isoelectric point of 6.0. Multiple- tissue Northern analysis indicates that EMSP1 is a tooth-specific prot ein. Gelatin enzymography shows a dramatic increase in EMSP1 activity in the transition-stage enamel matrix. EMSP1 is most homologous to kal likriens and trypsins.