Purification, characterization, and cloning of enamel matrix serine proteinase 1

J Dent Res. 1998 Feb;77(2):377-86. doi: 10.1177/00220345980770020601.

Abstract

The maturation of dental enamel succeeds the degradation of organic matrix. Inhibition studies have shown that this degradation is accomplished by a serine-type proteinase. To isolate and characterize cDNA clones 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 successive ammonium sulfate precipitations, hydroxyapatite HPLC, reverse-phase HPLC, DEAE ion exchange, and affinity chromatography with a Benzamidine Sepharose 6B column. The intact protein and lysylendopeptidase-generated cleavage products were characterized by amino acid sequence analyses. Degenerate oligonucleotide primers encoding two of the polypeptide sequences were synthesized. In a complementary strategy, degenerate oligonucleotide primers were designed against highly conserved active-site regions of chymotrypsin-like proteinases. Both approaches yielded PCR amplification products that served as probes for screening a porcine enamel organ epithelia-specific cDNA library. The longest full-length clone is 1133 nucleotides and encodes a preproprotein of 254 amino 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 protein. Gelatin enzymography shows a dramatic increase in EMSP1 activity in the transition-stage enamel matrix. EMSP1 is most homologous to kallikriens and trypsins.

Publication types

  • Research Support, Non-U.S. Gov't
  • Research Support, U.S. Gov't, P.H.S.

MeSH terms

  • Amelogenesis*
  • Amino Acid Sequence
  • Animals
  • Base Sequence
  • Cloning, Molecular
  • Dental Enamel / enzymology*
  • Dental Enamel Proteins / chemistry*
  • Dental Enamel Proteins / genetics
  • Dental Enamel Proteins / isolation & purification
  • Electrophoresis, Polyacrylamide Gel
  • Kallikreins*
  • Molecular Sequence Data
  • Molecular Weight
  • Organ Specificity
  • Polymerase Chain Reaction
  • Protein Processing, Post-Translational
  • Sequence Analysis
  • Serine Endopeptidases / chemistry*
  • Serine Endopeptidases / genetics
  • Serine Endopeptidases / isolation & purification
  • Swine

Substances

  • Dental Enamel Proteins
  • Kallikreins
  • Serine Endopeptidases
  • kallikrein 4

Associated data

  • GENBANK/U76256