Purification, Characterization, and Cloning of Enamel Matrix Serine Proteinase 1

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.

Identification and Characterization of KLK-L4, a New Kallikrein-like Gene That Appears to be Down-regulated in Breast Cancer Tissues

“…8). Phylogenetic analysis separated the classical kallikreins (KLK1, KLK2, and PSA) and grouped KLK-L4 with zyme, TLSP, KLK-L3, neuropsin, and NES1 genes, consistent with previously published studies (8,24,25) and indicating that this group of genes probably arose from a common ancestral gene by duplication.…”

“…The large size of the kallikrein gene family in other species, such as rat and mouse, where kallikreins are encoded by [13][14][15][16][17][18][19][20][21][22][23][24] genes (12)(13), and the recent identification of new kallikreinlike genes suggested that the human kallikrein gene family may be larger than previously thought. The rodent kallikrein genes are located in clusters on chromosome 7, and the region between two mouse kallikrein genes in a cluster can be as small as 3-7 kb (14).…”

Hypomaturation Enamel Defects in Klk4 Knockout/LacZ Knockin Mice

“…Klk4 is a serine protease that is secreted by transition and maturation stage ameloblasts but is not expressed by secretory stage ameloblasts (45,46). Klk4 might also be expressed by odontoblasts, the cells that form dentin (47).…”

The Use of Enamel Matrix Derivative in the Treatment of Periodontal Defects: a Literature Review and Meta-analysis

“…Since the enamelins were found to contain serum proteins (Limeback et al, 1989;Strawich and Glimcher, 1990), the more general term "nonamelogenin" is now commonly used to describe this high-molecular-weight fraction . It includes proline-rich enamelin (Fukae and Tanabe, 1987), tuftelin (Deutsch et al, 1991), and tuft proteins (Robinson et al, 1975).Three matrix proteins, corresponding to amelogenin (Hu et al, 1996), enamelin (Hu et al, 1997b), and sheathlin (also called ameloblastin or amelin) (Hu et al, 1997a), and 2 enzymes, corresponding to MMP-20 (Fukae et al, 1998) and EMSP1 (Simmer et al, 1998), have been purified and the cDNA cloned from developing porcine teeth. These proteins are all present in EMD.…”