Abstract
We have cloned a new human matrix metalloproteinase (MMP-28, epilysin) from human keratinocyte and testis cDNA libraries. Like most MMPs, epilysin contains a signal sequence, a prodomain with a PRCGVTD sequence, a zinc-binding catalytic domain with an HEIGHTLGLTH sequence, and a hemopexin-like domain. In addition, epilysin has a furin activation sequence (RRKKR) but has no transmembrane sequence. The exon-intron organization and splicing pattern of epilysin differ from that of other MMP genes. It has only 8 exons, and 5 exons are spliced at sites not used by other MMPs. Another novel feature of epilysin is that exon 4 is alternatively spliced to a transcript that does not encode the N-terminal half of the catalytic domain. Northern hybridization of tissue RNA indicated that epilysin is expressed at high levels in testis and at lower levels in lungs, heart, colon, intestine, and brain. RNase protection assay with various cell lines indicated that epilysin was selectively expressed in keratinocytes. Recombinant epilysin degraded casein in a zymography assay, and its proteolytic activity was inhibited by EDTA and by batimastat, a selective MMP inhibitor. Immunohistochemical staining showed expression of epilysin protein in the basal and suprabasal epidermis of intact skin. In injured skin, prominent staining for epilysin was seen in basal keratinocytes both at and some distance from the wound edge, a pattern that is quite distinct from that of other MMPs expressed during tissue repair. These findings suggest that this new MMP functions in several tissues both in tissue homeostasis and in repair.
Highlights
The matrix metalloproteinases (MMPs)1 compose a family of enzymes that share several common structural features and
Recombinant Epilysin Produced in E. coli and Casein Zymography—To express a fusion protein consisting of S. japonicum glutathione S-transferase (GST) and the pro- and catalytic domains of epilysin in Escherichia coli, a 802-bp fragment of the MMP-28 cDNA was amplified by PCR using primers 5Ј-CGGGATCCCAGCCCGCGGAGCGCGGA-3Ј and 5Ј-GGAATTCTCACCCATACAGGCTCTGCACGGCCAGC-3Ј, and the PCR product was digested with BamHI and EcoRI and cloned into the pGEX-6P-2 vector (Amersham Pharmacia Biotech)
Epilysin does not include domains characteristic of other metalloproteinases subfamilies, such as the disintegrin and thrombospondin-like domains found in ADAMs and tsADAMs, respectively, or a transmembrane domain as is found in most membrane-type MMPs [55]
Summary
Cloning of Human Epilysin cDNA, Exon/Intron Mapping—A search of the GenBankTM data base with the peptide string FDGXXXXLAHAXXPGXXXXGDXHFDXXEXW, which is conserved among MMPs, returned a homologous sequence within an 82-kb human genomic DNA clone (GenBankTM accession number AC006237). The resulting expression construct codes for a fusion protein of Schistosoma japonicum glutathione S-transferase (GST) and amino acid residues 123–520 of epilysin corresponding to the putative furin-activated enzyme This vector provides the signal peptide from the baculovirus protein gp to direct secretion of the fusion protein. Recombinant Epilysin Produced in E. coli and Casein Zymography—To express a fusion protein consisting of S. japonicum glutathione S-transferase (GST) and the pro- and catalytic domains of epilysin (amino acid residues 23–284, see Fig. 1) in Escherichia coli, a 802-bp fragment of the MMP-28 cDNA was amplified by PCR using primers 5Ј-CGGGATCCCAGCCCGCGGAGCGCGGA-3Ј (forward, epilysin sequence underlined) and 5Ј-GGAATTCTCACCCATACAGGCTCTGCACGGCCAGC-3Ј (reverse), and the PCR product was digested with BamHI and EcoRI and cloned into the pGEX-6P-2 vector (Amersham Pharmacia Biotech).
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