cDNA sequence coding for a rat glia-derived nexin and its homology to members of the serpin superfamily

Abstract

Rat glial cells release a neurite-promoting factor with serine protease inhibitory activity. By using a rat glioma cDNA clone as a probe, it was possible to isolate rat cDNAs containing the entire sequence coding for this neurite-promoting factor. The largest rat cDNA (approximately 2100 bp) was characterized by DNA sequencing. It contained the entire coding region, 135 bp of the 5' nontranslated region, and about 750 bp of the 3' nontranslated region. The open reading frame coded for 397 amino acids including a putative signal peptide of 19 amino acids. The correct identity of the coding sequence was substantiated by the fact that the sequence of tryptic peptides, derived from the purified rat factor, matched exactly with the deduced amino acid sequence. The rat protein sequence had 84% homology with the corresponding protein from human glioma cells. Both amino acid sequences indicated that the proteins belong to the protease nexins [Baker, B.J., Low, D. A., Simmer, R. L., & Cunningham, D.D. (1980) Cell (Cambridge, Mass.) 21, 37-45] and therefore can be defined as glia-derived nexins (GDNs). Further analysis showed that both rat and human GDN belong to the serpin superfamily and share 41%, 32%, and 25% homology with human endothelial-cell-type plasminogen activator inhibitor, antithrombin III, and alpha-1 proteinase inhibitor, respectively.

Protease-nexin I as an androgen-dependent secretory product of the murine seminal vesicle.

“…To obtain a cRNA probe for mouse PN-I, a 328 bp PstI fragment of a rat PN-I cDNA (Sommer et al, 1987) was subcloned in pGEMI. The plasmid was linearized with EcoRI and transcribed with SP6 RNA polymerase (Busso et al, 1986).…”

“…Using radiolabelled uPA, we have detected high levels of a uPA ligand in extracts of adult mouse and rat seminal vesicles. We have identified this ligand as protease nexin I (PN-I), a serpin released by human fibroblasts (Baker et al, 1980) and identical to glia-derived nexin (GDN) (Gloor et al, 1986;Sommer et al, 1987;McGrogan et al, 1988), first detected as a factor promoting neurite outgrowth in vitro (Guenther et al, 1985) that is found in different structures of the nervous system (Reinhard et al, 1988;Meier et al, 1989). PN-I reacts with and inhibits thrombin, uPA, tissuetype PA (tPA), trypsin and plasmin (Baker et al, 1980;Eaton and Baker, 1983;Guenther et al, 1985;Stone et al, 1987).…”

Endogenous Serine Protease Inhibitor Modulates Epileptic Activity and Hippocampal Long-Term Potentiation

“…This cassette was generated originally by deleting a DNA fragment (from the BanI site in exon 2, upstream of the translation start codon, to a XhoI site in exon 4) and inserting an XhoI linker. A blunted HindIII-EcoRI DNA fragment containing the rat PN-1 cDNA (Sommer et al, 1987) was inserted into the blunted XhoI site of the Thy 1 cassette. The PN-1 cDNA encompasses the authentic PN-1 translation initiation (ATG) codon, signal peptide, and termination codon (TGA).…”

“…Northern blots were hybridized to random-primed 32 P-labeled DNA probes. The following probes were used: a 750 bp XhoI-BamHI DNA fragment from exon 4 of the mouse Thy 1.2 gene (Ingraham et al, 1986) and a 1351 bp XhoI-XbaI DNA fragment carrying the rat PN-1 cDNA (Gloor et al, 1986;Sommer et al, 1987). For the thrombin inhibition assay, a previously described method (Nick et al, 1990) was adapted to microtiter plates.…”

“…Unlike the coagulation /fibrinolytic pathway where several different inhibitors are known, in the CNS only protease nexin-1 (PN-1; Guenther et al, 1985;Sommer et al, 1987) is present at significant levels (Mansuy et al, 1993;Sappino et al, 1993;Reinhard et al, 1994). PN-1 is expressed in both glial and neuronal cells of the developing and adult CNS (Mansuy et al, 1993;Sappino et al, 1993;Reinhard et al, 1994) and in glomeruli of the olfactory bulbs, where synapse formation and elimination remain an active process throughout life (Reinhard et al, 1988;Mansuy et al, 1993).…”