Cell surface isoforms of meprin A (EC 3.4.24.18) from mice and rats contain beta subunits that are type I integral membrane proteins and alpha subunits that are disulfide-linked to or noncovalently associated with membrane-anchored meprin subunits. Both alpha and beta subunits are synthesized with COOH-terminal domains predicted to be cytoplasmic, transmembrane, and epidermal growth factor-like; these domains are retained in beta subunits but are removed from alpha during maturation. The present studies establish that an inserted 56-amino acid domain (the "I" domain), present in alpha but not in beta, is necessary and sufficient for COOH-terminal proteolytic processing of the alpha subunit. This was demonstrated by expression of mutant meprin subunits (deletion mutants, chimeric alpha beta subunits, and beta mutants containing the I domain) in COS-1 cells. Mutations of two common processing sites present in the I domain (a dibasic site and a furin site) did not prevent COOH-terminal proteolytic processing, indicating that the proteases responsible for cleavage are distinct from those having these specificities. Deletion of the I domain from the alpha subunit resulted in accumulation of unprocessed subunits in a preGolgi compartment. Furthermore, COOH-terminal proteolytic processing of wild-type alpha subunits occurred before acquisition of endoglycosidase H resistance. Pulse-chase experiments and expression of an alpha subunit transcript containing a c-myc epitope tag, confirmed that proteolytic processing at the COOH terminus occurs in the endoplasmic reticulum. This work identifies the region of the alpha subunit that is essential for COOH-terminal processing and demonstrates that the differential processing of the evolutionarily-related subunits of meprin A that results in a structurally unique tetrameric protease begins in the endoplasmic reticulum.
Meprins are oligomeric, glycosylated cell surface or secreted metalloendopeptidases that are composed of multidomain disulfide-linked subunits. To investigate whether subunit oligomerization is critical for intracellular transport or for the enzymatic and/or physical properties of the proteinase, specific cysteine residues were mutated, and the mutants were expressed in 293 cells. Mutation of mouse meprin ␣ Cys-320 to Ala in the MAM domain (an extracellular domain found in meprin, A-5 protein, and receptor protein-tyrosine phosphatase ) resulted in expression of a monomeric form of meprin, as determined by SDS-polyacrylamide gel electrophoresis and nondenaturing gel electrophoresis. The monomeric subunits were considerably more vulnerable to proteolytic degradation and heat inactivation in vitro compared with the oligomeric form of the enzyme. Proteolytic activity of the monomeric meprin using a bradykinin analog or aminobenzoyl-Ala-Ala-Phe-p-nitroanilide as substrate was similar to that of disulfidelinked oligomeric meprin; however, activity against azocasein was markedly decreased. Mutation of another cysteine residue in the MAM domain (C289A), predicted to be involved in intrasubunit disulfide bridging, resulted in disulfide-linked oligomers and monomers. These results indicated that this mutant was capable of forming intersubunit disulfide bonds but less efficiently than wild-type meprin subunits. Mutant C289A also retained activity toward peptides but not the protein substrate and was more vulnerable to proteolytic degradation and heat inactivation compared with the wild-type enzyme. Both Cys mutants were expressed and secreted into the medium at levels comparable with the wild type and had slightly altered glycosylation. This work indicates that 1) Cys-320 of mouse meprin ␣ is most likely responsible for the covalent interactions of the subunits; 2) covalent dimerization of subunits is not essential for efficient biosynthesis, trafficking, or posttranslational processing of the secreted protease; and 3) mutations in the MAM domain affect noncovalent interactions of the subunits and the stability and activity of the protease domain, indicating that domain-domain interactions are critical for structure and function of the enzyme.Meprins are cell surface and secreted metalloendopeptidases that have been characterized in renal and intestinal brush border membranes of mice, rats, and humans (1-7). Active and latent forms of meprins have been found in mammalian tissues, and azocasein has generally been used as a good protein substrate for these enzymes (8). Meprin A (EC 3.4.24.18) from mouse and rat kidneys is the most thoroughly investigated isoform; it hydrolyzes a variety of biologically active peptides such as bradykinin, angiotensins, glucagon, luteinizing hormone-releasing hormone, parathyroid and melanocyte-stimulating hormone and is implicated in the processing and degradation of membrane-bound and matrix proteins (7-12).Meprins are proteases that are unique in their oligomeric structure. They are dimers...
Background: Glomerular mononuclear cell infiltration is associated with the development of a diffuse glomerulosclerosis in patients with diabetic nephropathy. Monocyte chemoattractant protein-1 (MCP-1) plays an important role in the recruitment and accumulation of monocytes and lymphocytes within the glomerulus. In the present study, we examined whether the ambient glucose concentration alters the expression of MCP-1 and its receptor CCR2 in primary human mesangial cells (HMC). Methods: MCP-1 mRNA expression was assessed by Northern blot and CCR2 mRNA expression by RT-PCR analysis. MCP-1 protein production was determined by ELISA. Migration studies were performed to assess functional MCP-1 receptor expression. Results: Exposure of HMC to 30 mMD-glucose led to a 30% increase in MCP-1 mRNA expression as compared to 5 mMD-glucose and osmotic controls while there was no difference in MCP-1 protein production. Simultaneously, CCR2 mRNA expression was down-regulated in HMC exposed to 30 mMD-glucose. 5 mMD-glucose primed HMC showed a dose-dependent migration towards MCP-1 that was dose-dependently inhibited by pertussis toxin, the broad-spectrum chemokine antagonist vMIP-II as well as the CCR2 receptor antagonist (1–8del)MCP-1 – demonstrating functional activity of MCP-1 receptor expression in primary HMC. In accordance with the downregulatory effects of 30 mMD-glucose on CCR2 mRNA expression no migratory response towards MCP-1 was observed under these conditions. The additional proinflammatory stimulus TNFα increased MCP-1 protein production in 30 as compared to 5 mMD-glucose primed HMC (2,194 ± 568 vs. 1,422 ± 379 pg MCP-1/104 cells × ml in 30 vs. 5 mMD-glucose primed HMC +24 h TNFα 500 U/ml, p = 0.002). However, this was not associated with an increased MCP-1 mRNA transcription. The 30 mMD-glucose induced downregulation of CCR2 mRNA expression was prevented in the presence of TNFα. Conclusion: High ambient glucose does not affect mesangial MCP-1 release and decreases its CCR2 receptor expression. However, in the presence of an inflammatory stimulus these effects of high glucose are reversed and an autocrine pathway of MCP-1 develops in mesangial cells.
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