Identification of the cysteine residues implicated in the formation of alpha(2) and alpha/beta dimers of rat meprin

Meprin (endopeptidase-24.18; EC 3.4.24.18) is a multisubunit zinc-metallopeptidase found in the brush-border membranes of rodent kidney and human intestine. The alpha and beta subunits of meprin are disulphide-linked to form either soluble alpha(2) homodimers or membrane-associated alpha/beta heterodimers. The aim of the present study was to identify the cysteine residue(s) implicated in the formation of alpha(2) and alpha/beta dimers and to investigate the effects of dimerization on intracellular transport and processing of the a subunit. Three cysteine residue candidates for the formation of disulphide bonds in the alpha subunit were selected by hydrophobic cluster analysis. These residues, located at positions 309, 560 and 562, were mutated to serine residues. When the resulting a subunit mutants were expressed alone in COS-1 cells, the alpha C560S and alpha C562S mutants were found to be secreted as alpha(2) homodimers whereas the alpha C309S mutant was found as monomers in the culture medium. In double-transfection experiments with the wild-type beta subunit, the alpha C560S and alpha C562S mutants behaved exactly as the wild-type alpha subunit and formed membrane-bound alpha/beta heterodimers. In contrast, the alpha C309S mutant was not retained at the cell surface but rather secreted as monomers in the culture medium, as was found in the simple transfection experiment. These results show that, despite the normal expression level and folding of the protein in a transport-competent form, the alpha C309S mutant is unable to form alpha(2) homodimers or alpha/beta heterodimers. This suggests that Cys(309) is the unique residue of the alpha subunit implicated in the alpha(2) and alpha/beta dimerizations. Hydrophobic cluster analysis of the alpha and beta subunit sequences predicts that Cys(309) is similar to Cys(306) of the beta subunit. We mutated the latter residue to a serine and expressed the beta C306S mutant and the wild-type alpha subunit in the same COS-1 cells. No beta(2) or alpha/beta dimers were observed on immunoblotting, showing that Cys(306) of the beta subunit is required for the formation of intermolecular disulphide bonds both in beta(2) homodimers and in alpha/beta heterodimers. Taken together, these results suggest that the alpha/beta heterodimeric form of meprin is held together by a single disulphide bond linking Cys(309) in the alpha subunit to Cys(306) in the beta subunit.