N-linked oligosaccharides on the meprin A metalloprotease are important for secretion and enzymatic activity, but not for apical targeting

The alpha and beta subunits of meprins, mammalian zinc metalloendopeptidase s, are extensively glycosylated; similar to 25% of the total molecular mass of the subunits is carbohydrate. The aim of this study was to investigate the roles of the N-linked oligosaccharides on the secreted form of mouse me prin A. Recombinant meprin cu and mutants in which one of the 10 potential Asn glycosylation sites was mutated to Can were all secreted and sorted exc lusively into the apical medium of polarized Madin-Darby canine kidney cell s, indicating that no specific N-linked oligosaccharide acts as a determina nt for apical targeting of meprin rw. Several of the mutant proteins had de creased enzymatic activity using a bradykinin analog as substrate, and degl ycosylation of the wildtype protein resulted in loss of 75-100% activity. S ome of the mutants were also more sensitive to heat inactivation. In studie s with agents that inhibit glycosylation processes in vivo, tunicamycin mar kedly decreased secretion of meprin, whereas castanospermine and swainsonin e had little effect on secretion, sorting, or enzymatic properties of mepri n, When all the potential glycosylation sites on a truncated form of meprin cy (alpha-(1-445)) were mutated, the protein was not secreted into the med ium, but was retained within the cells even after 10 h. These results indic ate that there is no one specific glycosylation site or type of oligosaccha ride thigh mannose- or complex-type) that determines apical sorting, but th at core N-linked carbohydrates are required for optimal enzymatic activity and for secretion of meprin alpha.