SITE-DIRECTED REMOVAL OF N-GLYCOSYLATION SITES IN THE BOVINE CATION-DEPENDENT MANNOSE 6-PHOSPHATE RECEPTOR - EFFECTS ON LIGAND-BINDING, INTRACELLULAR TARGETING AND ASSOCIATION WITH BINDING IMMUNOGLOBULIN PROTEIN

The bovine cation-dependent mannose 6-phosphate receptor (CD-MPR) cont ains five potential N-linked glycosylation sites, four of which are ut ilized. To evaluate the function of these oligosaccharides, site-direc ted mutagenesis was used to generate glycosylation-deficient CD-MPR mu tants lacking various potential glycosylation sites. The mutants were constructed in both a full-length and a soluble truncated (STOP155 con struct) form of the receptor and their properties were examined in tra nsfected COS-1 cells. The results showed that the presence of a single oligosaccharide chain, particularly at position 87, on the CD-MPR sig nificantly enhanced its mannose 6-phosphate (Man-6P)-binding ability w hen compared with non-glycosylated receptors. In addition, the presenc e of a single oligosaccharide chain at position 87, and to a lesser de gree at position 31 or 81, promoted the secretion of the STOP155 CD-MP R. Pulse-labelling of transfected COS-1 cells followed by immunoprecip itation with binding immunoglobulin protein (BiP)-specific and CD-MPR- specific antibodies indicated that BiP associated with the nonglycosyl ated forms of the receptor but not with the wild-type CD-MPR. Furtherm ore, the association of the various glycosylation-deficient forms of t he CD-MPR with BiP correlated inversely with their ability to bind Man -6-P. From these results we conclude that N-glycosylation of the bovin e CD-MPR facilitates the folding of the nascent polypeptide chain into a conformation that is conducive for intracellular transport and liga nd binding.