The carbohydrate of bovine interstitial retinol-binding protein

Pronase digestion of IRBP yielded one major glycopeptide (IRBP-GP1) of approximate Mr 2,500 IRBP-GP1 was heterogeneous by anion exchange chromatography, an observation that was attributed to the presence of varying numbers of sialic acid residues. Asialo-IRBP-GP1 was also heterogeneous by gel-filtration chromatography, possibly because of varying degrees of fucosylation. A minimum of four (possibly five) concanavalin A-binding glycopeptides was generated by cyanogen bromide cleavage of IRBP. These findings, in conjunction with earlier observations, suggest that bovine IRBP contains 4-5 N-linked oligosaccharide chains. These chains appear to have the same basic complex biantennary structure. They contain galactose, mannose and N-acetylglucosamine, in addition to sialic acid and fucose. Nearly all of the IRBP secreted by bovine retinas incubated with (3H)-leucine in the presence of tunicamycin was nonglycosylated and did not bind to concanavalin A. On SDS polyacrylamide gels non-glycosylated IRBP had an Mr that was 5,000-6,000 below that of the glycosylated protein. Non-glycosylated IRBP had an Mr of 250,000 on gel-filtration columns, a value that was identical with that of glycosylated IRBP. It is concluded that the oligosaccharide has little influence on the molecular conformation of IRBP, which is believed to be responsible for the anomalously high Mr seen on these columns. When mixed, the glycosylated and nonglycosylated forms of IRBP appeared to associate as stable aggregates. Bovine retinas incubated with (14C)-leucine and (3H)-fucose in the presence of castanospermine, an inhibitor of glucosidase I, secreted IRBP that appeared to have a reduced number of fucose residues. Swainsonine, an inhibitor of mannosidase II, effected only a marginal reduction in the degree of fucosylation of secreted IRBP.