STRUCTURE OF HUMAN TRANSFERRIN RECEPTOR OLIGOSACCHARIDES - CONSERVATION OF SITE-SPECIFIC PROCESSING

The human transferrin receptor (TfR) has three N-linked oligosaccharid es. A combination of site-directed mutagenesis and carbohydrate and pr otein chemistry was used to characterize the structures of the N-linke d oligoosaccharides and to map their locations. We find that the type of oligosaccharide at each position was unique for that particular sit e. Human TfR isolated from placentae was used to characterize the stru cture of the oligosaccharides found in the native TfR. Following diges tion of purified TfR with trypsin, individual peptides were obtained v ia RP-HPLC and were assayed for monosaccharides by strong acid hydroly sis and HPAE-PAD. Peptides containing carbohydrate were subjected to a mino acid sequencing to identify the specific Asn residue. The oligosa ccharides at Asn 251 are of the complex type. HPAE-PAD and FACE analys is suggests that they are triantennary and trisialylated with core fuc osylation. The glycopeptide containing the site at Asn 317 was obtaine d after limited tryptic digestion and RP-HPLC. FACE analysis reveals p redominantly a family of sialylated hybrid oligosaccharides. The conse nsus sequences for each N-linked site were mutated in various combinat ions and the resultant TfRs expressed in mouse 3T3 cells. Endoglycosid ase H digestion of the mutated TfRs indicates that the pattern of olig osaccharides is consistent with the type of oligosaccharides found at each position in human tissue and the glycosylation of one site does n ot directly affect the glycosylation of other sites. Previous studies indicated that the oliosaccharide at Asn 727 was high-mannose type [Ha yes, G. R., et al. (1995) Glycobiology 5, 227-232]. These results indi cate that the type of oligosaccharide found at each site is most depen dent on the environment surrounding it.