Structure of human transferrin receptor oligosaccharides: conservation of site-specific processing.
The human transferrin receptor (TfR) has three N-linked oligosaccharides. A combination of site-directed mutagenesis and carbohydrate and protein chemistry was used to characterize the structures of the N-linked oligosaccharides and to map their locations. We find that the type of oligosaccharide at each position was unique for that particular site. Human TfR isolated from placentae was used to characterize the structure of the oligosaccharides found in the native TfR. Following digestion of purified TfR with trypsin, individual peptides were obtained via RP-HPLC and were assayed for monosaccharides by strong acid hydrolysis and HPAE-PAD. Peptides containing carbohydrate were subjected to amino acid sequencing to identify the specific Asn residue. The oligosaccharides at Asn 251 are of the complex type. HPAE-PAD and FACE analysis suggests that they are triantennary and trisialylated with core fucosylation. The glycopeptide containing the site at Asn 317 was obtained after limited tryptic digestion and RP-HPLC. FACE analysis reveals predominantly a family of sialylated hybrid oligosaccharides. The consensus sequences for each N-linked site were mutated in various combinations and the resultant TfRs expressed in mouse 3T3 cells. Endoglycosidase H digestion of the mutated TfRs indicates that the pattern of oligosaccharides is consistent with the type of oligosaccharides found at each position in human tissue and the glycosylation of one site does not directly affect the glycosylation of other sites. Previous studies indicated that the oliosaccharide at Asn 727 was high-mannose type [Hayes, G. R., et al. (1995) Glycobiology 5, 227-232]. These results indicate that the type of oligosaccharide found at each site is most dependent on the environment surrounding it.
Hayes
G R
GR
Department of Biochemistry and Molecular Biology, State University of New York Health Sciences Center Syracuse, New York 13201, USA.
Williams
A M
AM
Lucas
J J
JJ
Enns
C A
CA
D016475:3T3 Cells; D000818:Animals; D002236:Carbohydrate Conformation; D002240:Carbohydrate Sequence; D002241:Carbohydrates / Q000737:chemistry; D002478:Cells, Cultured; D006596:Hexosaminidases / Q000378:metabolism; D006801:Humans; D051379:Mice; D008958:Models, Molecular; D008969:Molecular Sequence Data; D009844:Oligosaccharides / Q000737:chemistry*; D011990:Receptors, Transferrin / Q000737:chemistry*; D013329:Structure-Activity Relationship
D002241:Carbohydrates; D009844:Oligosaccharides; D011990:Receptors, Transferrin; D006596:Hexosaminidases