title: Site-specific glycosylation of recombinant rat and human soluble CD4 variants expressed in Chinese hamster ovary cells.
abstract: The rat and human forms of the T-cell surface glycoprotein CD4 share a common glycosylation site at the Asn270/271 position but differ with respect to the locations of the second glycosylation sites at Asn159 (rat) and Asn300 (human). The glycosylation of soluble recombinant forms of human and rat CD4 (sCD4) expressed in Chinese hamster ovary cells has been characterized. The most obvious differences between the rat and human sCD4 oligosaccharides were the greater abundance of oligomannose and hybrid oligosaccharides on rat sCD4 and the presence of oligosaccharides carrying a terminal alpha-galactose residue on human sCD4. This is the first report of the occurrence of alpha-galactose residues on a glycoprotein expressed in Chinese hamster ovary cells. Comparison of mutant rat sCD4 molecules with single glycosylation sites and glycopeptides indicated that site-specific and independent processing occurred at each glycosylation site. The glycosylation at the conserved site at Asn270 of rat sCD4 was identical to that seen for the equivalent site in human sCD4, and the oligomannose and hybrid structures were restricted to the nonconserved site at Asn159 in rat sCD4. However, there was more oligosaccharide processing at this site in a truncated form of rat sCD4 consisting of the two NH2-terminal domains. These results indicate that not only the local three-dimensional structure but also domain interactions can influence the processing at individual glycosylation sites.
authors.affiliation: Department of Biochemistry, Sir William Dunn School of Pathology, University of Oxford, United Kingdom.
authors.forename: D A
authors.initials: DA
authors.lastname: Ashford
authors.forename: C D
authors.initials: CD
authors.lastname: Alafi
authors.forename: V M
authors.initials: VM
authors.lastname: Gamble
authors.forename: D J
authors.initials: DJ
authors.lastname: Mackay
authors.forename: T W
authors.initials: TW
authors.lastname: Rademacher
authors.forename: P J
authors.initials: PJ
authors.lastname: Williams
authors.forename: R A
authors.initials: RA
authors.lastname: Dwek
authors.forename: A N
authors.initials: AN
authors.lastname: Barclay
authors.forename: S J
authors.initials: SJ
authors.lastname: Davis
authors.forename: C
authors.initials: C
authors.lastname: Somoza
mesh_terms: D000595:Amino Acid Sequence; D000818:Animals; D015704:CD4 Antigens / Q000096:biosynthesis / Q000235:genetics / Q000378:metabolism*; D016466:CHO Cells; D002236:Carbohydrate Conformation; D002240:Carbohydrate Sequence; D002850:Chromatography, Gel; D006224:Cricetinae; D006020:Glycopeptides / Q000302:isolation & purification; D006031:Glycosylation; D006801:Humans; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D009844:Oligosaccharides / Q000737:chemistry / Q000302:isolation & purification / Q000378:metabolism*; D011499:Protein Processing, Post-Translational; D051381:Rats; D011994:Recombinant Proteins / Q000378:metabolism*; D014162:Transfection
chemical_list: D015704:CD4 Antigens; D006020:Glycopeptides; D009844:Oligosaccharides; D011994:Recombinant Proteins