O-Glycan Sialylation and the Structure of the Stalk-like Region of the T Cell Co-receptor CD8

• Published in: The Journal of biological chemistry Vol. 278; no. 29; pp. 27119 - 27128
• Main Authors: Merry, Anthony H., Gilbert, Robert J.C., Shore, David A., Royle, Louise, Miroshnychenko, Olga, Vuong, Mai, Wormald, Mark R., Harvey, David J., Dwek, Raymond A., Classon, Brendan J., Rudd, Pauline M., Davis, Simon J.
• Format: Journal Article
• Language: English
• Published: United States Elsevier Inc 18.07.2003; American Society for Biochemistry and Molecular Biology
• Subjects: Amino Acid Sequence; Animals; Carbohydrate Conformation; Carbohydrate Sequence; CD8 antigen; CD8 Antigens - chemistry; CD8 Antigens - genetics; CHO Cells; Chromatography, High Pressure Liquid; Cricetinae; Glycosylation; In Vitro Techniques; Mice; Models, Molecular; Molecular Sequence Data; Molecular Structure; Polysaccharides - chemistry; Protein Conformation; Rats; Recombinant Fusion Proteins - chemistry; Recombinant Fusion Proteins - genetics; Sialic Acids - chemistry; sialylation; Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization
• ISSN: 0021-9258; 1083-351X
• DOI: 10.1074/jbc.M213056200

Studies of mucins suggest that the structural effects of O-glycans are restricted to steric interactions between peptide-linked GalNAc residues and adjacent polypeptide residues. It has been proposed, however, that differential O-glycan sialylation alters the structure of the stalk-like region of the T cell co-receptor, CD8, and that this, in turn, modulates ligand binding (Daniels, M. A., Devine, L., Miller, J. D., Moser, J. M., Lukacher, A. E., Altman, J. D., Kavathas, P., Hogquist, K. A., and Jameson, S. C. (2001) Immunity 15, 1051–1061; Moody, A. M., Chui, D., Reche, P. A., Priatel, J. J., Marth, J. D., and Reinherz, E. L. (2001) Cell 107, 501–512). We characterize the glycosylation of soluble, chimeric forms of the αα- and αβ-isoforms of murine CD8 containing the O-glycosylated stalk of rat CD8αα, and we show that the stalk O-glycans are differentially sialylated in CHO K1 versus Lec3.2.8.1 cells (82 versus ∼6%, respectively). Sedimentation analysis indicates that the Perrin functions, Pexp, which reflect overall molecular shape, are very similar (1.61 versus 1.54), whereas the sedimentation coefficients (s) of the CHO K1- and Lec3.2.8.1-derived proteins differ considerably (3.73 versus 3.13 S). The hydrodynamic properties of molecular models also strongly imply that the sialylated and non-sialylated forms of the chimera have parallel, equally highly extended stalks (∼2.6 Å/residue). Our analysis indicates that, as in the case of mucins, the overall structure of O-glycosylated stalk-like peptides is sialylation-independent and that the functional effects of differential CD8 O-glycan sialylation need careful interpretation.