O-glycan sialylation and the structure of the stalk-like region of the T cell co-receptor CD8
Merry AH, Gilbert RJ, Shore DA, Royle L, Miroshnychenko O, Vuong M, Wormald MR, Harvey DJ, Dwek RA, Classon BJ, Rudd PM, Davis SJ. (2003), J Biol Chem. 278, 27119-28
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 alphaalpha- and alphabeta-isoforms of murine CD8 containing the O-glycosylated stalk of rat CD8alphaalpha, and we show that the stalk O-glycans are differentially sialylated in CHO K1 versus Lec22.214.171.124 cells (82 versus approximately 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 Lec126.96.36.199-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 (approximately 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.
Key figure: Molecular models of sCD8αα used to simulate hydrodynamic parameters, and the results of the simulations
a, the model of sCD8ααE is based on the crystal structure of human sCD8αα from the sCD8αα-class I MHCp complex (14). The sCD8ααLec (b) and sCD8ααK1 (c) structures are based on the crystal structure of murine sCD8αα from the sCD8αα-class I MHCp complex (15), the amino acid sequence of the stalks, and the glycan analysis described herein. An extension of 2.6 Å per residue was used to model the stalk region. s and P are the calculated sedimentation coefficients and Perrin functions derived for the models (see text for details of the calculation), whereas sexp and Pexp are the experimentally determined sedimentation coefficients and Perrin functions corrected for hydration using a value of δ = 0.3 g/g (see Table V). In a, b, and c, the protein (purple) and N– and O-glycans (small red and dark blue spheres) are shown as “bead” models (assemblages of larger, transparent spheres). d, to demonstrate the extent to which the modeling is sensitive to the structure of CD8, the hydrodynamic properties are shown for models of sCD8ααLec (shown schematically) in which the stalk is fully extended as in b (model 1), absent to different degrees (i.e. by 50 and 80% in models 2 and 3) or not fully extended (models 4–6).