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

Louise Royle,David A. Shore,Olga Miroshnychenko,Raymond A. Dwek,Mai Vuong,Pauline M. Rudd,Mark R. Wormald,Anthony H. Merry,David J. Harvey,Robert J.C. Gilbert,Brendan J. Classon,Simon J. Davis

doi:10.1074/jbc.m213056200

Abstract

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 Lec3.2.8.1 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 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 ( approximately 2.6 A/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.

Highlights

The early responses of T lymphocytes are determined by interactions of both the T cell receptor (TCR)1 and CD8 or CD4
We characterize the glycosylation of soluble, chimeric forms of the ␣␣- and ␣␤-isoforms of murine CD8 containing the Oglycosylated stalk of rat CD8␣␣, and we show that the stalk O-glycans are differentially sialylated in Chinese hamster ovary (CHO) K1 versus Lec3.2.8.1 cells (82 versus ϳ6%, respectively)
More family; NP-High Performance Liquid Chromatography (HPLC), normal phase-high performance liquid chromatography; MALDI-TOF MS, matrix-assisted laser desorption/ionization time-of-flight mass spectrometry; MHC, major histocompatibility complex; MHCp, MHC-peptide complex; Q-TOF, quadrupole time-of-flight mass spectrometry; sCD8, soluble form of CD8; sCD8␣␣K1, murine sCD8␣␣ expressed in CHO K1 cells; sCD8␣␣Lec, murine sCD8␣␣ expressed in Lec3.2.8.1 cells; sCD8␣␣E, human CD8␣␣ expressed in E. coli cells; 2-AB, 2-aminobenzamide; HexNAc, N-acetylhexosamine; PNGaseF, peptide N-glycosidase F; S, Svedberg units

Summary

Introduction

The early responses of T lymphocytes are determined by interactions of both the T cell receptor (TCR)1 and CD8 or CD4. By taking these ratios into account, and the results of the NPHPLC analysis (Fig. 4, a and c; Table IV), ϳ70% of the Oglycans present on Lec3.2.8.1-derived CD8 consisted of single GalNAc residues; the remainder were extended with ␤1,3-galactose to form type 1 core Gal-␤1,3GalNAc, and only 18% of these (ϳ6% of the O-glycans overall) were sialylated.

Results

Conclusion