Composition and sequence specific resonance assignments of the heterogeneous N-linked glycan in the 13.6 kDa adhesion domain of human CD2 as determined by NMR on the intact glycoprotein.
CD2, a T cell specific surface adhesion receptor, is critically important for T lymphocytes to mediate their regulatory and effector functions. The amino terminal domain of human CD2 is responsible for cell adhesion, binding to CD58 on antigen-presenting cells or target cells. This adhesion domain in human CD2 contains a single high-mannose N-glycan. This carbohydrate or part of it appears to be required to maintain the native conformation of the polypeptide and its ability to bind CD58. To better understand the structural aspects that regulate human CD2 adhesion functions, we had previously determined the solution structure of the protein part of the N-glycosylated adhesion domain of human CD2 (hu-sCD2(105); MW approximately 13.6 kDa) by NMR spectroscopy. Here, we have identified protein--carbohydrate and carbohydrate--carbohydrate interactions and, in combination with previous knowledge from electrospray ionization mass spectrometry, have determined the composition of the heterogeneous high-mannose glycan in hu-sCD2(105). These contacts clearly define the carbohydrate's orientation with respect to the protein. The NMR data further suggest that one arm of the glycan is folded toward the trisaccharide core consisting of GlcNAc1-GlcNAc2-Man3. A detailed comparison between chemical shift data of free model oligosaccharides with those of the glycomers present in our hu-sCD2(105) sample reveals that only the resonances of the two GlcNAc residues are significantly different from those of free high-mannose glycans. This work was based on a new strategy to achieve sequential assignments of the 1H and 13C resonances of the heterogeneous high-mannose carbohydrate [(Man)nGlcNAc2, n = 5-8] in hu-sCD2(105) on the intact glycoprotein using a combination of homonuclear 1H-1H and heteronuclear 1H-13C NMR experiments at natural abundance.
Wyss
D F
DF
Department of Biological Chemistry and Molecular Pharmacology, Harvard Medical School, Boston, Massachusetts 02115.
Choi
J S
JS
Wagner
G
G
D018801:CD2 Antigens / Q000737:chemistry*; D002240:Carbohydrate Sequence; D002448:Cell Adhesion; D006801:Humans; D009682:Magnetic Resonance Spectroscopy; D008968:Molecular Conformation; D008969:Molecular Sequence Data; D013601:T-Lymphocytes; D014312:Trisaccharides / Q000737:chemistry*
D018801:CD2 Antigens; D014312:Trisaccharides