The glycosylation of the complement regulatory protein, human erythrocyte CD59

Rudd PM, Morgan BP, Wormald MR, Harvey DJ, van den Berg CW, Davis SJ, Ferguson MA, Dwek RA. (1997), J Biol Chem. 272, 7229-44

Abstract

Human erythrocyte CD59 contains N- and O-glycans and a glycosylphosphatidylinositol (GPI) anchor, all of which have been analyzed in this study. The anchor consists principally of the minimum core glycan sequence Manalpha1-2Manalpha1-6Manalpha1-4GlcN-linked to a phosphatidylinositol moiety with the structure sn-1-O-alkyl(C18:0 and C18:1)-2-O-acyl(C20:4)glycerol-3-phospho-1-(2-O-palmitoyl(C16:0))myo- inositol. This structure is essentially identical to that of human erythrocyte cholinesterase (Deeg, M. A., Humphrey, D. R., Yang, S. H. , Ferguson, T. R., Reinhold, V. N., and Rosenberry, T. L. (1992) J. Biol. Chem. 267, 18573-18580). This first comparison of GPI anchors from different proteins expressed in the same tissue suggests that human reticulocytes produce only one type of anchor structure. The N- and O-glycans were sequenced using a novel approach involving digestion of the total glycan pool with multiple enzyme arrays. The N-glycan pool contained families of bi-antennary complex-type structures with and without lactosamine extensions and outer arm fucose residues. The predominant O-glycans were NeuNAcalpha2-3Galbeta1-3GalNAc and Galbeta1-3[NeuNAcalpha2-3]GalNAc. Inspection of a molecular model of CD59, based on the NMR solution structure of the extracellular domain and the structural data from this study, suggested several roles for the glycans, including spacing and orienting CD59 on the cell surface and protecting the molecule from proteases. This work completes the initial structural analysis of CD59, providing the most complete view of any cell surface glycoprotein studied to date.

Key figure: A molecular model of CD59 based on the protein coordinates from the solution structure

a and b, two orthogonal views of the model drawn in space-filling format using the program RasMol (59). Active site residues D24, W40, R53, and E56² are shown in purple The glycan anchor (G, green), is modeled with a trimannosyl core (gold), an ethanolamine bridge at Man3, and additional ethanolamine groups at Man1 and Man2. Two lipids are attached to inositol via phosphate, and the third is attached directly to the inositol ring through an ester linkage. A trisialylated, tetra-antennary complex N-glycan (N,light blue) is shown attached to Asn¹⁸. The structure of the N-glycosidic linkage is based on a study by Wormald et al. (60). The O-glycan (O, dark blue), NeuNAc2,3Galβ1-3GalNAc, is attached to Thr⁵¹ to indicate one of the possible linkage positions. To illustrate the probable relationship between CD59 and the erythrocyte cell surface, the plasma membrane is depicted by two parallel red lines; the distance between these lines reflects the maximum dimensions of the bilayer based on the work of Hauser et al. (61). Views c and d, which are slightly enlarged with respect to views a and b, are orthogonal to the plane of the membrane which, for clarity, is not depicted. In view c, all residues are colored as in views a and b, whereas in view d, additional potential (exposed) O-glycosylation sites, rather than active site residues, are shown in purple and are labeled.