Oligosaccharide analysis and molecular modeling of soluble forms of glycoproteins belonging to the Ly-6, scavenger receptor, and immunoglobulin superfamilies expressed in Chinese hamster ovary cells
Rudd PM, Wormald MR, Harvey DJ, Devasahayam M, McAlister MS, Brown MH, Davis SJ, Barclay AN, Dwek RA. (1999), Glycobiology. 9, 443-58
Most cell surface molecules are glycoproteins consisting of linear arrays of globular domains containing stretches of amino acid sequence with similarities to regions in other proteins. These conserved regions form the basis for the classification of proteins into superfamilies. Recombinant soluble forms of six leukocyte antigens belonging to the Ly-6 (CD59), scavenger receptor (CD5), and immunoglobulin (CD2, CD48, CD4, and Thy-1) superfamilies were expressed in the same Chinese hamster ovary cell line, thus providing an opportunity to examine the extent to which N-linked oligosaccharide processing might vary in a superfamily-, domain-, or protein-dependent manner in a given cell. While we found no evidence for superfamily-specific modifications of the glycans, marked differences were seen in the types of oligosaccharides attached to individual proteins within a given superfamily. The relative importance of local protein surface properties versus the overall tertiary structure of the molecules in directing this protein-specific variation was examined in the context of molecular models. These were constructed using the 3D structures of the proteins, glycan data from this study, and an oligosaccharide structural database. The results indicated that both the overall organization of the domains and the local protein structure can have a large bearing on site-specific glycan modification of cells in stasis. This level of control ensures that the surface of a single cell will display a diverse repertoire of glycans and precludes the presentation of multiple copies of a single oligosaccharide on the cell surface. The glycans invariably shield large regions of the protein surfaces although, for the glycoproteins examined here, these did not hinder the known active sites of the molecules. The models also indicated that sugars are likely to play a role in the packing of the native cell surface glycoproteins and to limit nonspecific protein-protein interactions. In addition, glycans located close to the cell membrane are likely to affect crucially the orientation of the glycoproteins to which they are attached.
(a) Schematic diagrams of the leukocyte cell surface antigens forming the basis of this study. Membrane attached rat Thy-1, human CD2, rat CD48, rat CD4, human CD5, and human CD59 are depicted with shaded or unshaded ovals, squares, and triangles representing IgSF domains, SCRC domains and Ly-6 (L6) superfamily domains, respectively. N-Glycans are represented by solid circles joined to the domains by thin lines, and O-glycans by single thick lines. The IgSF domains are designated as V-set (V) or C2-set (C2) on the basis of sequence analysis (Williams and Barclay, 1988). Sites of truncation are indicated with horizontal arrows and GPI anchors with vertical arrows. (b) The soluble derivatives of these glycoproteins characterized in this study. These are represented diagrammatically with the abbreviations used in the text to refer to the constructs, shown underneath. (c) Schematic diagrams illustrating the topology of the leukocyte antigens, where this is known, and the location of the glycosylation sites on these molecules. The classes of the major oligosaccharide populations at each site is given where this information is available. The positions of the β-strands are indicated by the parallel lines with arrows showing the direction of each strand. The loops between the strands are indicated by the connecting lines. The strands are labeled according to the convention established for immunoglobulin domains (see Williams and Barclay, 1988). Data from CD54 is from Bloom et al., 1996.