Structural and functional analyses of glycosylation on the distinct molecules of human GM‐CSF receptors

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Abstract
We have previously demonstrated that granulocyte‐macrophage colony‐stimulating factor (GM‐CSF) receptors are composed of at least two molecules of 80 and 135 kDa, which were denoted α‐ and β‐chains, respectively [Chiba, S., Shibuya, K., Piao, Y.‐F., Tojo, A., Sasaki, N., Matsuki, S., Miyagawa, K., Miyazono, K. & Takaku, F. (1990) Cell Regul. 1, 327–335]. In this paper, we describe an investigation of the biochemical disparity noted between the α‐ and β‐chains of GM‐CSF receptors using proteolytic and deglycosidic enzymes, and further demonstrate the potential importance of carbohydrate structures of the GM‐CSF receptors using different lectins and glycoprotein synthesis inhibitors. Cross‐linked α‐ and β‐chains with 125I‐GM‐CSF were digested by Staphylococcus aureus V8 protease and gave a different pattern. Furthermore, the size of the α‐chain was reduced by 25 kDa by the removal of the N‐linked oligosaccharides with peptidase: N‐glycosidase F treatment, whereas that of the β‐chain remained unmodified by the enzyme. These results suggest that the α‐chain of GM‐CSF receptors agrees with the recently cloned low‐affinity GM‐CSF receptor [Gearing, D. P., King, J. A., Gough, N. M. & Nicola, N. A. (1989) EMBO J. 8, 3667–3676] having approximately 30% N‐linked oligosaccharides and is biochemically different from the αβ‐chain. By analyses using lectins, some of the oligosaccharides in the α‐chain seem to be the complex‐type and/or hybrid‐type, because wheat germ agglutinin and leukoagglutinating phytohemagglutinin inhibited both GM‐CSF‐induced proliferation and GM‐CSF binding to its receptors. Further analyses using glycoprotein synthesis inhibitors showed that N‐linked processing of the α‐chain, especially glucose removal by glucosidase I and II (whose activities are inhibited by deoxynojirimycin), appeared to be required for the expression onto the cell surface although the β‐chain expression was little affected by their inhibitors. Thus the β‐chain, probably located near the α‐chain on the cell surface, was associated with a high‐affinity class of GM‐CSF receptors.