Structural and functional analyses of glycosylation on the distinct molecules of human GM-CSF receptors.
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 alpha- and beta-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 alpha- and beta-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 alpha- and beta-chains with 125I-GM-CSF were digested by Staphylococcus aureus V8 protease and gave a different pattern. Furthermore, the size of the alpha-chain was reduced by 25 kDa by the removal of the N-linked oligosaccharides with peptidase: N-glycosidase F treatment, whereas that of the beta-chain remained unmodified by the enzyme. These results suggest that the alpha-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 alpha beta-chain. By analyses using lectins, some of the oligosaccharides in the alpha-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 alpha-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 beta-chain expression was little affected by their inhibitors. Thus the beta-chain, probably located near the alpha-chain on the cell surface, was associated with a high-affinity class of GM-CSF receptors.
Shibuya
K
K
Third Department of Internal Medicine, Faculty of Medicine, University of Tokyo, Japan.
Chiba
S
S
Miyagawa
K
K
Kitamura
T
T
Miyazono
K
K
Takaku
F
F
D002455:Cell Division / Q000187:drug effects; D002460:Cell Line; D006031:Glycosylation; D016178:Granulocyte-Macrophage Colony-Stimulating Factor / Q000378:metabolism; D006801:Humans; D007700:Kinetics; D037102:Lectins / Q000494:pharmacology; D046911:Macromolecular Substances; D008970:Molecular Weight; D010449:Peptide Mapping; D011500:Protein Synthesis Inhibitors / Q000494:pharmacology; D016187:Receptors, Granulocyte-Macrophage Colony-Stimulating Factor / Q000187:drug effects / Q000378:metabolism*; D011994:Recombinant Proteins / Q000378:metabolism
D037102:Lectins; D046911:Macromolecular Substances; D011500:Protein Synthesis Inhibitors; D016187:Receptors, Granulocyte-Macrophage Colony-Stimulating Factor; D011994:Recombinant Proteins; D016178:Granulocyte-Macrophage Colony-Stimulating Factor