The Mannose 6-Phosphate/Insulin-like Growth Factor II Receptor Is a Nanomolar Affinity Receptor for Glycosylated Human Leukemia Inhibitory Factor

Frédéric Blanchard,Sylvie Raher,Laurence Duplomb,Patricia Vusio,Vincent Pitard,Jean-Luc Taupin,Jean-François Moreau,Bernard Hoflack,Stéphane Minvielle,Yannick Jacques,Anne Godard

doi:10.1074/jbc.273.33.20886

Abstract

Comparison of the binding properties of non-glycosylated, glycosylated human leukemia inhibitory factor (LIF) and monoclonal antibodies (mAbs) directed at gp190/LIF-receptor beta subunit showed that most of the low affinity (nanomolar) receptors expressed by a variety of cell lines are not due to gp190. These receptors bind glycosylated LIF produced in Chinese hamster ovary cells (CHO LIF) (Kd = 6.9 nM) but not Escherichia coli-derived LIF or CHO LIF treated with endoglycosidase F. CHO LIF binding to these receptors is neither affected by anti-gp190 mAbs nor by anti-gp130 mAbs and is specifically inhibited by low concentrations of mannose 6-phosphate (Man-6-P) (IC50 = 40 microM), suggesting that they could be related to Man-6-P receptors. The identity of this LIF binding component with the Man-6-P/insulin-like growth factor-II receptor (Man-6-P/IGFII-R) was supported by several findings. (i) It has a molecular mass very similar to that of the Man-6-P/IGFII-R (270 kDa); (ii) the complex of LIF cross-linked to this receptor is immunoprecipitated by a polyclonal anti-Man-6-P/IGFII-R antibody; (iii) this antibody inhibits LIF and IGFII binding to the receptor with comparable efficiencies; (iv) soluble Man-6-P/IGFII-R purified from serum binds glycosylated LIF (Kd = 4.3 nM) but not E. coli LIF. The potential role of Man-6-P/IGFII-R in LIF processing and biological activity is discussed.

Highlights

Kin-6 (IL-6) subfamily of helical cytokines ( including IL-11, oncostatin M (OSM), ciliary neurotrophic factor (CNTF), and cardiotrophin-1 (CT-1)) [1,2,3]
With the recent availability of such anti-leukemia inhibitory factor (LIF)-R␤ monoclonal antibodies (mAbs) [13, 14], we show that the low affinity LIF receptor expressed by a number of cell lines is unrelated to gp190
U266 cells Express a LIF Receptor That Does Not Involve gp190 —In the course of studies investigating the expression of gp190, gp130 and LIF receptors by various cell lines, a surprising observation was that the plasmocytoma cell line U266 expressed LIF receptors but did not bind anti-gp190 mAbs

Summary

EXPERIMENTAL PROCEDURES

Cell Lines, Antibodies, and Cytokines—The human myeloma U266 cell line and the human choriocarcinoma JAR cell line were obtained from the American Type Culture Collection (ATCC, Rockville, MD). A natural form of LIF was purified from A375 melanoma cells by affinity chromatography on polyclonal anti-LIF antibodies coupled to agarose beads (Affi-Gel, Bio-Rad). Cells were incubated as described above with a constant and non-saturating concentration of radioactive LIF (with respect to its equilibrium dissociation constant) and increasing concentration of unlabeled competitor ligand (antibodies, cytokines, or monosaccharides) in a final volume of 50 ␮l. After incubating under agitation overnight at 4 °C, agarose beads were centrifuged, washed in solubilization buffer, and submitted to SDSPAGE and autoradiography as described above. Binding of CHO LIF, E. coli LIF, or IGFII was assayed at different concentrations from 5 nM to 1 ␮M in Hepes-buffered saline (BIACore) and at a flow rate of 10 ␮l/min. The resulting sensorgrams were analyzed using the BIAEvaluation (BIACore) software

RESULTS

NDa ND ND ND ND

DISCUSSION