Neuroglycan C Is a Novel Midkine Receptor Involved in Process Elongation of Oligodendroglial Precursor-like Cells

Keiko Ichihara-Tanaka,Atsuhiko Oohira,Martin Rumsby,Takashi Muramatsu

doi:10.1074/jbc.m602228200

Abstract

Midkine is a heparin-binding growth factor that promotes cell attachment and process extension in undifferentiated bipolar CG-4 cells, an oligodendroglial precursor cell line. We found that CG-4 cells expressed a non-proteoglycan form of neuroglycan C, known as a part-time transmembrane proteoglycan. We demonstrated that neuroglycan C before or after chondroitinase ABC treatment bound to a midkine affinity column. Neuroglycan C lacking chondroitin sulfate chains was eluted with 0.5 m NaCl as a major fraction from the column. We confirmed that CG-4 cells expressed two isoforms of neuroglycan C, I, and III, by isolating cDNA. Among three functional domains of the extracellular part of neuroglycan C, the chondroitin sulfate attachment domain and acidic amino acid cluster box domain showed affinity for midkine, but the epidermal growth factor domain did not. Furthermore, cell surface neuroglycan C could be cross-linked with soluble midkine. Process extension on midkine-coated dishes was inhibited by either a monoclonal anti-neuroglycan C antibody C1 or a glutathione S-transferase-neuroglycan C fusion protein. Finally, stable transfectants of B104 neuroblastoma cells overexpressing neuroglycan C-I or neuroglycan C-III attached to the midkine substrate, spread well, and gave rise to cytoskeletal changes. Based on these results, we conclude that neuroglycan C is a novel component of midkine receptors involved in process elongation.

Highlights

Preferentially expressed in the kidney after birth [1, 2]
CG-4 Cells Express neuroglycan C (NGC)-I and NGC-III as a Non-PG Form— First, we examined the expression of NGC in bipolar CG-4 cells by immunofluorescence microscopy using monoclonal antiNGC antibody C5 (Fig. 2A)
To study whether NGC expressed in CG-4 cells carries chondroitin sulfate chains, NGC was immunoprecipitated from lysate of CG-4

Summary

Introduction

Preferentially expressed in the kidney after birth [1, 2]. MK is a 13-kDa protein with about 50% sequence identity to pleiotrophin (PTN), which is called heparin-binding growth-associated molecule [3, 4]. NGC-overexpressing B104 cells and mock-transfected cells were incubated in Dulbecco’s modified Eagle’s medium overnight, trypsinized, suspended with 0.1% soybean trypsin inhibitor in PBS, washed with PBS two times, seeded on the wells of Falcon 1147 plates coated with mMK, and analyzed for process extension. Cells with polyclonal anti-NGC antibodies, incubated with chondroitinase ABC or with buffer alone, and analyzed by Western blotting (Fig. 2B).

Results

Conclusion