Glycosylation Site for Chondroitin Sulfate on the Neural Part-time Proteoglycan, Neuroglycan C

Sachiko Aono,Yoshihito Tokita,Takuya Shuo,Shinobu Yamauchi,Fumiko Matsui,Keiko Nakanishi,Kanako Hirano,Mamoru Sano,Atsuhiko Oohira

doi:10.1074/jbc.m403263200

Sachiko Aono, Yoshihito Tokita + Show 7 more

Open Access

https://doi.org/10.1074/jbc.m403263200

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Abstract

Neuroglycan C (NGC) is a membrane-spanning chondroitin sulfate (CS) proteoglycan that is expressed predominantly in the central nervous system (CNS). NGC dramatically changed its structure from a proteoglycan to a nonproteoglycan form with cerebellar development, whereas a small portion of NGC molecules existed in a nonproteoglycan form in the other areas of the mature CNS, suggesting that the CS glycosylation of NGC is developmentally regulated in the whole CNS. As primary cultured neurons and astrocytes from cerebral cortices expressed NGC in a proteoglycan form and in a nonproteoglycan form, respectively, CS glycosylation seems to be regulated differently depending on cell type. To investigate the glycosylation process, cell lines expressing a proteoglycan form of NGC would be favorable experimental models. When a mouse NGC cDNA was transfected into COS 1, PC12D, and Neuro 2a cells, only Neuro 2a cells, a mouse neuroblastoma cell line, expressed NGC bearing CS chains. In PC12D cells, although three intrinsic CS proteoglycans were detected, exogenously expressed NGC did not bear any short CS chains just like NGC in the mature cerebellum. This suggests that the addition of CS chains to the NGC core protein is regulated in a manner different from that of other CS proteoglycans. As the first step in investigating the CS glycosylation mechanism using Neuro 2a cells, we determined the CS attachment site as Ser-123 on the NGC core protein by site-directed mutagenesis. The CS glycosylation was not necessary for intracellular trafficking of NGC to the cell surface at least in Neuro 2a cells.

Highlights

Proteoglycans are a group of proteins that bear covalently bound sulfated glycosaminoglycan chains
In P7 mice, Neuroglycan C (NGC) was recognized as a broad band, which is characteristic of a proteoglycan, at around 150 kDa in homogenates of all parts examined (Fig. 2)
We demonstrated that the chondroitin sulfate (CS) glycosylation of NGC occurred depending on cell type as well as on developmental stage of the central nervous system (CNS)

Summary

EXPERIMENTAL PROCEDURES

Cell Culture—COS 1 cells and Neuro 2a cells (Dainippon Pharmaceutical Co., Osaka, Japan) were maintained in high glucose Dulbecco’s modified Eagle’s medium (DMEM), 10% fetal calf serum (FCS; Sigma), 50 units/ml penicillin G potassium, and 50 ␮g/ml streptomycin sulfate. 4 ϫ 106 cells were suspended in 10 ml of high glucose DMEM containing 0.4% HS, 50 units/ml penicillin G, 25 ␮g/ml streptomycin, and 10 ␮l/ml N2 supplement (Invitrogen) and plated onto a 100-mm poly-L-lysine-coated plastic dish [23]. Proteoglycans were precipitated from the cell lysate by adding 3 volumes of 95% ethanol containing 1.3% potassium acetate at 0 °C and collected by centrifugation at 10,000 rpm for 10 min at 4 °C. All CS proteoglycans including NGC on the blots were detected with monoclonal antiproteoglycan ⌬Di-0S, ⌬Di-4S, and ⌬Di-6S antibodies (Seikagaku Co.) after treatment with CHase ABC These three monoclonal antibodies react with the CS “stub” that remains on core proteins after extensive CHase ABC digestion. The precipitated beads were washed twice with 200 ␮l of Chondroitin Sulfate of NGC

Mutation site

RESULTS

DISCUSSION