Retinal ganglion cell survival in vitro maintained by a chondroitin sulfate proteoglycan from the superior colliculus carrying the HNK-1 epitope.
We recently reported evidence implicating a superior colliculus-derived chondroitin sulfate proteoglycan (SCCP) in the trophic support of cultured retinal ganglion cells (Schulz et al., 1990). In the present work we show preparations of the SCCP to be reactive with an antibody (CS-56) to chondroitin sulfate types A and C and with the HNK-1 antibody. Reaction with the HNK-1 antibody allowed us partially to purify the native proteoglycan by immunoaffinity chromatography. HNK-1 reactive material was further processed by a combination of molecular sieve chromatography in the presence of 4M guanidine HCL followed by anion exchange chromatography to yield a product that migrated electrophoretically as a single band in polyacrylamide gel with an apparent molecular weight of not less than 400 k. The SCCP, when added to a fully defined culture medium, maintained the survival of the vast majority (80%) of the ganglion cells over a 16 hr culture period with 86% of these cells showing a profusion of processes; few ganglion cells (10%) survived in the absence of the proteoglycan. Electrophoretic analysis of nonreduced preparations of the molecule did not reveal any low molecular weight silver stained components that may have remained associated with the molecule after guanidine HCL treatment. However, two bands corresponding to molecular weights of around 60 and 80 k were reproducibly observed on polyacrylamide gels following electrophoresis of the molecule in the presence of beta-mercaptoethanol. Our findings provide further evidence suggesting a role for a chondroitin sulfate proteoglycan carrying the HNK-1 epitope in the trophic support of central neurones.
Nichol
K A
KA
Department of Physiology, University of Sydney, N.S.W., Australia.
Everett
A W
AW
Schulz
M
M
Bennett
M R
MR
D000818:Animals; D000911:Antibodies, Monoclonal / Q000276:immunology; D015703:Antigens, CD / Q000276:immunology*; D000945:Antigens, Differentiation, T-Lymphocyte / Q000276:immunology*; D018959:CD57 Antigens; D002470:Cell Survival / Q000187:drug effects; D002478:Cells, Cultured; D011508:Chondroitin Sulfate Proteoglycans / Q000276:immunology / Q000302:isolation & purification / Q000494:pharmacology*; D002852:Chromatography, Ion Exchange; D004591:Electrophoresis, Polyacrylamide Gel; D004797:Enzyme-Linked Immunosorbent Assay; D000939:Epitopes / Q000032:analysis*; D008970:Molecular Weight; D009414:Nerve Growth Factors / Q000276:immunology / Q000302:isolation & purification / Q000494:pharmacology*; D009419:Nerve Tissue Proteins / Q000276:immunology / Q000302:isolation & purification / Q000494:pharmacology*; D016501:Neurites / Q000648:ultrastructure; D051381:Rats; D017208:Rats, Wistar; D012165:Retinal Ganglion Cells / Q000187:drug effects* / Q000648:ultrastructure; D013477:Superior Colliculi / Q000737:chemistry*
D000911:Antibodies, Monoclonal; D015703:Antigens, CD; D000945:Antigens, Differentiation, T-Lymphocyte; D018959:CD57 Antigens; D011508:Chondroitin Sulfate Proteoglycans; D000939:Epitopes; D009414:Nerve Growth Factors; D009419:Nerve Tissue Proteins