Isolation and Characterization of a Large, Neurite-Associated Glycoconjugate from Neuroblastoma Cells

• Published in: The Journal of cell biology 1983-03, Vol.96 (3), p.661-668
• Main Authors: Ellen A. G. Chernoff, Maresh, Grace A., Culp, Lloyd A.
• Format: Article
• Language: English
• Subjects: Animals; Axons - ultrastructure; Cell adhesion; Cell Line; Cell lines; Cells; Chemical Phenomena; Chemistry; Chromatography; Chromatography, Affinity; Cultured cells; Glycoconjugates; Glycoproteins - analysis; Glycoproteins - isolation & purification; Glycosaminoglycans - analysis; Mannose - analysis; membrane glycoproteins; Membrane Proteins - analysis; Membrane Proteins - isolation & purification; Molecular Weight; Neurites; Neuroblastoma; neuroblastoma cells; Neurons; Neurons - analysis; Neurons - ultrastructure; Polysaccharides; Rats
• ISSN: 0021-9525; 1540-8140
• DOI: 10.1083/jcb.96.3.661

A high molecular weight glycoconjugate has been isolated from neurite-producing neuronal tumor cells in culture and has been designated as Iobased on its elution characteristics in gel filtration chromatography. This molecule cannot be found in a variety of nonneuronal cells. Iois found in the substratum-attached material or cell fraction of neurite-producing neuroblastoma cells, depending upon culture conditions. It is found in the substratum-bound fraction of B104 rat neuroblastoma cells during serum starvation and in the EGTA-detached cell fraction of B104 cells grown in chemically defined N2 medium. It occurs only in the cell fraction of the human neuroblastoma line Platt. Examination of behavioral variants of the B104 rat line further strengthens the association of Iowith neurite production; the constitutive neurite-producing ERB9 variant contains Iowhile the non-neurite-producing ERA11 variant does not. Iois large, eluting in the void volume of Sepharose-CL2B columns. Radioiodination of intact cells with lactoperoxidase shows Ioto be a cell surface component. Metabolic radiolabeling studies show that it contains a high proportion of polysaccharide to protein, does not contain mannose, and is unsulfated. Alkaline borohydride reduction releases two size classes of large polysaccharide chains. The alkaline reduction results, along with the mannose incorporation studies, show the presence of O-glycosidic linkages and few, if any, N-linkages. Resistance to nitrous acid deamination, insensitivity to glycosaminoglycan lyases, and the absence of sulfation, indicate that Iodoes not contain the glycosaminoglycans hyaluronic acid, chondroitin-, dermatan-, or heparan-sulfates. Affinity column chromatography reveals high binding affinity of Ioto polyornithine and no binding to gelatin (collagen) or the glycosaminoglycans hyaluronate and heparin. These studies describe a unique high molecular weight glycoconjugate on the surface of neurite-producing neuroblastoma cell lines from two species.