Isolation and characterization of a large, neurite-associated glycoconjugate from neuroblastoma cells

Abstract

A high molecular weight glycoconjugate has been isolated from neurite-producing neuronal tumor cells in culture and has been designated as Io based on its elution characteristics in gel filtration chromatography. This molecule cannot be found in a variety of nonneuronal cells. Io is 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 8104 rat line further strengthens the association of Io with neurite production; the constitutive neurite-producing ERB9 variant contains Io while the non-neurite-producing ERA11 variant does not. Io is large, eluting in the void volume of Sepharose-CL2B columns. Radioiodination of intact cells with lactoperoxidase shows 10 to 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 10 does not contain the glycosaminoglycans hyaluronic acid, chondroitin-, dermatan-, or heparan-sulfates. Affinity column chromatography reveals high binding affinity of Io to 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.Several approaches have been used to examine mechanisms in neuronal cells underlying growth cone adhesion and neurite outgrowth. The effects of physical/chemical properties of the tissue culture substratum on strength of adhesion and rate of neurite outgrowth were initially explored by Letourneau (1). Substrata coated with polycationic polypeptides, especially polyornithine (PORN) and polylysine, were found to optimize growth cone adhesion and neurite outgrowth from embryonic sensory ganglia. A complementary approach has involved a search for factors from cells or their environment that stimulate neuronal differentiation. Studies using embryonic ciliary ganglia have shown the existence of substances in extracts of target tissues (2, 3), conditioned medium from heart cultures (4, 5) and a variety of other tissues (6) that stimulate neurite outgrowth. These substances are active only when bound to PORN-coated substrata (designated PORN-bound neurite-1~4[: /OURN,~L OF CEtL 8fOLOGY-VOtUME 96 MARCH 1983 661-668

Heparan sulfate proteoglycans in the substratum adhesion sites of human neuroblastoma cells: Modulation of affinity binding to fibronectin

“…The substratum adhesion sites of rat B104 neuroblastoma cells were first shown to be enriched in microfilament and intermediate filament subunit proteins, cellular fibronectin, and considerable amounts of heparan sulfate which had not been characterized until this current study. In contrast to similar studies in fibroblasts (Culp et al, 19791, there was very little hyaluronic acid or dermatan sulfate but small amounts of chondroitin sulfate in neuroblastoma adhesion sites, including Platt human neuroblastoma sites Chernoff et al, 1983). A partially purified heparan sulfate proteoglycan from bovine corneal endothelial cells was shown to promote neurite outgrowth of rat sympathetic neurons (Lander et al, 1982), although this was later shown to be due to laminin in the extract (Lander et al, 1985).…”

“…It has previously been shown that SDS quantitatively solubilizes protein and polysaccharide from the substratum adhesion sites of neuroblastoma cells Chernoff et al, 1983). Other extraction conditions were then sought which would give a very high recovery of proteoglycan from these sites, using reagents which would only reversibly denature these molecules.…”

“…Most of these molecules appeared to be small in size with only two to four chains per monomer (Maresh et al, 1984). Furthermore, the pattern of cytoskeletal protein and cell surface glycoprotein distribution in neuroblastoma adhesion sites is much simpler than that observed in the EGTA-detached cell fraction Chernoff et al, 1983;Maresh et al, 1984).…”

“…MATERIALS AND METHODS Cell growth and isolation of substratum-attached material Mycoplasma-free Platt human neuroblastoma cells were grown as described previously (Chernoff et al, 1983) in Dulbecco's-modified Eagle's medium supplemented with 10% neonatal calf serum, 10 mM HEPES buffer, 250 U/ml penicillin, and 250 pg/ml streptomycin sulfate. For radiolabeling, 1 x lo6 cells were seeded into each of 30 x 100 mm plates for 72 hr of exponential growth in 7 ml of the same medium without streptomycin sulfate and pencillin but with 50 pCi/ml of [35Sl-Na2S04 (for a final sulfate concentration of 0.91 mM, which is above the sulfate-limiting conditions for these and various other cells) (Humphries et al, 1986) or, in some cases, with 10 pCi/ml of L-[4,5 3Hl-leucine.…”

Inverse relationship between invasiveness and differentiative capacity in different human neuroblastoma cell lines

“…LaN1 (Seeger et al, 1977) and Platt (Chernoff et al, 1983) neuroblastoma lines were kindly provided by Dr. L.A. Culp, Case Western Reserve University, Cleveland, OH. The SY5Y neuroblastoma line was obtained through the courtesy of Dr. M. Olivotto of our Institute.…”