Glycopeptides from Surface Membranes of Neuroblastoma Cells

Abstract

Sequential removal of surface glycopeptides was achieved by subjection of mouse neuroblastoma cells to a two-step trypsin treatment under different conditions. The glycopetides released by each trypsinization step were digested by Pronase and examined on columns of Sephadex G-50. Different chromatographic patterns were found for the two digests. Thus, several groups of glycopeptides can be distinguished by the trypsinization procedure. One group is readily removed and appears to be at a more accessible location on the cell surface. Among the four neuroblastoma clones examined, the glycopeptide patterns from axon-forming cells differed from those of axon-minus cells.It is generally accepted that the conductance of impulse in nerve cells depends on specific changes in the ionic permeability of the surface membrane. These membranes are involved in the formation of axons and dendrites, the specificity of neuron junctions, and the formation of the synaptic structure.Glycoproteins appear to be on the outermost surface of all cells examined (1-3) and have been implicated in cell recognition (4, 5) and contact (6, 7). It has been suggested that sialic acid, a terminal component of glycoproteins and glycolipids may be fundamental in processes occurring during excitation of nerve cells (8).As part of an approach to these problems, we have examined the glycoproteins from the cell surface of clones isolated from a mouse neuroblastoma, C-1300. These clones differ in their capacity to extend neurites, to synthesize various neurotransmitters, and to respond to electrical stimuli (9). In this study a trypsinization procedure has been developed by which several groups of glycopeptides are distinguished. One group is readily removed and appears to be at a more accessible location on the cell surface. A preliminary report has been published (10). Cell Cultures. Clones N-18, NS-20, N-1, and NlA-103 derived from a mouse neuroblastoma C-1300 (9), were obtained from Dr. M. Nirenberg, NIH, Bethesda, Md. Clone N-18, which is inactive with respect to tyrosine hydroxylase (EC 1.14.3a) and choline acetyltransferase (EC 2.3.1.6), contains acetylcholinesterase (EC 3.1.1.7) and is able to extend neurites in the absence of serum. Clone NS-20, a cholinergenic clone, contains choline acetyltransferase and acetylcholinesterase and is able to form neurites but does not contain detectable activity of tyrosine hydroxylase. Clone N-1 contains low activity of tyrosine hydroxylase, choline acetyltransferase, and acetylcholinesterase and does not form neurites. Clone NlA-103, a subelone of N-1, is almost devo'd of tyrosine hydroxylase and choline acetyltransferase activities, has a low acetylcholinesterase activity, and is an axon-minus line (9).The cells were grown in Dulbecco modified Eagle's medium (Grand Island Biological Co., Grand Island, N.Y.) containing 1.2 g of NaHCO3 per liter and supplemented with 10% fetalcalf serum (Colorado Serum Co.), penicillin (100 U/ml), and streptomycin (100 ,ug/ml), in an atmosphere of 5% C02-95% air with 100...

Design of universal cancer vaccines using natural tumor vessel-specific antigens (SANTAVAC)

“…However, several important peculiarities of vaccine development involving the use of proteolytically cleaved cell surface antigens need to be emphasized. Although the protease-based isolation of cell surface molecules [55][56][57][58] and the use of cancer cells for vaccination have long been described, the approach has some important limitations in terms of protease purity, protease-mediated cellular damage, and antigen preparations.…”

Alterations of Membrane Glycopeptides in Human Colonic Adenocarcinoma

“…These changes apparently occur in both internally and externally located membranes (14,15). The majority of studies were performed on cultured cell systems in which the effects of various tumorigenic agents can be measured easily.…”

“…Differences in composition of membrane glycoproteins resulting from viral transformation of cells and in the metabolic systems which elaborate and degrade them have been reported (9)(10)(11)(12)(13)(14)(15)(16)(17). Although a consensus has not been reached, the studies uniAbbreviations: con A, concanavalin A; a1AGP-NANA, a,-acid glycoprotein minus sialic acid; OSM-NANA.…”

Carbohydrate structure and cell differentitation: unique properties of fucosyl-glycopeptides isolated from embryonal carcinoma cells.

“…For labeling with radioactive sugars, the cells were plated in 10-cm tissue culture dishes. After 24 hr, when the cells reached exponential growth phase, 50 gCi of [6-3H]fucose (10)(11)(12)(13)(14)(15)(16)(17)(18)(19)(20)(21)(22)(23)(24)(25) Ci/mmol, Commissariat a l'Energie Atomique) was added to each dish. The cells were cultured for 24 hr, washed once with Earle's solution lacking Mg2+ and Ca2 , and harvested from tissue culture dishes by EDTA (6).…”