Interaction of wild-type and variant mouse 3T3 cells with lectins from Bandeiraea simplicifolia seeds.

Abstract

An isolectin derived from Bandeiraea simplicifolia seeds and s ecific for terminal a->-galactopyranosyl groups was found tobe cytotoxic to Swiss 3T3 mouse cells. Alter mutagenesis and selection with BS I-B4, a variant clonal cell line resistant to both this isolectin and the a-D-and f-Dfalactose-binding lectin abrin was isolated. The parental cell fine showed homogeneous and noninteracting binding sites for BS I-B4, whereas the variant cells exhibited a curved plot with a reduced number of binding regions. Another lectin, BS II, which is derived from the same seeds but specific for terminal N-acetyl-D-glucosaminyl groups, was cytotoxic to the variant but not the parental cells. These results suggest a possible lesion in the bios nthesis of cell surface structures resulting in the exposure of subterminal N-acetyl-D-glucosaminyl moieties in the variant line.Carbohydrate-containing structures found on animal cell surface membranes play a vital role in the regulation of many important cellular events such as cell division, differentiation, immune response, intercellular communication, and normal and abnormal cell growth (1,2). Lectins, which interact with specific carbohydrate moieties, have been used to probe the structural and functional roles of cell surface carbohydrates (3). In addition, several investigators have isolated cell mutants resistant to the cytotoxic action of some lectins (4-8). The 3T3 mouse cell line which exhibits contact inhibition of cell division has served as a model in investigations of cell surface properties after transformation. Mutants of 3T3 cells displaying altered membrane phenotypes would provide an opportunity to determine what changes, if any, in normal membrane structure are important to or associated with changes observed in transformed or tumor cells.Murphy and Goldstein (9) described an isolectin, BS I-B4, derived from Bandeiraea simplicifolia seeds, that is specific for binding a-D-galactopyranosyl groups. As an initial step in understanding the molecular organization of the membrane components of 3T3 cells, we report here information concerning the interactions of this lectin with Swiss 3T3 cells and a variant 3T3 cell line with impaired ability to bind this lectin.MATERIALS AND METHODS Cell Culture. Swiss 3T3 mouse fibroblasts were obtained from Myron Levine (Department of Human Genetics, University of Michigan Medical School). Wild-type and BS I-B4-resistant variant cells were grown in a modified Eagle's medium (10) supplemented with either 5% or 10% fetal calf serum (GIBCO), penicillin (200 units/ml), and streptomycin (200 gg/ml) or gentamicin sulfate (50 gg/ml, Schering). Unless otherwise indicated, experiments were conducted at 36-37°C in a 5% C02-enriched air atmosphere.Mutagenesis and Selection. After 7 hr of incubation at 370C of 106 3T3 cells in each of three 16-ounce (473 ml) glass bottles (Brockway), ethyl methanesulfonate was added to one bottle at a final concentration of 250 ,tg/ml and to another at 400 ,4g/ml. After 16 hr, the medium was replaced with...

Mammalian glycosylation mutants as tools for the analysis and reconstitution of protein transport

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Retroviral Vector Producer Cell Killing in Human Serum Is Mediated by Natural Antibody and Complement: Strategies for Evading the Humoral Immune Response

“…Recently, we have demonstrated that both CHO and BHK cells are resistant to killing in 40% human seram (data not shown). Altematively, the selection of glycosylation mutants from preexisting VPC lines based on IB4 lectin or human serum sensitivity could be utilized (Stanley et al, 1979;Tsuruoka et al, 1993). These strategies of generating human serum-resistant VPC and retroviral particles are currently underway.…”

Enzyme-linked lectin assay (ELLA)

“…The human erythrocytes were provided by W. John Judd, Department of Pathology, University of Michigan, and were obtained from normal blood donors. The parent Swiss 3T3 cells and a variant line of 3T3 cells deficient in terminal a-D-galactopyranosyl groups (3T3 Bdr) were obtained from Dr Barry Peters, Department of Pharmacology, University of Michigan, and have been described in a previous report [6]. Murine L-929 cells, and a variant of the L-929 cells deficient in surface a-o-galactopyranosyl end groups (L-929 Abn') were obtained from Dr E. H. Y. Chu.…”