Abstract
Mouse mammary carcinoma mutant cell line FUA169, characterized by high GM3 ganglioside content, was established from parent cell line FM3A/F28-7, which has high lactosyl ceramide (LacCer) content but no GM3. FUA169 displays no changes in protein glycosylation, and is a typical glycolipid mutant differing from its parent in that it contains high quantities of GM3 and GlcCer, but no LacCer (see accompanying paper; Tsuruoka, T., Tsuji, T., Nojiri, H., Holmes, E. H., Hakomori, S. (1993) J. Biol. Chem. 268, 2211-2216). In contrast to parent F28-7 cells, FUA169 cells showed clear adhesion to fibronectin (FN). Several lines of evidence indicate that adhesion of FUA169 cells to FN requires the presence of GM3, which supports the function of integrin receptor. (i) Both FUA169 and F28-7 cells express the same quantity of FN integrin receptor, which consists of alpha 5 beta 1 (sensitive to RGDS peptide) and alpha 4 beta 1 (sensitive to CS1 peptide). However, adhesion to FN-coated plates, regardless of type of FN, was much higher for FUA169 than for F28-7 cells. (ii) F28-7 cells, which normally lack GM3 and adhere only weakly to FN, acquired GM3 during incubation in GM3-containing medium, and subsequently adhered strongly to FN. (iii) Cholesterol-lecithin liposomes (cholesterol was 14C-labeled) incorporating alpha 5 beta 1 receptor isolated from human placenta showed clear adhesion to FN-coated plates, and this adhesion was completely inhibited by RGDS peptide and by anti-beta 1 mAb ZH1. When liposomes included a moderate quantity of GM3 (0.22-0.44 micrograms (0.2-0.4 nmol)/55 micrograms of phosphatidylcholine, 33 micrograms of cholesterol, 5 micrograms of alpha 5 beta 1 in liposome), adhesion was enhanced significantly. In contrast, adhesion was greatly reduced below control level for alpha 5 beta 1 liposomes containing a higher quantity (2.2 micrograms; > 2 nM) of GM3. Adhesion to FN was also inhibited, but never enhanced, for alpha 5 beta 1 liposomes with similar composition but containing 0.4 nmol (or other quantities) of LacCer or GlcCer instead of GM3. These findings suggest that the greater adhesion to FN by FUA169 cells, relative to parent F28-7 cells, is due to functional support by GM3 of alpha 5 beta 1 integrin receptor.
Highlights
From $The Biomembrane Institute, Seattle, Washington 98119, the Departments of §Pathobiology and **Chemistry, University of Washington, Seattle, Washington 98195,liMelj.i Seika Co., Morooka-cho, Kohoku-ku, Yokohama 222, Japan, and the IIFaculty of Pharmaceutical Sciences, University of Tokyo, Bunkyo-ku, Tokyo 113, Japan
We have shown that cells expressing GMs1 ganglioside adhere to cells expressing Gg3or lactosyl ceramide (LacCer), based on direct involvement of G M i~n cell adhesion, i.e. interaction of G Mw~ith these GSLs [5,6].Other observations, as follows, indicate that gangliosides help control cell adhesion through modulationof integrin receptor function. (i) In 1979, before integrin receptors were discovered, Kleinmanet al. [7] reported that adhesioonf cells to FN was inhibited by various gangliosides, polysialo
We employed mouse mammary carcinoma mutant cell line FUA169 and parent cell line FM3A/F28-7,as described inthe accompanying ing a higher quantity(2.2 pg; >2 nM) of G M ~A.dhesion paper [25].Our results indicate thGaMt 3 supports the function to FN was inhibited, but never enhanced, cfyo5r81 of FN receptor and perhaps other typeosf integrin receptor
Summary
FUA169, characterized by high G Mg~anglioside con- of cell adhesion haveclarified one “phase”of adhesion involvtent, was established from parceenllt line FM3A/F28- ing integrin receptors andpericellular adhesive proteins We employed mouse mammary carcinoma mutant cell line FUA169 (characterized by high GM3 and GlcCer content) and parent cell line FM3A/F28-7 (which has high duced below control level forcy501 liposomes contain- LacCer content bunto GM3),as described inthe accompanying ing a higher quantity(2.2 pg; >2 nM) of G M ~A.dhesion paper [25].Our results indicate thGaMt 3 supports the function to FN was inhibited, but never enhanced, cfyo5r81 of FN receptor and perhaps other typeosf integrin receptor. Cell Adhesion Assay"FUA169 and F28-7 cells were labeled with TLPHPNLHGPEILDVPSTVQKY, spanning the CS1 region of the ["Hlthymidine (1 pCi/ml, 24 h), washed 3 times, and suspended in IIICS domain of FN, was synthesized using an Applied Biosystems medium supplemented with 0.1% heat-denatured BSA. We modified the method for inclusion of cholesterol and G M ~ (or LacCer or GlcCer), asfollows
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