New globoseries glycosphingolipids in human teratocarcinoma reactive with the monoclonal antibody directed to a developmentally regulated antigen, stage-specific embryonic antigen 3.

R Kannagi,S B Levery,F Ishigami,S Hakomori,L H Shevinsky,B B Knowles,D Solter

doi:10.1016/s0021-9258(18)32147-1

Abstract

Glycolipids in a cultured human teratocarcinoma cell line (2102Ep) were investigated. The major glycolipids in these cells are globoseries glycolipids having the following structures: (formula; see text) Synthesis of these structures by serial addition of galactose, fucose, and N-acetylneuraminic acid to globoside (Gb4) in this teratocarcinoma is obvious, although further elongation of Gb4 in human cells and tissues has not been previously found with the exception of the presence of a small quantity of Forssman glycolipid in some tissues in the human population (Fs+ group) and in some human cancers. The latter four glycolipids (b-e), with the common internal structure R leads to 3GalNAc beta 1 leads to 3Gal alpha 1 leads to 4R', were all reactive to a monoclonal antibody directed to the 4- to 8-cell stage of murine embryos, known as the stage-specific embryonic antigen 3 (SSEA-3 (Shevinsky, L. H., Knowles, B. B., Damjanov, I., and Solter, D. (1982) Cell 30, 697-705]; structure (c) showed the strongest reactivity. These findings, together with the demonstration of the glycolipid nature of SSEA-1 antigen (Kannagi, R., Nudelman, E., Levery, S. B., and Hakomori, S. (1982) J. Biol. Chem. 257, 14865-14874), indicate that cell surface glycolipids play significant roles as differentiation antigens during the course of embryogenesis.

Highlights

Glycolipids in a cultured human teratocarcinoma cellcarried by lipids and/or by protein molecules
It has been previously reported that humanteratocarcinoma cell lines express an embryonic antigen, SSEA-3, de
Thecell-binding inhibition test was performed as follows: 5 X lo4 2102Ep cells were incubated for 1 h a t 37 "C with a 1:lOOO dilution of SSEA-3 antibody

Summary

MATERIALS AND METHODS

Purified glycolipids GL-1 to GL-4, GL-6, and GL-s7howed a single spot on TLC after acetylatioAn.cetylated GL-5 was separated into and isolated bygel filtrationon a column of SephadexLH-20 in three spotshaving very similar RFvalues, 0.67, 0.64, and 0.61 chloroform-methanol(1:l).Permethylatedneutral glycolipids were hydrolyzed in 90% acetic acid containing 0.5 N sulfuric acid a t 80 "C for 6-8 h [19]. After treatment with PBS containing 5%bovine serum albumin, the well was reacted successively with 1:500 diluted monoclonal SSEA-3 antibody (IgM), 1:lOOO diluted rabbit anti-rat IgM, and "'I-protein A solution. Inhibition test was performed as follows: 5 X lo4 2102Ep cells were incubated for 1 h a t 37 "C with a 1:lOOO dilution of SSEA-3 antibody (50 plltube) in the presence or absenceof liposome suspensions (50 pl/tube) containing various amounts of glycolipids.

RESULTS

Lt n

DISCUSSION

Findings

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