Günter Rosenfelder

Glycosphingolipid-blotting: an immunological detection procedure after separation by thin layer chromatography

A method for detecting glycosphingolipids (GSL) in situ after thin layer chromatography is described. The separated GSL are transferred by diffusion to nitrocellulose. The replica is incubated with poly- or monoclonal antibodies and bound antibodies are detected with second antibodies coupled to peroxidase. Advantages of the procedure are its speed, the non-radioactive detection method, and its suitability for screening applications. In addition, small scale affinity purification of antibodies from the replicas is possible. The presence of Forssman antigen in mouse tissues and the reaction of monoclonal antibodies with human GSL is demonstrated.

Glycolipids as markers of murine T and B lymphoblastoid tumour cell lines

Cell surface glycosphingolipids are thought to be implicated in processes of cell-cell recognition and growth regulation, receptor function, and malignant transformation [l-3]. The identification and the structural analysis of these membrane components are also expected to afford an insight into the process of thymusderived (T) and bone marrow-derived (B) lymphocyte differentiation, since activated T and B lymphocytes express different sets of glycosphingolipids f4,5]. It is, therefore, not unre~onable to assume that tumour celi lines of T and of B cell origin will each yield characteristic glycosphingolipid distribution patterns relating either to the expression of tumour-associated moieties orto components typical for certain functional states during differentiation and/or maturation. Here, spontaneous or chemically induced, transplantable murine T and B lymphoblastoid tumours, adapted to in vitro conditions of growth [6,7] were used in a comparative qualitative and quantitative study of ganglioside and neutral glycolipid distribution patterns revealed by hip-performance thin-layer chromatography (HPTLC) after biosynthetic sugar labelhng [ 51. We document here differences not only between T and B cell lines but also among T and 3 cell lines that may be used as biochemical markers.

Simultaneous determination of sugar incorporation into glycosphingolipids and glycoproteins.

We assessed inhibitors of glycosylation by simultaneous determination of [14C]Gal incorporated into glycosphingolipids and glycoproteins as well as of [3H]Leu incorporated into proteins of intact cells. After metabolic labeling in 96-well plates in the presence or absence of a test substance, cells were collected on glass-fiber filters. The lipid components were extracted from the filter and radioactivities of both extract and filter determined. The reliability of the procedure was tested with different drugs. Using the glucocerebroside synthetase inhibitor 1-phenyl-2-decanoylamino-3-morpholino-1-propanol (PDMP; 5 microM), glycolipid biosynthesis was shown to be reduced to 50% in the murine T-cell EL-4 6.1 line, whereas glycosylation of proteins was not disturbed. With 0.5 microM tunicamycin, the glycosylation of proteins was 50% of that in the control. The procedure was also able to detect various specific effects: the inhibition of protein glycosylation with D-glucosamine and castanospermine, the inhibition of glycosphingolipid biosynthesis with L-cycloserine, and a slight enhancement of glycosphingolipid biosynthesis with conduritol B epoxide and castanospermine. Within a series of N-acyl homologs of PDMP the inhibitory potency increased with chain length. In contrast, these homologs were equipotent by enzymatic in vitro assays.

High Sensitivity Analysis of Amino Acids, Peptides and Proteins by Color Labelling Techniques

Summary High sensitivity (piconol) analysis of amino acids, peptides and proteins can be achieved by pre-column derivatization of those compounds with color chromophore. Three chromophoric reagents, dimethylaminoazobenzene sulfonyl chloride (DABS-Cl, specific for amino, phenol and imidazole groups), dimethylaminoazobenzene isothiocyanate (DABITC, specific for amino group) and dimethylaminoazobenzene iodoacetamide (DABIA, specific for sulfhydryl group) were selected for the microanalysis of amino acids and polypeptides. DAB labelled derivatives were readily separated by HPLC and have detection limit of lower than 1 pmol at the visible region.

Glycosphingolipid Patterns of Established Human Hematopoietic Cell Lines

Abstract Radioactive glycosphingolipid (GSL) labelling patterns from a representative collection of human B-, T-, “null”, myeloid and early erythroid cell lines have been analysed by high performance thin layer chromatography (HPTLC) and high performance liquid chromatography (HPLC). The GSL were tentatively identified by cochromatography with reference GSL and by partial hydrolysis. About 120 different molecular species of GSL could be separated on silica gel plates, e.g. in the case of the promyelocytic HL-60 line. The predominant GSL of all lines were GM3 gangliosides. Each group of cell lines showed a characteristic pattern of additional GSL. “Null” cells proved to be the most homogeneous group, B-cells showed a high degree of heterogeneity. GSL can be expected to serve as additional biochemical markers to establish the origin of a cell line or to classify pathological cell populations.

Monoclonal Antibody Secreting Mouse Hybridoma Clones Express Different Sets of Membrane Glycosphingolipids in Different Antigenic Systems

Abstract B-cell hybridomas developed for the secretion of monoclonal antibodis are also a rich source of membrane material for biochemical analyses of other than Ig,H-2 and Ia-antigens. Here, the glycosphingolipid labelling patterns of hybridomas are reported, which produced antibodies against streptococcal group A polysaccharides (ACHO), azobenzene arsonate (ABA) or phosphorylcholine (PC). While the Sp2/O tumour cell line revealed by high performance thin layer chromatography (HPTLC) only 3 ganglioside bands, hybridomas exhibited up to 50 additional GSL. In the streptococcal system at least three different GSL-patterns could be distinguished. These were independent of H-2, Ig-allotype, -class, and -subclass. Distinct GSL patterns were found for hybridomas of the ABA and PC systems without correlation to the major idiotypes. However, there is the indication that IgM and IgG antibody producing hybridomas show as a result of differentiation unique but distinct GSL patterns.

Monoclonal Antibodies Against Membrane Glycolipids of an Anti-Streptococcal Group a b-Cell Hybridoma

Abstract A B-cell hybridoma producing monoclonal antibody specific for Streptococcal Group A polysaccharide was used as a source of murine lymphocyte membrane glycolipids. Mouse monoclonal antibodies were raised against purified gangliosides and neutral glycosphingolipids and were detected using an ELISA assay. We report the preliminary characterization of the neutral glycosphingolipid antigens recognized by two hybridomas.