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This communication is concerned with the binding specificity of the leukocyte-adhesion molecule L-selectin (leukocyte homing receptor) towards structurally defined sulphated oligosaccharides of the blood group Le(a) and Le(x) series, and of the glycosaminoglycan series heparin, chondroitin sulphate and keratan sulphate. The recombinant soluble form of the rat L-selectin (L-selectin-IgG Fc chimera) investigated here was shown previously to bind to lipid-linked oligosaccharides 3-O, 4-O and 6-O sulphated at galactose, such as sulphatides and a mixture of 3-sulphated Le(a)/Le(x) type tetrasaccharides isolated from ovarian cystadenoma, as well as to the HNK-1 glycolipid with 3-O sulphated glucuronic acid. In the present study, the L-selectin investigated in both chromatogram binding and plastic microwell binding experiments using neoglycolipids was found to bind to the individual 3-sulphated Le(a) and Le(x) sequences (penta-, tetra- and trisaccharides), and with somewhat lower intensities to their non-fucosylated analogues. Glycosaminoglycan disaccharides of keratan sulphate, heparin and chondroitin sulphate types were also bound by L-selectin in one or both assay systems, leading to the conclusion that clustered glycosaminoglycan oligosaccharides with 6-O sulphation of N-acetylgalactosamine, N-acetylglucosamine or glucosamine, 4-O sulphation of N-acetylgalactosamine, 2-O sulphation of uronic acid, N-sulphation of glucosamine and, to a lesser extent, the non-sulphated uronic acid-containing disaccharides, can support L-selectin adhesion. As inflammatory chemokines (short-range stimulators of lymphocyte migration which trigger integrin activation) are known to bind to endothelial glycosaminoglycans, we propose that the binding of the lymphocyte membrane L-selectin to endothelial glycosaminoglycans may provide a link between the selectin-mediated and integrin-mediated adhesion systems in leukocyte extravasation cascades. The possibility is also raised that lymphocyte L-selectin interactions with glycosaminoglycans may contribute to pathologies of glycosaminoglycan-rich tissues, e.g. cartilage loss in rheumatoid arthritis and inflammatory lesions of the cornea.

The L5 antigenic determinant was previously suggested to be a carbohydrate epitope present on murine cell recognition molecules in the developing brain and to be an early neural marker in the chick embryo. Here, we show that L5 immunoreactivity is associated with complex-type N-glycosidic oligosaccharides. To identify the carbohydrate structure recognized by the L5 antibody, we investigate its binding to N-linked oligosaccharides derived from L5 glycoproteins and to known glycans. Results of mass spectrometric analyses of L5positive neoglycolipids prepared from L5 glycoproteins are consistent with those for N-glycans containing a 3-fucosyl N-acetyllactosamine sequence. We also investigate L5 binding to structurally defined, lipid-linked oligosaccharides based on the blood group type I and II backbones. Chromatogram binding assays, ELISA, and inhibition studies show that the antibody reacts strongly with carbohydrate chains presenting the 3-fucosyl N-acetyllactosamine sequence [Lewisx(Lex) or X-hapten] also recognized by anti-SSEA-1 and anti-CD15. Histochemical studies with different antibodies recognizing the L&< sequence show partially overlapping patterns of immunoreactivity during early neural development in the chick embryo. Therefore, we suggest that the epitope recognized by L5 antibody is closely related to those for anti-SSEA-1 and anti-CD15. Key Words: CD1 5-Chick embryo-L5_Lewisx_Neural induction-SSEA-1. J. Neurochem. 66, 834-844 (1996).Among the monoclonal antibodies raised to glycoproteins of the murine nervous system, the L5 antibody has been shown to recognize neurons and astrocytes in the cerebellum (Streit et al., 1990(Streit et al., , 1993. The L5 antigenic determinant is expressed on multiple glycoproteins, the recognition molecule LI, Thy-I, the chondroitin sulphate proteoglycan astrochondrin, and several components not yet characterized (Streit et al., 1990). Based on results of in vitro experiments, it was proposed that the L5 epitope might participate in the outgrowth of astrocyte processes on extracellular matrix (Streit et al., 1993). secondary ion mass spectrometry; MFLNH, monofucolacto-N-hexaosc: MonoLe', biantennary N-glycosidic oligosaccharides containing Le' on one antenna; PBS, phosphate-buffered saline; 3SLNFP-1l, 3 '-sialyllacto-N-fucopentaose 11; 3SLNFP-l1I and 6SLNFP-l11. 3' and 6'-sialyllacto-N-fucopentaose Ill ceramides, respectively; SuLe'Tetra, sulphated Lewis'-tetrasaccharide.

A second generation of lipid-linked oligosaccharide probes, fluorescent neoglycolipids, has been designed and synthesized for ligand discovery within highly complex mixtures of oligosaccharides. The aminolipid 1,2-dihexadecyl-sn-glycero-3-phosphoethanolamine (DHPE), which has been used extensively to generate neoglycolipids for biological and structural studies, has been modified to incorporate a fluorescent label, anthracene. This new lipid reagent, N-aminoacetyl-N-(9-anthracenylmethyl)-1,2-dihexadecyl-sn-glycero-3-phosphoethanolamine (ADHP), synthesized from anthracenaldehyde and DHPE gives an intense fluorescence under UV light. Fluorescent neoglycolipids derived from a variety of neutral and acidic oligosaccharides by conjugation to ADHP, by reductive amination, can be detected and quantified by spectrophotometry and scanning densitometry, and resolved by TLC and HPLC with subpicomole detection. Antigenicities of the ADHPneoglycolipids are well retained, and picomole levels can be detected using monoclonal carbohydrate sequencespecific antibodies. Among O-glycans from an ovarian cystadenoma mucin, isomeric oligosaccharide sequences, sialyl-Le a -and sialyl-Le x -active, could be resolved by HPLC as fluorescent neoglycolipids, and sequenced by liquid secondary-ion mass spectrometry. Thus the neoglycolipid technology now uniquely combines high sensitivity of immuno-detection with a comparable sensitivity of chemical detection. Principles are thus established for a streamlined technology whereby an oligosaccharide population is carried through ligand detection and ligand isolation steps, and sequence determination by mass spectrometry, enzymatic sequencing and other state-of-the-art technologies for carbohydrate analysis.Keywords: carbohydrate recognition; fluorescence; oligosaccharide ligands; oligosaccharide probes; neoglycolipids.Neoglycolipids, derived by chemical conjugation of oligosaccharides to lipid, were introduced [1] and developed as oligosaccharide probes to address the need for a microprocedure for direct binding studies with glycans released from glycoproteins and glycolipids [2±5]. Neoglycolipids are generated by conjugation of oligosaccharide to an aminolipid such as 1,2-dihexadecyl-sn-glycero-3-phosphoethanolamine (DHPE) by reductive amination. As with natural glycolipids, the hydrophobic lipid enables the oligosaccharides to be coated onto matrices for solid phase binding experiments. Through clustering of the lipid moieties, the oligosaccharides are presented in an oligomeric state, which generates the avidities required for readily detectable binding. Neoglycolipids have the advantage that they contain a single lipid moiety, contrasting with the heterogeneous lipids of natural glycolipids. Following conjugation of mixtures, each oligosaccharide remains a discrete entity, rather than there being a population of oligosaccharides conjugated to a macromolecular carrier. Thus mixtures of neoglycolipids are amenable to resolution by TLC for binding experiments on chromatograms. The excellent ioni...

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