The Le^x Carbohydrate Sequence Is Recognized by Antibody to L5, a Functional Antigen in Early Neural Development

Abstract

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.

Lewisxand α2,3-Sialyl Glycans and Their Receptors TAG-1, Contactin, and L1 Mediate CD24-Dependent Neurite Outgrowth

“…Polyclonal antibodies to mouse L1 (Chen et al, 1999), NCAM (Kadmon et al, 1992), Contactin (R&D Systems), TAG-1 [TG3, Traka et al, (2003) and from R&D Systems], Caspr1 (Santa Cruz Biotechnology), and Caspr2 (Santa Cruz Biotechnology) and monoclonal antibodies to mouse CD24 [79; Kadmon et al (1992)], oligomannosides, HNK-1 (for reference, see Heller et al, 2003), and Lewis x (Streit et al, 1996) were used. Carbohydrates and glycoconjugates were purchased from Dextra Laboratories, peptide N-glycosidase F (PNGase F) from Roche Molecular Biochemicals, and O-sialoglycoprotein endopeptidase (OSGE) of Mannheimia hemolytica from Cedarline.…”

The Unique 473HD-Chondroitinsulfate Epitope Is Expressed by Radial Glia and Involved in Neural Precursor Cell Proliferation

“…The following primary antibodies were used in this study. The monoclonal antibodies were as follows: 473HD (rat IgM) (Faissner et al, 1994), 487 (L5 epitope/LeX, rat IgM) (Streit et al, 1996), RC2 (mouse IgM; Developmental Studies Hybridoma Bank, University of Iowa, Iowa City, IA), O4 (mouse IgM) (Sommer and Schachner, 1981), polysialic-acid-containing neural cell adhesion molecule (PSA-NCAM) (Rougon et al, 1986), anti-nestin (mouse IgG; Chemicon, Temecula, CA), anti-␤III-tubulin (mouse IgG; Sigma, St. Louis, MO), and anti-5-bromo-2-deoxyuridine (BrdU) (mouse IgG; Roche Products, Welwyn Garden City, UK). The polyclonal antibodies (all rabbit) were against the following: DSD-1-PG/phosphacan [referred to as pk-anti-phosphacan, batch KAF 13(4)] (Faissner et al, 1994), GFAP (Dako, High Wycombe, UK), NG2 (Chemicon), brain lipid-binding protein (BLBP; gift from Dr. N. Heintz, Rockefeller University, New York, NY), and GLAST (gift from Dr. D. Pow, University of Queensland, Brisbane, Australia; or Chemicon).…”

“…2 N) cells compared with the nonselected cell population (Table 1). We also investigated the expression of the adult NSC marker LeX (Capela and Temple, 2002) as detected with mAb 487 (Streit et al, 1996). The 473HD-positive cells were significantly enriched in 487/ LeX-positive cells (Fig.…”

Dermatan sulfotransferase Chst14/D4st1, but not chondroitin sulfotransferase Chst11/C4st1, regulates proliferation and neurogenesis of neural progenitor cells

“…The monoclonal antibodies 487 and 473HD (kindly provided by Andreas Faissner, Ruhr University Bochum, Bochum, Germany), and polyclonal antibody against the glutamate aspartate transporter (GLAST) (kindly provided by Niels Danbolt, University of Oslo, Oslo, Norway) have been described (Faissner et al, 1994;Lehre et al, 1995;Streit et al, 1996). All commercial primary antibodies are listed in supplementary material Table S2.…”