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To link to full-text access for this article, visit this link: http://dx.doi.org/10.1016/j.ydbio.2007.04.013 Byline: Ya-Wen Chen (a), Johannes W. Pedersen (b), Hans H. Wandall (b), Steven B. Levery (c), Sandrine Pizette (a), Henrik Clausen (c), Stephen M. Cohen (a) Keywords: Egghead; Brainiac; Glycosyltransferase; Glycolipid; Glycosylation; CG8536; CG14517; CG17223; CG5878 Abstract: Glycosphingolipids (GSL) are glycosylated polar lipids in cell membranes essential for development of vertebrates as well as Drosophila. Mutants that impair enzymes involved in biosynthesis of GSL sugar chains provide a means to assess the functions of the sugar chains in vivo. The Drosophila glycosyltransferases Egghead and Brainiac are responsible for the 2nd and 3rd steps of GSL sugar chain elongation. Mutants lacking these enzymes are lethal and the nature of the defects that occur has suggested that GSL might impact on signaling by the Notch and EGFR pathways. Here we report on characterization of enzymes involved in the 4th and 5th steps of GSL sugar chain elongation in vitro and explore the biological consequences of removing the enzymes involved in step 4 in vivo. Two [beta]4-N-Acetylgalactosyltransferase enzymes can carry out step 4 ([beta]4GalNAcTA and [beta]4GalNAcTB), and while they may have overlapping activity, the mutants produce distinct phenotypes. The [beta]4GalNAcTA mutant displays behavioral defects, which are also observed in viable brainiac mutants, suggesting that proper locomotion and coordination primarily depend on GSL elongation. [beta]4GalNAcTB mutant animal shows ventralization of ovarian follicle cells, which is caused by defective EGFR signaling between the oocyte and the dorsal follicle cells to specify dorsal fate. GSL sequentially elongated by Egh, Brn and [beta]4GalNAcTB in the oocyte contribute to this signaling pathway. Despite the similar enzymatic activity, we provide evidence that the two enzymes are not functionally redundant in vivo, but direct distinct developmental functions of GSL. Author Affiliation: (a) Developmental Biology Program, European Molecular Biology Laboratory, Meyerhofstrasse 1, 69117 Heidelberg, Germany (b) Institute of Cellular and Molecular Medicine, University of Copenhagen, Blegdamsvej 3, 2200 Copenhagen N, Denmark (c) Department of Chemistry, University of New Hampshire, Durham, NH 03824, USA Article History: Received 31 January 2007; Revised 27 March 2007; Accepted 11 April 2007

Enzymatic analyses of native, recombinant, and truncated forms of alpha1--2 fucosyltransferase of rat hepatoma reveal that while both native and recombinant display full activity, the truncated enzyme requires detergent for activity expression. Data suggest that missing N-terminal transmembrane domain in the truncated enzyme is important for enzyme's conformation or its interaction with acceptor glycolipids.

The role of the P histo-blood group-related glycosphingolipid, sialosyl galactosyl globoside (SGG), as a binding receptor for uropathogenic Escherichia coli was investigated. To this end, SGG from normal human kidney was purified and structurally characterized. Experimental results showed that the primary structure of SGG is identical to a globo-series glycolipid that was previously characterized and developmentally regulated. The role of SGG in the pathogenesis of urinary tract infections was discussed.

A study was conducted to characterize the structure elucidation of Paracoccidioides brasiliensis glycosphingolipids Pb-1 and Pb-2 with respect to their glycan and ceramide moieties. The lipophilic components were eluted from the C18-silica cartridge with MeOH and CHCl3-MeOH. 1H nuclear magnetic spectroscopy was carried out with a Bruker AM-500. Results suggested a number of common features of fungal GIPC biosynthesis which may represent potential targets related to fungal disease.

E-selectin binding gangliosides from myelogenous leukemia HL60 cells were studied to examine their E-selectin binding pattern and to compare it with that of human neutrophils. Both E-selectin nonbinding and nonbinding fractions were analyzed using a variety of techniques. The major gangliosides present in each fraction are presented. The characteristic structures of the gangliosides are also provided.

Gangliosides extracted from myelogenous leukemia HL60 cells and human leukocytes were extensively fractionated and analyzed to examine the E-selectin binding ability of the respective fractions. High performance thin layer chromatography revealed that only monosialogangliosides having a polylactosamine core with less than 10 monosaccharide units exhibited E-selectin binding. None of the fractions exhibited P-selectin binding. The structures of the major gangliosides of HL60 cells are provided.

The antigens Lea-Lex are produced enzymatically and are compared with Lea-Lea to characterize the epitope structure of the tumor-associated antigen, which is defined by mAb 43-9F. The evaluation is performed in a side-by-side immunoassay by the application of ST-421, mAbs43-9F and anti-Lea mAbCA3-F4. The hydrogen-nuclear magnetic resonance spectroscopy identifies the presence of enzymatically prepared Lea-Lex glycosphingolipids, which reveal the nature of the characterization.

The structures of x2 and sialosyl-x2 glycosphingolipid (GSL) were isolated and characterized using fast-atom bombardment mass spectrosopy, gas chromatography mass spectrometry and 1-D H-nuclear magnetic resonance. A monoclonal antibody (TH2) specific for the terminal trisaccharide of x2 glycosphingolipid (GSL) confirmed the existence of extended structures of the GSL isotopes. The amount of x2, sialosyl-x2 and extended x2 were found to be the same in blood cells and in various tissues. However, the amount of x2 and x2-derived structures were higher in blood samples of rare blood group p individuals.

A study was conducted to isolate and characterize glycosphingolipids possessing long-chain cyclic acetals. The compound was found to use a unique type of natural glycan modification. 16:0, 18:0 and 18:1 fatty aldehydes were also observed to have been produced by the compound. The compound also exhibited a structure consistent with those of galactocerebroside having 3,4- and 4,6-linked cyclic plasma substituents.

A study using 1H nuclear magnetic resonance spectroscopywas conducted on the biosynthetic pathways associated with glycosphingolipids bearing GlcNAcbeta1->3 and GalNAcbeta1->4 linked to beta-Gal of lactosylceramide. A GalNAcbeta1->4 transferase preparation was usedto synthesize the lacto-ganglio hybrid core structure LcGg4Cer from Lc3Cer. Theproduct was found to have GalNacbeta1->4-(GlcNAcbeta1->3)Galbeta1->4Glcbeta1->1Cer as primary structure. A propsed three-dimensional LcGg4Cer structure, which has an unusual terminal N-acetylhexosamine residue arrangement, is presented.