Establishment of a monoclonal antibody directed against Gb3Cer/CD77: a useful immunochemical reagent for a differentiation marker in Burkitt's Iymphoma and germinal centre B cells

A new monoclonal antibody (TU-1) directed against the Galα1-4Galβ1-4Glc residue of the Gb3Cer/CD77 antigen was prepared by the hybridoma technique following immunization of mice with an emulsion composed of monophosphoryl lipid A, trehalose dimycolate, and Gb3Cer isolated from porcine erythrocytes. TU-1 showed reactivity towards Gb3Cer and lyso-Gb3Cer (Galα1-4Galβ1-4Glcβ1-1′Sph), although the reactivity towards lyso-Gb3Cer was about 10-fold lower than that to Gb3Cer. But it did not react with other structurally-related glycolipids, such as LacCer (Galβ1-4Glcβ1-1′Cer), Gg3Cer, Gg4Cer, Gb4Cer (GalNAcβ1-3Galα1-4Galβ1-4Glcβ1-1′Cer), galactosylparagloboside (Galα1-3Galβ1-4GlcNAcβ1-3Galβ1-4Glcβ1-1′Cer), sulfatide (HSO3-3Galβ1-1′Cer), other gangliosides (GM3, GM2, GM1a, GD1a and GT1b), or P1 antigen (Galα1-4Galβ1-4GlcNAcβ1-3Galβ1-4Glcβ1-1′Cer) among neutral glycolipids prepared from P1 phenotype red blood cells. Furthermore, TU-1 reacted with viable lymphoma cells, such as human Burkitt lymphoma cell line, Daudi, and Epstein-Barr virus (EBV)-transformed B cells by the immunofluorescence method, and also with germinal centre B cells in human tonsil and vessel endothelial cells in human thymus histochemically. These results indicate that TU-1 is a monoclonal antibody directed against Gb3Cer/CD77 antigen and can be utilized as a diagnostic reagent for Burkitt's lymphoma and also for detection of the blood group Pk antigen in glycolipid extracts of erythrocytes. Abbreviations: ATL, adult T-cell leukaemia; BSA, bovine serum albumin; Cer, ceramide; DPPC, L-α-dipalmitoylphosphatidylcholine; EBV, Epstein-Barr virus; FCS, fetal calf serum; GalCer, Galβ1-1′Cer; GlcCer, Glcβ1-1′Cer; LacCer, Galβ1-4Glcβ1-1′Cer; Gb3Cer, Galα1-4Galβ1-4Glcβ1-1′Cer; Iyso-Gb3Cer, Galα1-4Galβ1-4Glc1-1′Sph; Gb4Cer, GalNAcβ1-3Galα1-4Galβ1-4Glc1-1′Cer; galactosylparagloboside, Galα1-3Galβ1-4GlcNAcβ1-3Galβ1-4Glcβ1-1′Cer; Gg3Cer, GalNAcβ1-4Galβ1-4Glcβ1-1′Cer; Gg4Cer, Galβ1-3GalNAcβ1-4Galβ1-4Glcβ1-1′Cer; GM3, Neu5Acα2-3Galβ1-4Glcβ1-1′Cer; GM2, GalNAcβ1-4(Neu5Acα2-3) Galβ1-4Glcβ1-1′Cer; GM1a, Galβ1-3GalNAcβ1-4(Neu5Acα2-3)Galβ1-4Glcβ1-1′Cer; GD1a, Neu5Acα2-3Galβ1-3GalNAcβ1-4(Neu5Acα2-3)Galβ1-4Glcβ1-1′Cer; GD1b, Galβ1-3GalNAcβ1-4(Neu5Acα2-8Neu5Acα2-3)Galβ1-4Glcβ1-1′Cer; GT1b, Neu5Acα2-3Galβ1-3GalNAcβ1-4(Neu5Acα2-8Neu5Acα2-3) Galβ1-4Glcβ1-1′Cer; HRP, horseradish peroxidase; LDH, lactate dehydrogenase; MAb, monoclonal antibody; MPL, monophosphoryl lipid A; P1 antigen, Galα1-4Galβ1-4GlcNAcβ1-3Galβ1-4Glcβ1-1′Cer; PVP, polyvinylpyrolidone; Sph, sphingosine; sulfatide, HSO3-Galβ1-1′Cer; TDM, trehalose dimycolate; TLC, thin-layer chromatography This revised version was published online in November 2006 with corrections to the Cover Date.     

Convenient and rapid analysis of linkage isomers of fucose-containing oligosaccharides by matrix-assisted laser desorption/ionization quadrupole ion trap time-of-flight mass spectrometry

Matrix-assisted laser desorption/ionization quadrupole ion trap time-of-flight mass spectrometry (MALDI-QIT-TOF MS) was used to analyze three pyridylamino (PA)-fucosyloligosaccharides isolated from human milk: lacto-N-fucopentaose (LNFP) I [Fucα1-2Galβ1-3GlcNAcβ1-3Galβ1-4Glc-PA], LNFP II [Galβ1-3(Fucα1-4)GlcNAcβ1-3Galβ1-4Glc-PA], and LNFP III [Galβ1-4(Fucα1-3)GlcNAcβ1-3Galβ1-4Glc-PA]. These oligosaccharides are linkage isomers. MALDI-QIT-TOF MS provides MSⁿ spectra, which we used to characterize these PA-oligosaccharides. MS/MS/MS analysis of the non-reducing end tri-saccharide ions generated by MS/MS was able to distinguish these oligosaccharide isomers. The MALDI-QIT-TOF MS is a very convenient and rapid method, therefore, it would be useful for high throughput structural analyses of various types of pyridylaminated oligosaccharide isomers.     

Neutral glycosphingolipids and gangliosides from spleen T lymphoblasts of genetically different inbred mouse strains

The gangliosides GM1b, GalNAc-GM1b and GD1α are typical compounds of concanavalin A stimulated splenic T lymphoblasts of CBA/J inbred mice. Their structural characterization has been described in previous studies. The intention of this work was the comparative TLC immunostaining analysis of the glycosphingolipid composition of lectin stimulated splenic T lymphoblasts obtained from six genetically different inbred mouse strains. The strains examined were AKR, BALB/c, C57BL/6, CBA/J, DBA/2 and WHT/Ht, which are commonly used for biochemical and immunological studies. The neutral glycosphingolipid GgOse4Cer, the precursor for GM1b-type gangliosides, was expressed by all six strains investigated. AKR, C57BL/6 and DBA/2 showed high and BALB/c, CBA/J and WHT/Ht diminished expression in T lymphoblasts, based on single cell calculation. The gangliosides GM1b and GalNAc-GM1b, elongation products of GgOse4Cer, displayed strain-specific differences in their intensities, which were found to correlate with the intensities of GgOse4Cer expression of the same strains. Concerning sialic acid substitution of gangliosides, GM1b and GalNAc-GM1b predominantly carry N-acetylneuraminic acid, whereas choleragenoid receptors GM1a and Gal-GalNAc-GM1b, which are also expressed by all six strains, are characterized by dominance of N-glycolylneuraminic acid. Two highly polar gangliosides, designated with X and Y, which have not been previously recognized in murine lymphoid tissue, were detected by positive anti-GalNAc-GM1b antibody and choleragenoid binding, respectively. Both gangliosides were restricted to AKR, DBA/2 and C57BL/6 mice. The other three strains BALB/c, CBA/J and WHT/Ht are lacking these structures. In summary, the GM1b-type pathway is quite active in all six strains analysed in this study. Strain-specific genetic variations in T lymphoblast gangliosides were observed with the occurrence of gangliosides X and Y. This study and data from other groups strongly indicate for GM1b-type gangliosides a functional association with T cell activation and leukocyte mediated reactions. Abbreviations: ConA, concanavalin A; GSL(s), glycosphingolipid(s); HPTLC, high-performance thin-layer chromatography; NeuAc, N-acetylneuraminic acid; NeuGc, N-glycolylneuraminic acid. The designation of the following glycosphingolipids follows the IUPAC-IUB recommendations (1977) [48] and the ganglioside nomenclature system of Svennerholm [49] for GM1a-type gangliosides. Glucosylceramide or GlcCer, Glcβ1-1Cer; lactosylceramide or LacCer, Galβ1-4Glcβ1-1Cer; gangliotriaosylceramide or GgOse3Cer or Gg3, GalNAcβ1-4Galβ1-4Glcβ1-1Cer; gangliotetraosylceramide or GgOse4Cer or Gg4, Galβ1-3GalNAcβ1-4Galβ1-4Glcβ1-1Cer; gangliopentaosylceramide or GgOse5Cer, GalNAcβ1-4Galβ1-3GalNAcβ1-4Galβ1-4Glcβ1-1Cer; gangliohexaosylceramide or GgOse6Cer, Galβ1-3GalNAcβ1-4Galβ1-3GalNAcβ1-4Galβ1-4Glcβ1-1Cer. GM3, II3NeuAc-LacCer; GM1 or GM1a, II3NeuAc-GgOse4Cer; GM1b, IV3NeuAc-GgOse4Cer; GalNAc-GM1b, IV3NeuAc-GgOse5Cer; GD1a, IV3NeuAc, II3NeuAc-GgOse4Cer; GD1b, II3(NeuAc)2-GgOse4Cer; GD1c, IV3(NeuAc)2-GgOse4Cer; GD1α, IV3NeuAc, III6NeuAc-GgOse4Cer. Only NeuAc-substituted gangliosides are presented in this list of abbreviations This revised version was published online in November 2006 with corrections to the Cover Date.     

Structure determination of a sulfated N-glycans, candidate for a precursor of the selectin ligand in bovine lung

To clarify the structure of non-sialic acid anionic residue on N-glycans in the mammalian tissues, we have isolated sialidase-resistant anionic residue on N-glycans from bovine lung. Analyses by partial acid hydrolysis and glycosidase digestions combined with a two-dimensional HPLC mapping method revealed that the major sialidase-resistant anionic N-glycan had a fucosylbianntenary core structure. The anionic residue was identified as a sulfate ester by methanolysis, anion-exchange chromatography, and mass spectrometry. The linkage position of the sulfate ester was the 6-position of the GlcNAc residue on the Manα1-6 branch. This conclusion was based on the results of glycosidase digestions followed by two-dimensional HPLC mapping. Furthermore, the disialylated form of this sulfated glycan was dominant, and no asialo form was detected. The structure of the major anionic N-glycan prepared from bovine lung and having a sulfate was proposed to be the pyridylamino derivative of Siaα2-3Gαlβ1-4(HSO₃-6)GlcNAcβ1-2Manα1-6(Siaα2-3Galβ1-4GlcNAcβ1-2Manα1-3)Manβ1-4GlcNAcβ1-4(Fucα1-6)GlcNAc.     

Perspectives on the significance of altered glycosylation of glycoproteins in cancer

No abstract Abbreviations: Sia, sialic acid, type unspecified; Tn antigen, GalNAcα 1-O-Ser/Thr; T antigen, Galβ1-3GalNAcα-O-Ser/Thr; Sialyl LewisX, Siaα2-3Galβ1-4(Fucα1-3)GlcNAc; Sialyl Lewisa, Siaα2-3Galβ1-3(Fucα1-4)GlcNAc; Sialyl-Tn antigen, Siaα2-6GalNAcα1-O-Ser/Thr; FucT, fucosyltransferase; ST, sialyltransferase. This revised version was published online in November 2006 with corrections to the Cover Date.     

The study of the substrate specificity of rat-brain fucosyltransferase using synthetic acceptors

The substrate specificity of fucosyltransferase (FT) from rat forebrain and cerebellum was studied using synthetic acceptors. Of 16 acceptors tested, only those containing the Galβ1-4GlcNAcβ1-R fragment were subjected to enzymic fucosylation. The isomer with a 1–3 bond as well as lactose and oligosaccharides with an additional Neu5Ac residue attached to Gal or a Fuc residue attached to GlcNAc were not fucosylated, whereas Fucα1-2Galβ1-4GlcNAc displayed the same substrate properties as Galβ1-4GlcNAc. FT from the cerebellum and forebrain was shown to have a specificity similar to that of mammalian FT IV. The activity of the cerebellum FT with all types of substrates was higher than that of FT isolated from the forebrain, the specificity profiles being similar. This communication is dedicated to the 70th birthday of Prof. A.Ya. Khorlin.     

Unique disialosyl gangliosides from salmon kidney: Characterization of V3αFuc, IV3βGalNAc, II3(αNeuAc)2-Gg4Cer and its analogue with 4-O-acetyl-N-acetylneuraminic acid

Four unidentified acidic glycolipids (X3-X6) were isolated from the kidney of the Pacific salmon on an anion exchange column and by high performance liquid chromatography using a silica bead (Iatrobeads) column. Based on methylation analysis, chemical and enzymatic degradation, proton nuclear magnetic resonance spectroscopy and mass spectrometry, the glycon structure of X5 and X6 was identified as a unique disialosyl fucosyl-N-acetylgalactosaminyl ganglio-N-tetraose: Fucα3GalNAcβ3Galβ3GalNAcβ4[NeuAcα8NeuAcα3] Galβ4Glcβ1Cer. NMR showed that X3 and X4 were analogues of X5 and X6 and contained O-acetyl groups on C4 of the outer N-acetylneuraminic acid, first disialosyl gangliosides containing 4-O-acetyl-N-acetylneuraminic acid. The ceramides of X3 and X5 contained predominantly C24: 1, and X4 and X6 contained saturated fatty acids (C14: 0, C16: 0 and C18: 0), whereas the long chain base was exclusively sphingenine. The concentrations of X3 and X4 were 0.13 and 0.16 nmol/g of kidney respectively and those of X5 and X6, were 0.07 nmol/g each.     

Di-<Emphasis Type="Italic">tert</Emphasis>-butylsilylene-directed α-selective synthesis of <Emphasis Type="Italic">p</Emphasis>-nitrophenyl T-antigen analogues

Seven analogues of p-nitrophenyl T-antigen [Galβ(1→3)GalNAcα(1→O)PNP] have been synthesized as potential substrates for elucidation of the substrate specificity of endo-α-N-acetylgalactosaminidase. These compounds, which are commercially unavailable, include: GlcNAcβ(1→3){GlcNAcβ(1→6)}GalNAcα(1→O)PNP [core 4 type], GalNAcα(1→3)GalNAcα(1→O)PNP [core 5 type], GlcNAcβ(1→6)GalNAcα(1→O)PNP [core 6 type], GalNAcα(1→6)GalNAcα(1→O)PNP [core 7 type], Galα(1→3)GalNAcα(1→O)PNP [core 8 type], Glcβ(1→3)GalNAcα(1→O)PNP and GalNAcβ(1→3)GalNAcα(1→O)PNP. The assembly of these synthetic probes was accomplished efficiently, based on di-tert-butylsilylene(DTBS)-directed α-galactosylation as a key reaction.     

Design and efficient synthesis of novel GM2 analogues with respect to the elucidation of the function of GM2 activator

To elucidate the mechanism underlying the hydrolysis of the GalNAcβ1→4Gal linkage in ganglioside GM2 [GalNAcβ1→4(NeuAcα2→3)Galβ1→4Glcβ1→1′ Cer] by β-hexosaminidase A (Hex A) with GM2 activator protein, we designed and synthesized two kinds of GM2 linkage analogues—6′-NeuAc-GM2 and α-GalNAc-GM2. In this paper, the efficient and systematic synthesis of these GM2 analogues was described. The highlight of our synthesis process is that the key intermediates, newly developed sialyllactose derivatives, were efficiently prepared in sufficient quantities; these derivatives directly served as highly reactive glycosyl acceptors and coupled with GalNTroc donors to furnish the assembly of GM2 tetrasaccharides in large quantities.     

Alpha-Galactosyl trisaccharide epitope: Modification of the 6-primary positions and recognition by human anti-αGal antibody

Galactose oxidase (EC 1.1.3.9, GAO) was used to convert the C-6′ OH of Galβ(1 → 4)Glcβ–OBn (5) to the corresponding hydrated aldehyde (7). Chemical modification, through dehydratative coupling and reductive amination, gave rise to a small library of Galβ(1 → 4)Glcβ–OBn analogues (9a–f, 10, 11). UDP-[6-³H]Gal studies indicated that α1,3-galactosyltransferase recognized the C-6′ modified Galβ(1 → 4)Glcβ–OBn analogues (9a–f, 10, 11). Preparative scale reactions ensued, utilizing a single enzyme UDP-Gal conversion as well as a dual enzymatic system (GalE and α1,3GalT), taking full advantage of the more economical UDP-Glc, giving rise to compounds 6, 15–22. Galα(1 → 3)Galβ(1 → 4)Glcβ–OBn trisaccharide (6) was produced on a large scale (2 g) and subjected to the same chemoenzymatic modification as stated above to produce C-6″ modified derivatives (23–30). An ELISA bioassay was performed utilizing human anti-αGal antibodies to study the binding affinity of the derivatized epitopes (6, 15–30). Modifications made at the C-6′ position did not alter the IgG antibody's ability to recognize the unnatural epitopes. Modifications made at the C-6″ position resulted in significant or complete abrogation of recognition. The results indicate that the C-6′ OH of the αGal trisaccharide epitope is not mandatory for antibody recognition. Published in 2004. This revised version was published online in July 2006 with corrections to the Cover Date.     

Distinct contributions of β4GalNAcTA and β4GalNAcTB to <Emphasis Type="Italic">Drosophila</Emphasis> glycosphingolipid biosynthesis

Drosophila melanogaster has two β4-N-acetylgalactosaminyltransferases, β4GalNAcTA and β4GalNAcTB, that are able to catalyse the formation of lacdiNAc (GalNAcβ,4GlcNAc). LacdiNAc is found as a structural element of Drosophila glycosphingolipids (GSLs) suggesting that β4GalNAcTs contribute to the generation of GSL structures in vivo. Mutations in Egghead and Brainaic, enzymes that generate the β4GalNAcT trisaccharide acceptor structure GlcNAcβ,3Manβ,4GlcβCer, are lethal. In contrast, flies doubly mutant for the β4GalNAcTs are viable and fertile. Here, we describe the structural analysis of the GSLs in β4GalNAcT mutants and find that in double mutant flies no lacdiNAc structure is generated and the trisaccharide GlcNAcβ,3Manβ,4GlcβCer accumulates. We also find that phosphoethanolamine transfer to GlcNAc in the trisaccharide does not occur, demonstrating that this step is dependent on prior or simultaneous transfer of GalNAc. By comparing GSL structures generated in the β4GalNAcT single mutants we show that β4GalNAcTB is the major enzyme for the overall GSL biosynthesis in adult flies. In β4GalNAcTA mutants, composition of GSL structures is indistinguishable from wild-type animals. However, in β4GalNAcTB mutants precursor structures are accumulating in different steps of GSL biosynthesis, without the complete loss of lacdiNAc, indicating that β4GalNAcTA plays a minor role in generating GSL structures. Together our results demonstrate that both β4GalNAcTs are able to generate lacdiNAc structures in Drosophila GSL, although with different contributions in vivo, and that the trisaccharide GlcNAcβ,3Manβ,4GlcβCer is sufficient to avoid the major phenotypic consequences associated with the GSL biosynthetic defects in Brainiac or Egghead.     

Immunization of mice with fucosyl-GM1 conjugated with keyhole limpet hemocyanin results in antibodies against human small-cell lung cancer cells

Fucosyl-GM1 (Fuc-GM1) [Fucα1 → 2Galβ1 → 3GalNAcβ1 → 4(NeuAcα2-3)Galβ1 → 4Glcβ1 → O-Cer] is a small-cell-lung-cancer (SCLC)-associated ganglioside initially defined by the murine monoclonal antibody F12. On the basis of its known distribution, Fuc-GM1 is a potential target for active immunotherapy in SCLC patients. Fuc-GM1 has been extracted and purified from bovine thyroid. The immunogenicity of Fuc-GM1 was tested in mice either alone, mixed with carrier protein keyhole limpet hemocyanin (KLH) or covalently linked with KLH, plus immunological adjuvant QS-21. The Fuc-GM1-KLH conjugate plus QS-21 adjuvant was found to be optimal. It induced consistent IgM and IgG enzyme-linked immunosorbent assay (ELISA) titers against Fuc-GM1. These antibodies were strongly reactive with the strongly Fuc-GM1-positive rat hepatoma cell line H4-II-E, and they were moderately reactive with the moderately positive human SCLC cell line H146 by flow cytometry and complement-mediated lysis. Both ELISA and fluorescence-activated cell sorting (FACS) reactions were inhibited with Fuc-GM1or H4-II-E but not with the structurally related ganglioside GM1 or Fuc-GM1-negative colon cancer cell line LS-C. On the basis of these results, a vaccine comprising Fuc-GM1-KLH plus QS-21 is being prepared for testing in patients with SCLC. Received: 25 March 1999 / Accepted: 5 August 1999     

Assessment of glycosaminoglycan-protein linkage tetrasaccharides as acceptors for GalNAc- and GlcNAc-transferases from mouse mastocytoma

Two glycosaminoglycan-protein linkage tetrasaccharide-serine compounds, GlcAβ1-3Galβ1-3Galβ1-4Xylβ1-O-Ser and GlcAβ1-3Gal(4-O-sulfate)β1-3Galβ1-4Xylβ1-O-Ser, were tested as hexosamine acceptors, using UDP-[3H]GlcNAc and UDP-[3H]GalNAc as sugar donors, and solubilized mouse mastocytoma microsomes as enzyme source. The nonsulfated Ser-tetrasaccharide was found to function as an acceptor for a GalNAc residue, whereas the Ser-tetrasaccharide containing a sulfated galactose unit was inactive. Characterization of the radio-labelled product by digestion with α-N-acetylgalactosaminidase and β-N-acetylhexosaminidase revealed that the [3H]GalNAc unit was α-linked, as in the product previously synthesized using serum enzymes, and not β-linked as found in the chondroitin sulfate polymer. Heparan sulfate/heparin biosynthesis could not be primed by either of the two linkage Ser-tetrasaccharides, since no transfer of [3H]GlcNAc from UDP-[3H]GlcNAc could be detected. By contrast, transfer of a [3H]GlcNAc unit to a [GlcAβ1-4GlcNAcα1-4]2-GlcAβ1-4-aMan hexasaccharide acceptor used to assay the GlcNAc transferase involved in chain elongation, was readily detected. These results are in agreement with the recent proposal that two different N-acetylglucosaminyl transferases catalyse the biosynthesis of heparan sulfate. Although the mastocytoma system contains both the heparan sulfate/heparin and chondroitin sulfate biosynthetic enzymes the Ser-tetrasaccharides do not seem to fulfil the requirements to serve as acceptors for the first HexNAc transfer reactions involved in the formation of these polysaccharides. This revised version was published online in November 2006 with corrections to the Cover Date.     

Glycotope analysis in miracidia and primary sporocysts of Schistosoma mansoni: Differential expression during the miracidium-to-sporocyst transformation

Fucosylated carbohydrate epitopes (glycotopes) expressed by larval and adult schistosomes are thought to modulate the host immune response and possibly mediate parasite evasion in intermediate and definitive hosts. While previous studies showed glycotope expression is developmentally and stage-specifically regulated, relatively little is known regarding their occurrence in miracidia and primary sporocysts. In this study, previously defined monoclonal antibodies were used in confocal laser scanning microscopy, standard epifluorescence microscopy and Western blot analyses to investigate the developmental expression of the following glycotopes in miracidia and primary sporocysts of Schistosoma mansoni: GalNAcβ1-4GlcNAc (LDN), GalNAcβ1-4(Fucα1-3)GlcNAc (LDN-F), Fucα1-3GalNAcβ1-4GlcNAc (F-LDN), Fucα1-3GalNAcβ1-4(Fucα1-3)GlcNAc (F-LDN-F), GalNAcβ1-4(Fucα1-2Fucα1-3)GlcNAc (LDN-DF), Fucα1-2Fucα1-3GalNAcβ1-4(Fucα1-2Fucα1-3)GlcNAc (DF-LDN-DF), Galβ1-4(Fucα1-3)GlcNAc (Lewis X) and the truncated trimannosyl N-glycan Manα1-3(Manα1-6)Manβ1-4GlcNAcβ1-4GlcNAcβ1-Asn (TriMan). All but Lewis X were variously expressed by miracidia and sporocysts of S. mansoni. Most notably, α3-fucosylated LDN (F-LDN, F-LDN-F, LDN-F) was prominently expressed on the larval surface and amongst glycoproteins released during larval transformation and early sporocyst development, possibly implying a role for these glycotopes in snail–schistosome interactions. Interestingly, Fucα2Fucα3-subsituted LDN (LDN-DF, DF-LDN-DF) and LDN-F were heterogeneously surface-expressed on individuals of a given larval population, particularly amongst miracidia. In contrast, LDN and TriMan primarily localised in internal somatic tissues and exhibited only minor surface expression. Immunoblots indicate that glycotopes occur on overlapping but distinct protein sets in both larval stages, further demonstrating the underlying complexity of schistosome glycosylation. Additionally, sharing of specific larval glycotopes with Biomphalaria glabrata suggests an evolutionary convergence of carbohydrate expression between schistosomes and their snail host.     

En Bloc Duplications, Mutation Rates, and Densities of Amino Acid Changes Clarify the Evolution of Vertebrate α-1,3/4-Fucosyltransferases

Numerous vertebrates have four α-1,3/4-fucosyltransferase genes (FUT9, FUT7, FUT4, and FUT Lewis) belonging to the same family. Until now, studies on the evolution of this family have mainly focused on Lewis genes but how the other α-1,3/4-fucosyltransferases have emerged from a common ancestor is not well known. In order to define the respective roles of duplications and mutations, we have compared amino acid sequences representative of bony fish (Takifugu rubripes), amphibians (Xenopus laevis), birds (Gallus gallus), and mammals (Bos taurus). The FUT tree has two fundamental branches, each split into two subfamilies. We found evidence for two duplication events, dated around 710–760 Myr and 590–640 Myr, respectively, compatible with the hypothesis of two rounds of whole genome duplications in chordate genomes, before the emergence of bony vertebrates. Based on the Homo sapiens (human) physical map, we identified blocks of paralogues belonging to regions of FUT9 (6q16), FUT4 (11q21), FUT7 (9q34), and FUT Lewis (19p13) and to a region on HSA1p that is devoid of any FUT. In zebrafish (Danio rerio), an orthologue region of HSA1 harbors an FUT9 specific to bony fish, showing that duplications are not restricted to a single FUT gene but involve blocks of paralogues. In addition, sets of genes within each block clarify the order of duplication events and, as a result, the order of α-1,3/4-fucosyltransferase gene emergence. We have also determined the mutation rates and the density of amino acid changes along protein sequences in each α-1,3/4-fucosyltransferase subfamily during the main vertebrate transitions. After the emergence of tetrapods, the mutation rate of FUT9 decreased dramatically, suggesting the early acquisition of a crucial fucosyltransferase activity in the first stages of development. The FUT7 mutation rate, which in tetrapod ancestors is about half that in amniote ancestors, may be related to the role of this gene in immune systems. In contrast to other subfamilies, we found a constant mutation rate in FUT Lewis and a rather homogeneous amino acid density change, independently of the vertebrate transition, suggesting that hitherto Lewis epitopes have dispensable functions. [Reviewing Editor: Dr. Gail Simmons]     

Immunogenicity of synthetic conjugates of Lewisy oligosaccharide with proteins in mice: towards the design of anticancer vaccines

 Many human carcinomas overexpress the Lewis^y (Le^y) blood-group epitope [Fucα1→2Galβ1→4 (Fucα1→3)GlcNAcβ1→3Gal-]. With a view to developing Le^y based vaccines we have examined the immunogenicity of Le^y-protein conjugates in mice. Le^y pentasaccharide was synthesized as its allyl glycoside and coupled to keyhole limpet hemocyanin (KLH) by reductive amination or by a novel method utilizing a maleido-derivitized alkyl carboxyhydrazide as a bridging group to 2-iminothiolane-derivitized KLH. Le^y oligosaccharide was also coupled to bovine serum albumin by reductive amination. Immunization of groups of mice with the three conjugates, together with the immunological adjuvant QS21, showed that Le^y oligosaccharide directly coupled to KLH was the most efficient conjugate for eliciting IgG and IgM antibody responses to naturally occurring forms of Le^y epitopes carried on mucins and glycolipids. These antibodies were also reactive with and cytotoxic to a human breast cancer cell line expressing Le^y (MCF-7). These experiments suggest that Le^y-KLH antigen and QS21 adjuvant could be considered as an immunogenic therapeutic vaccine in carcinoma patients. Received: 28 March 1997 / Accepted: 2 September 1997     

Expression of a unique globo-series glycolipid in cultured rat dorsal root ganglion neurons: Relationship with neuronal development

Previous studies from this laboratory demonstrated the presence of a UDP-galactose:Gb3Cer α1-3galactosyltansferase activity responsible for the synthesis of a unique glycosphingolipid (GSL), Galα1-3Gb3Cer, in cultured PC12 pheochromocytoma cells (21). In this investigation, we examined the presence of this enzyme activity in isolated rat embryonic dorsal root ganglion neurons (DRGN), which, like pheochromocytoma cells, originate from the neural crest cells. DRGN exhibited the α-galactosyltransferase activity and the activity was comparable to that of the PC12 cells while several other rat tissues, with the exception of kidney, showed minimal activity. In order to define the spatial and temporal expression of Galα1-3Gb3Cer in DRGN, we examined the expression of Galα1-3Gb3Cer in cultured DRGN derived from embryonic day 16 rat embryos. Using a polyclonal antibody raised against Galα1-3Gb3Cer, we examined the localization of this glycolipid in DRGN cells after, 5, 8, 12, and 15 days in culture. Immunostaining was restricted to the neurons while Schwann cells were negative. At day 5, the immunostaining was weak and confined to the cell body of the DRGN, though neurites were present at this stage. The period between days 5 and 15 represented a period of rapid neuritic growth and continued enlargement of the cell bodies. Immunoreactivity in the cell bodies increased dramatically by day 8. By day 12, immunoreactivity was present in neurites, and by day 15, was strong in both cell bodies and neurites. The expression of Galα1-3Gb3Cer in vivo was confirmed by immunostaining of frozen sections of dorsal root ganglia. Our present studies which demonstrate neuron-specific expression of Galα1-3Gb3Cer during neurotigenesis combined with previous observations for its expression in PC12 cells, strongly implicates this GSL in neuronal development. This paper is dedicated to Dr. Marion Smith.     

Isolation and structural analysis of three neutral glycosphingolipids from a mixed population of Caenorhabditis elegans (Nematoda: Rhabditida)

The free-living nematode, Caenorhabditis elegans, has been proposedand analyzed as a prototypic model for parasitic nematodes.In order to study whether there is a structural basis for theproposed analogy with respect to nematode glycoconjugates, wehave analyzed Caenorhabditis elegans glycosphingolipids. Three,simple neutral glycosphingo-lipid components of the neutralglycolipid fraction were isolated by high-performance liquidchromatography. Structural analysis was performed by methylationanalysis, exoglycosidase cleavage, matrix-assisted laser desorption/ionization time-of-flight mass spectrometry, and ceramide analysis.The chemical structures have been determined as Glcß1Cer,Manß4Glcß1Cer and GlcNAcß3Manp4Glcß1Cer;that are characterized as belonging to the arthro-series ofprotostomial glycosphingolipids. The ceramide moiety of theparent glycosphingolipid-ceramide mono-hexoside was dominatedby 2-hydroxy fatty acids, and a d17:1 sphingoid-base with aniso- or anteiso-branched chain. The chemical composition ofthe three glycosphingolipids from Caenorhabditis elegans displayedclose structural coincidence with the equivalent structuresfrom the porcine parasitic nematode, Ascaris suum (G.Lochnit,R.D. Dennis, U.Zähringer, and R.Geyer, Glycoconjugate J.,1997), in support of this organism as a prototypic glyco-sphingolipidmodel for parasitic nematodes. Arthro-series glycosphingolipids Caenorhabditis elegans d17:1 branched sphingoid-bases MALDI-TOF-MS nematode neutral glycosphingolipids