Absorption of anti-blood group A antibodies on P-selectin glycoprotein ligand-1/immunoglobulin chimeras carrying blood group A determinants: core saccharide chain specificity of the Se and H gene encoded alpha1,2 fucosyltransferases in different host cells

To specifically eliminate recipient anti-blood group ABO antibodies prior to ABO-incompatible organ or bone marrow transplantation, an efficient absorber of ABO antibodies has been developed in which blood group determinants may be carried at high density and by different core saccharide chains on a mucin-type protein backbone. The absorber was made by transfecting different host cells with cDNAs encoding a P-selectin glycoprotein ligand-1/mouse immunoglobulin G(2b) chimera (PSGL-1/mIgG(2b)), the H- or Se-gene encoded alpha1,2-fucosyltransferases (FUT1 or FUT2) and the blood group A gene encoded alpha1,3 N-acetylgalactosaminyltransferase (alpha1,3 GalNAcT). Western blot analysis of affinity-purified recombinant PSGL-1/mIgG(2b) revealed that different precursor chains were produced in 293T, COS-7m6, and Chinese hamster ovary (CHO)-K1 host cells coexpressing FUT1 or FUT2. FUT1 directed expression of H type 2 structures mainly, whereas FUT2 preferentially made H type 3 structures. None of the host cells expressing either FUT1 or FUT2 supported expression of H type 1 structures. Furthermore, the highest A epitope density was on PSGL-1/mIgG2(2b) made in CHO-K1 cells coexpressing FUT2 and the alpha1,3 GalNAcT. This PSGL-1/mIgG(2b) was used for absorption of anti-blood group A antibodies in human blood group O serum. At least 80 times less A trisaccharides on PSGL-1/mIgG(2b) in comparison to A trisaccharides covalently linked to macroporous glass beads were needed for the same level of antibody absorption. In conclusion, PSGL-1/mIgG(2b), if substituted with A epitopes, was shown to be an efficient absorber of anti-blood group A antibodies and a suitable model protein for studies on protein glycosylation.     

Pichia pastoris-produced mucin-type fusion proteins with multivalent O-glycan substitution as targeting molecules for mannose-specific receptors of the immune system

Mannose-binding proteins like the macrophage mannose receptor (MR), the dendritic cell-specific intercellular adhesion molecule-3 grabbing non-integrin (DC-SIGN) and mannose-binding lectin (MBL) play crucial roles in both innate and adaptive immune responses. Immunoglobulin fusion proteins of the P-selectin glycoprotein ligand-1 (PSGL-1/mIgG(2b)) carrying mostly O-glycans and, as a control, the α1-acid glycoprotein (AGP/mIgG(2b)) carrying mainly N-linked glycans were stably expressed in the yeast Pichia pastoris. Pichia pastoris-produced PSGL-1/mIgG(2b) was shown to carry O-glycans that mediated strong binding to mannose-specific lectins in a lectin array and were susceptible to cleavage by α-mannosidases including an α1,2- but not an α1,6-mannosidase. Electrospray ionization ion-trap mass spectrometry confirmed the presence of O-glycans containing up to nine hexoses with the penta- and hexasaccharides being the predominant ones. α1,2- and α1,3-linked, but not α1,6-linked, mannose residues were detected by (1)H-nuclear magnetic resonance spectroscopy confirming the results of the mannosidase cleavage. The apparent equilibrium dissociation constants for binding of PNGase F-treated mannosylated PSGL-1/mIgG(2b) to MR, DC-SIGN and MBL were shown by surface plasmon resonance to be 126, 56 and 16 nM, respectively. In conclusion, PSGL-1/mIgG(2b) expressed in P. pastoris carried O-glycans mainly comprised of α-linked mannoses and with up to nine residues. It bound mannose-specific receptors with high apparent affinity and may become a potent targeting molecule for these receptors in vivo.     

PSGL-1 from the murine leukocytic cell line WEHI-3 is enriched for core 2-based O-glycans with sialyl Lewis x antigen

Leukocyte trafficking involves specific recognition between P-selectin and L-selectin and PSGL-1 containing core 2-based O-glycans expressing sialyl Lewis x (SLe(x)) antigen. However, the structural identity of the glycan component(s) displayed by murine neutrophil PSGL-1 that contributes to its P-selectin counter-receptor activity has been uncertain, since these cells express little if any SLe(x) antigen, and because there have been no direct studies to examine murine PSGL-1 glycosylation. To address this uncertainty, we studied PSGL-1 glycosylation in the murine cell line WEHI-3 using metabolic-radiolabeling with (3)H-monosaccharide precursors to detect low-abundance O-glycan structures. We report that PSGL-1 from WEHI-3 cells expresses a di-sialylated core 2 O-glycan containing the SLe(x) antigen. This fucosylated O-glycan is scarce on PSGL-1 and essentially undetectable in total leukocyte glycoproteins from WEHI-3 cells. These results demonstrate that WEHI-3 cells selectively fucosylate PSGL-1 to generate functionally important core 2-based O-glycans containing the SLe(x) antigen.     

Core saccharide dependence of sialyl Lewis X biosynthesis

The sialyl-Lewis X (SLe^x) determinant is important in leukocyte extravasation, metastasis and bacterial adhesion. The role of the protein, N-glycan and O-glycan core structures for the biosynthesis of SLe^x in vivo by fucosyltransferases (FucTs) is not known. Immunoglobulin G (IgG) Fc fusion proteins of α₁-acid glycoprotein (AGP), P-selectin glycoprotein ligand-1 (PSGL-1) or CD43 were used to probe the specificity of FucT-III-VII expressed alone in 293T and COS cells or together with O-glycan core enzymes in Chinese hamster ovary (CHO)-K1 cells. Western blotting with the monoclonal antibodies CSLEX and KM93 showed that FucT-III and V-VII produced SLe^x on core 2 in CHO cells. Only FucT-V, -VI and, with low activity, -VII worked on core 3 on CD43/IgG, but no SLe^x was detected with CSLEX on PSGL-1/IgG with core 3. KM93 stained SLe^x on core 2, but was not reactive with SLe^x on core 3. FucT-III, V-VII made SLe^x on N-glycans of AGP/IgG in CHO, but not in COS and 293T cells, even though the same FucTs could make SLe^x on CD43/IgG and PSGL-1/IgG in these cells. Our results define the specificities of FucT-III-VII in SLe^x biosynthesis on O-glycans with different core structures and the fine specificity of the widely used anti-SLe^x monoclonal antibody, KM93.     

Glycosyltransferases involved in type 1 chain and Lewis antigen biosynthesis exhibit glycan and core chain specificity

Sialyl Lewis A (SLe(a)), Lewis A (Le(a)), and Lewis B (Le(b)) have been studied in many different biological contexts, for example in microbial adhesion and cancer. Their biosynthesis is complex and involves beta1,3-galactosyltransferases (beta3Gal-Ts) and a combined action of alpha2- and/or alpha4-fucosyltransferases (Fuc-Ts). Further, O-glycans with different core structures have been identified, and the ability of beta3Gal-Ts and Fuc-Ts to use these as substrates has not been resolved. Therefore, to examine the in vivo specificity of enzymes involved in SLe(a), Le(a), and Le(b) synthesis, we have transiently transfected CHO-K1 cells with relevant human glycosyltransferases and, on secreted reporter proteins, detected the resulting Lewis antigens on N- and O-linked glycans using western blotting and Le-specific antibodies. beta3Gal-T1, -T2, and -T5 could synthesize type 1 chains on N-linked glycans, but only beta3Gal-T5 worked on O-linked glycans. The latter enzyme could use both core 2 and core 3 precursor structures. Furthermore, the specificity of FUT5 and FUT3 in Le(a) and Le(b) synthesis was different, with FUT5 fucosylating H type 1 only on core 2, but FUT3 fucosylating H type 1 much more efficient on core 3 than on core 2. Finally, FUT1 and FUT2 were both found to direct alpha2-fucosylation on type 1 chains on both N- and O-linked structures. This knowledge enables us to engineer recombinant glycoproteins with glycan- and core chain-specific Lewis antigen substitution. Such tools will be important for investigations on the fine carbohydrate specificity of Le(b)-binding lectins, such as Helicobacter pylori adhesins and DC-SIGN, and may also prove useful as therapeutics.     

Avian influenza H5 hemagglutinin binds with high avidity to sialic acid on different O-linked core structures on mucin-type fusion proteins

The interaction between P-selectin glycoprotein ligand-1/mouse IgG_2b (PSGL-1/mIgG_2b) fusion protein carrying multiple copies of the influenza hemagglutinin receptor Siaα2-3Gal on different O-glycan chains and recombinant human influenza H5N1 A/Vietnam/1203/04 hemagglutinin was investigated with a Biacore biosensor. The fusion protein was produced by stable cell lines in large scale cultures and purified with affinity- and gel filtration chromatography. The C-P55 and 293-P cell lines were established by transfecting the Chinese hamster ovary (CHO)-K1 and Human embryonic kidney (HEK)-293 cell lines with plasmids encoding the PSGL-1/mIgG_2b fusion protein, while the C-PSLex cell line was engineered by transfecting CHO-K1 cells with the plasmids encoding the core 2 β1,6GnT-I and FUT-VII glycosyltransferases. Glycosylation was characterized by lectin Western blotting of the proteins and liquid chromatography - mass spectrometry of released non-derivatized O-glycans. Biacore experiments revealed that PSGL-1/mIgG_2b is a good binding partner of H5. The binding curves displayed a slow dissociation indicating a multivalent binding. The H5 hemagglutinin binds with similar strength to PSGL-1/mIgG_2b carrying mostly sialylated core 1 (clone C-P55), a mix of sialylated core 1 and sialylated lactosamine (clone 293-P) or mainly sialylated lactosamine (clone C-PSLex) O-glycans, indicating that this hemagglutinin is unable to discriminate between these structures. The potential use of the large, flexible PSGL-1/mIgG_2b mucin-type fusion protein carrying Siaα2-3Gal as a multivalent inhibitor of influenza virus is discussed.     

Immunogenicity of influenza virus vaccine is increased by anti-gal-mediated targeting to antigen-presenting cells

This study describes a method for increasing the immunogenicity of influenza virus vaccines by exploiting the natural anti-Gal antibody to effectively target vaccines to antigen-presenting cells (APC). This method is based on enzymatic engineering of carbohydrate chains on virus envelope hemagglutinin to carry the alpha-Gal epitope (Gal alpha 1-3Gal beta 1-4GlcNAc-R). This epitope interacts with anti-Gal, the most abundant antibody in humans (1% of immunoglobulins). Influenza virus vaccine expressing alpha-Gal epitopes is opsonized in situ by anti-Gal immunoglobulin G. The Fc portion of opsonizing anti-Gal interacts with Fc gamma receptors on APC and induces effective uptake of the vaccine virus by APC. APC internalizes the opsonized virus to transport it to draining lymph nodes for stimulation of influenza virus-specific T cells, thereby eliciting a protective immune response. The efficacy of such an influenza vaccine was demonstrated in alpha 1,3galactosyltransferase (alpha 1,3GT) knockout mice, which produce anti-Gal, using the influenza virus strain A/Puerto Rico/8/34-H1N1 (PR8). Synthesis of alpha-Gal epitopes on carbohydrate chains of PR8 virus (PR8(alpha gal)) was catalyzed by recombinant alpha1,3GT, the glycosylation enzyme that synthesizes alpha-Gal epitopes in cells of nonprimate mammals. Mice immunized with PR8(alpha gal) displayed much higher numbers of PR8-specific CD8(+) and CD4(+) T cells (determined by intracellular cytokine staining and enzyme-linked immunospot assay) and produced anti-PR8 antibodies with much higher titers than mice immunized with PR8 lacking alpha-Gal epitopes. Mice immunized with PR8(alpha gal) also displayed a much higher level of protection than PR8 immunized mice after being challenged with lethal doses of live PR8 virus. We suggest that a similar method for increasing immunogenicity may be applicable to avian influenza vaccines.     

Fucosyltransferase substrate specificity and the order of fucosylation in invertebrates

Core alpha1,6-fucosylation is a conserved feature of animal N-linked oligosaccharides being present in both invertebrates and vertebrates. To prove that the enzymatic basis for this modification is also evolutionarily conserved, cDNAs encoding the catalytic regions of the predicted Caenorhabditis elegans and Drosophila melanogaster homologs of vertebrate alpha1,6-fucosyltransferases (E.C. 2.4.1.68) were engineered for expression in the yeast Pichia pastoris. Recombinant forms of both enzymes were found to display core fucosyltransferase activity as shown by a variety of methods. Unsubstituted nonreducing terminal GlcNAc residues appeared to be an obligatory feature of the substrate for the recombinant Caenorhabditis and Drosophila alpha1,6-fucosyltransferases, as well as for native Caenorhabditis and Schistosoma mansoni core alpha1,6-fucosyltransferases. On the other hand, these alpha1,6-fucosyltransferases could not act on N-glycopeptides already carrying core alpha1,3-fucose residues, whereas recombinant Drosophila and native Schistosoma core alpha1,3-fucosyltransferases were able to use core alpha1,6-fucosylated glycans as substrates. Lewis-type fucosylation was observed with native Schistosoma extracts and could take place after core alpha1,3-fucosylation, whereas prior Lewis-type fucosylation precluded the action of the Schistosoma core alpha1,3-fucosyltransferase. Overall, we conclude that the strict order of fucosylation events, previously determined for fucosyltransferases in crude extracts from insect cell lines (core alpha1,6 before core alpha1,3), also applies for recombinant Drosophila core alpha1,3- and alpha1,6-fucosyltransferases as well as for core fucosyltransferases in schistosomal egg extracts.     

Molecular basis of leukocyte rolling on PSGL-1. Predominant role of core-2 O-glycans and of tyrosine sulfate residue 51

Interactions between the leukocyte adhesion receptor L-selectin and P-selectin glycoprotein ligand-1 play an important role in regulating the inflammatory response by mediating leukocyte tethering and rolling on adherent leukocytes. In this study, we have examined the effect of post-translational modifications of PSGL-1 including Tyr sulfation and presentation of sialylated and fucosylated O-glycans for L-selectin binding. The functional importance of these modifications was determined by analyzing soluble L-selectin binding and leukocyte rolling on CHO cells expressing various glycoforms of PSGL-1 or mutant PSGL-1 targeted at N-terminal Thr or Tyr residues. Simultaneous expression of core-2 beta1,6-N-acetylglucosaminyltransferase and fucosyltransferase VII was required for optimal L-selectin binding to PSGL-1. Substitution of Thr-57 by Ala but not of Thr-44, strongly decreased L-selectin binding and leukocyte rolling on PSGL-1. Substitution of Tyr by Phe revealed that PSGL-1 Tyr-51 plays a predominant role in mediating L-selectin binding and leukocyte rolling whereas Tyr-48 has a minor role, an observation that contrasts with the pattern seen for the interactions between PSGL-1 and P-selectin where Tyr-48 plays a key role. Molecular modeling analysis of L-selectin and P-selectin interactions with PSGL-1 further supported these observations. Additional experiments showed that core-2 O-glycans attached to Thr-57 were also of critical importance in regulating the velocity and stability of leukocyte rolling. These observations pinpoint the structural characteristics of PSGL-1 that are required for optimal interactions with L-selectin and may be responsible for the specific kinetic and mechanical bond properties of the L-selectin-PSGL-1 adhesion receptor-counterreceptor pair.     

Characterization of the rat alpha(1,3)galactosyltransferase: evidence for two independent genes encoding glycosyltransferases that synthesize Galalpha(1,3)Gal by two separate glycosylation pathways

The important xenoepitope Galalpha(1,3)Gal was thought to be exclusively synthesized by a single alpha(1,3)galactosyltransferase. However, the cloning of the distant family member rat iGb3 synthase, which is also capable of synthesizing Galalpha(1,3)Gal as the glycolipid structure iGb3, challenges the notion that alpha(1,3)galactosyltransferase is the sole Galalpha(1,3)Gal-synthesizing enzyme. We describe the cloning of the rat homolog of alpha(1,3)galactosyltransferase, showing that indeed the rat expresses two distinct alpha(1,3)galactosyltransferases, alpha(1,3)GT and iGb3 synthase. Rat alpha(1,3)galactosyltransferase shows a high amino acid sequence identity with the alpha(1,3)galactosyltransferase of mouse (90%), pig (76%), and ox (75%), in contrast to the low amino acid sequence identity (42%) with iGb3 synthase. The rat alpha(1,3)galactosyltransferase is expressed in heart, brain, spleen, kidney, and liver and has a similar intron/exon structure to the mouse alpha(1,3)galactosyltransferase. Transfection studies show that in contrast to the iGb3 synthase, rat alpha(1,3)galactosyltransferase can synthesize Galalpha(1,3)Gal on glycoproteins but cannot synthesize the glycolipid iGb3, defining two separate glycosylation pathways for the synthesis of Galalpha(1,3)Gal. Furthermore iGb3 synthase was found to be distinct from alpha(1,3)GT with its ability to synthesize poly-alpha-Gal glycolipid structures.     

The possibility of reducing xenoantigen levels with a novel gal 3'-sulfotransferase (GP3ST)

Glycoprotein-3-sulfotransferase (GP3ST) is a key enzyme in downregulating the expression of Galalpha1,3Galbeta1,4GlcNAc-R (the alpha-Gal epitope), via enzymatic competition with an alpha1,3 galactosyltransferase (alpha1,3GT), such as alpha2,6 sialyltransferase (alpha2,6ST). In this study, we report the dominance of GP3ST over alpha1,3GT using transfected pig endothelial cell (PEC) lines. The introduction of the GP3ST gene into PEC suppresses its antigenicity with respect to normal human pooled serum (NHS), including the alpha-Gal epitope and the Hanganutziu-Deicher (H-D) antigen, and, in addition, reduces the susceptibility to NHS in complement-mediated cell lysis. Western and lectin blot analyses of the products of parental PEC and its transfectants indicated that proteins smaller than 66 kDa have a diminished reactivity with NHS and the IB4 lectin. The levels of the alpha-Gal epitope in neutral glycosphingolipids were also decreased in the GP3ST transfectants as detected in thin layer chromatography by immunostaining. These data indicate that GP3ST is very effective in reducing xenoepitope levels.     

The wbbD gene of E. coli strain VW187 (O7:K1) encodes a UDP-Gal: GlcNAc{alpha}-pyrophosphate-R {beta}1,3-galactosyltransferase involved in the biosynthesis of O7-specific lipopolysaccharide

In this work, we demonstrate that the wbbD gene of the O7 lipopolysaccharide (LPS) biosynthesis cluster in Escherichia coli strain VW187 (O7:K1) encodes a galactosyltransferase involved in the synthesis of the O7-polysaccharide repeating unit. The galactosyltransferase catalyzed the transfer of Gal from UDP-Gal to the GlcNAc residue of a GlcNAc-pyrophosphate-lipid acceptor. A mutant strain with a defective wbbD gene was unable to form O7 LPS and lacked this specific galactosyltransferase activity. The normal phenotype was restored by complementing the mutant with the cloned wbbD gene. To characterize the WbbD galactosyltransferase, we used a novel acceptor substrate containing GlcNAcalpha-pyrophosphate covalently bound to a hydrophobic phenoxyundecyl moiety (GlcNAc alpha-O-PO(3)-PO(3)-(CH(2))(11)-O-phenyl). The WbbD galactosyltransferase had optimal activity at pH 7 in the presence of 2.5 mM MnCl(2). Detergents in the assay did not increase glycosyl transfer. Digestion of enzyme product by highly purified bovine testicular beta-galactosidase demonstrated a beta-linkage. Cleavage of product by pyrophosphatase and phosphatase, followed by HPLC and NMR analyses, revealed a disaccharide with the structure Gal beta1-3GlcNAc. Our results conclusively demonstrate that WbbD is a UDP-Gal: GlcNAcalpha-pyrophosphate-R beta1,3-galactosyltransferase and suggest that the novel synthetic glycolipid acceptor may be generally applicable to characterize other bacterial glycosyltransferases.     

Functional analysis of the combined role of the O-linked branching enzyme core 2 beta1-6-N-glucosaminyltransferase and dimerization of P-selectin glycoprotein ligand-1 in rolling on P-selectin

Leukocyte P-selectin glycoprotein ligand-1 (PSGL-1) is expressed as a homodimer and mediates leukocyte rolling through interactions with endothelial P-selectin. Previous studies have shown that PSGL-1 must be properly modified by specific glycosyltransferases including alpha1,3-fucosyltransferase-VII, core 2 beta1-6-N-glucosaminyltransferase (C2GlcNAcT-I), one or more alpha2,3-sialytransferases, and a tyrosulfotransferase. In addition, dimerization of PSGL-1 through its sole extracellular cysteine (Cys(320)) is essential for rolling on P-selectin under shear conditions. In this report, we measured the contributions of both C2GlcNAcT-I glycosylation and dimerization of PSGL-1 to adhesive bonds formed during tethering and rolling of transfected cell lines on purified P-selectin. Tethering to P-selectin under flow increased with dimerization compared with cells expressing monomeric PSGL-1 (referred to as C320A). The rolling defects (decreased cellular accumulation, PSGL-1/P-selectin bond strengths and tethering rates, and increased velocities and skip distance) demonstrated by transfectants expressing monomeric PSGL-1 could be overcome by increasing the substrate P-selectin site density and by overexpressing C2GlcNAcT-I in C320A transfectants. Two molecular weight variants of PSGL-1 were isolated from cell lines transfected with PSGL-1, C320A, and/or C2GlcNAcT-I cDNAs, and these differences in electrophoretic mobility appeared to correlate with C2GlcNAcT-I expression. C320A transfectants expressing low molecular weight PSGL-1 had lower C2GlcNAcT-I levels (measured by reactivity to core 2 specific linkage antibody, CHO-131) and compromised rolling on P-selectin (regardless of site density) compared with C320A cells with high levels of C2GlcNAcT-I and high molecular weight PSGL-1. Both C2GlcNAcT-I glycosylation and PSGL-1 dimerization increased the rate of tethering to P-selectin under flow, whereas C2GlcNAcT-I levels primarily influenced tether bond strength.     

Dimerization of P-Selectin Glycoprotein Ligand-1 (PSGL-1) Required for Optimal Recognition of P-Selectin

Interactions between P-selectin, expressed on endothelial cells and activated platelets, and its leukocyte ligand, a homodimer termed P-selectin glycoprotein ligand-1 (PSGL-1), mediate the earliest adhesive events during an inflammatory response. To investigate whether dimerization of PSGL-1 is essential for functional interactions with P-selectin, a mutant form of PSGL-1 was generated in which the conserved membrane proximal cysteine was mutated to alanine (designated C320A). Western blotting under both denaturing and native conditions of the C320A PSGL-1 mutant isolated from stably transfected cells revealed expression of only a monomeric form of PSGL-1. In contrast to cells cotransfected with α1-3 fucosyltransferase-VII (FucT-VII) plus PSGL-1, K562 cells expressing FucT-VII plus C320A failed to bind COS cells transfected with P-selectin in a low shear adhesion assay, or to roll on CHO cells transfected with P-selectin under conditions of physiologic flow. In addition, C320A transfectants failed to bind chimeric P-selectin fusion proteins. Both PSGL-1 and C320A were uniformly distributed on the surface of transfected K562 cells. Thus, dimerization of PSGL-1 through the single, conserved, extracellular cysteine is essential for functional recognition of P-selectin.     

Poly-N-acetyllactosaminyl O-glycans attached to leukosialin. The presence of sialyl Le(x) structures in O-glycans.

Poly-N-acetyllactosamine extension has been found in O-glycans in addition to N-glycans and glycosphingolipids. Attempts were made in HL-60 and K562 cells to determine the amount of poly-N-acetyllactosaminyl O-glycans in the major sialoglycoprotein, leukosialin. Leukosialin was immunoprecipitated from [3H]glucosamine-labeled HL-60 and K562 cells. Glycopeptides were prepared by Pronase digestion, and O-glycan-containing glycopeptides were isolated by affinity chromatography using Jacalin-agarose. The glycopeptides bound to Jacalin-agarose and those unbound were treated with alkaline borohydride, and the released O-glycans were fractionated by Bio-Gel P-4 filtration. Sequential glycosidase digestion of the O-glycans, with or without pretreatment by fucosidase or neuraminidase, revealed the following conclusions. 1) Leukosialin from HL-60 cells contains about 1-2 poly-N-acetyllactosaminyl O-glycan chains/molecule. 2) About 50% of these poly-N-acetyllactosaminyl O-glycans contain sialyl Le(x) termini, NeuNAc alpha 2-->3Gal beta 1-->4 (Fuc alpha 1-->3)GlcNAc beta 1-->R. The amount of sialyl Le(x) structure in leukosialin is roughly equivalent to that on cell surfaces of HL-60 cells. 3) Leukosialin from K562 cells, on the other hand, contains no detectable amount of poly-N-acetyllactosaminyl O-glycans. 4) The presence of poly-N-acetyllactosamine in O-glycans is dependent on the core 2 beta 1,6-N-acetylglucosaminyl transferase. 5) Jacalin-agarose binds to sialylated small oligosaccharides such as NeuNAc alpha 2-->3Gal beta 1-->3(NeuNAc alpha 2-->6) GalNAc but not the hexasaccharide NeuNAc alpha 2-->3Gal beta 1-->3(NeuNAc alpha 2-->3Gal beta 1-->4GlcNAc beta 1-->6) GalNAc. These results indicate that the formation of polylactosaminyl O-glycans and sialyl Le(x) structure in O-glycans is dependent on the core 2 formation.     

Expression of human alpha-l-fucosyltransferase gene homologs in monkey kidney COS cells and modification of potential fucosyltransferase acceptor substrates by an endogenous glycosidase

Previous investigations on the monkey kidney COS cell line demonstrated the weak expression of fucosylated cell surface antigens and presence of endogenous fucosyltransferase activities in cell extracts. RT-PCR analyses have now revealed expression of five homologs of human fucosyltransferase genes, FUT1, FUT4, FUT5, FUT7, and FUT8, in COS cell mRNA. The enzyme in COS cell extracts acting on unsialylated Type 2 structures is closely similar in its properties to the alpha1,3- fucosyltransferase encoded by human FUT4 gene and does not resemble the product of the FUT5 gene. Although FUT1 is expressed in the COS cell mRNA, it has not been possible to demonstrate alpha1,2- fucosyltransferase activity in cell extracts but the presence of Le(y) and blood-group A antigenic determinants on the cell surface imply the formation of H-precursor structures at some stage. The most strongly expressed fucosyltransferase in the COS cells is the alpha1,6-enzyme transferring fucose to the innermost N -acetylglucosamine unit in N - glycan chains; this enzyme is similar in its properties to the product of the human FUT8 gene. The enzymes resembling the human FUT4 and FUT8 gene products both had pH optima of 7.0 and were resistant to 10 mM NEM. The incorporation of fucose into asialo-fetuin was optimal at 5.5 and was inhibited by 10 mM NEM. This result initially suggested the presence of a third fucosyltransferase expressed in the COS cells but we have now shown that triantennary N- glycans with terminal nonreducing galactose units, similar to those present in asialo-fetuin, are modified by a weak endogenous beta-galactosidase in the COS cell extracts and thereby rendered suitable substrates for the alpha1,6- fucosyltransferase.