Molecular cloning of a fourth member of a human alpha (1,3)fucosyltransferase gene family. Multiple homologous sequences that determine expression of the Lewis x, sialyl Lewis x, and difucosyl sialyl Lewis x epitopes.

We and others have previously described the isolation of three human alpha (1,3)fucosyltransferase genes which form the basis of a nascent glycosyltransferase gene family. We now report the molecular cloning and expression of a fourth homologous human alpha (1,3)fucosyltransferase gene. When transfected into mammalian cells, this fucosyltransferase gene is capable of directing expression of the Lewis x (Gal beta 1-->4[Fuc alpha 1-->3]GlcNAc), sialyl Lewis x (NeuNAc alpha 2-->3Gal beta 1-->4 [Fuc alpha 1-->3]GlcNAc), and difucosyl sialyl Lewis x (NeuNAc alpha 2-->3Gal beta 1-->4[Fuc alpha 1-->3]GlcNAc beta 1-->3 Gal beta 1-->4[Fuc alpha 1-->3]GlcNAc) epitopes. The enzyme shares 85% amino acid sequence identity with Fuc-TIII and 89% identity with Fuc-TV but differs substantially in its acceptor substrate requirements. Polymerase chain reaction analyses demonstrate that the gene is syntenic to Fuc-TIII and Fuc-TV on chromosome 19. Southern blot analyses of human genomic DNA demonstrate that these four alpha (1,3)fucosyltransferase genes account for all DNA sequences that cross-hybridize at low stringency with the Fuc-TIII catalytic domain. Using similar methods, a catalytic domain probe from Fuc-TIV identifies a new class of DNA fragments which do not cross-hybridize with the chromosome 19 fucosyltransferase probes. These results extend the molecular definition of a family of human alpha (1,3)fucosyltransferase genes and provide tools for examining fucosyltransferase gene expression.     

Reduction of the major xenoantigen on glycosphingolipids of swine endothelial cells by various glycosyltransferases

The effect of the various glycosyltransferases on glycosphingolipids was examined, using transfected swine endothelial cell (SEC) lines. The reactivity of parental SEC to normal human serum (NHS) and Griffonia simplicifolia IB(4) (GSIB4) lectin, which binds to the Gal alpha1-3 Gal beta 1-4 GlcNAc-R (alpha-galactosyl epitope), was reduced by approximately 20% by the treatment with D-PDMP (D-threo-1-phenyl-2-decan- oylamino-3-morpholino-1-propanol), suggesting that glycosphingolipids contained by SEC have a considerable amount of the alpha-galactosyl epitope. The overexpression of two different types of glycosyltransferase, N-acetylglucosaminyl transferase III (GnT-III), as well as alpha2, 6-sialyltransferase (ST6Gal I), alpha2,3-sialyltransferase (ST3Gal III), and alpha1,2-fucosyltransferase (alpha1,2FT), suppresses the total antigenicity of SEC significantly. However, the reduction in reactivities toward NHS and GSIB4 lectin in the case of GnT-III transfectants was milder than those in other transfectants. Western blot analysis indicated that the glycoproteins in all transfectants had diminished reactivity to NHS and GSIB4 lectin to approximately the same extent. Therefore, the neutral glycosphingolipids of these transfectants were separated by thin layer chromatography, followed by immunostaining with NHS and GSIB4 lectin. The levels of the alpha-galactosyl epitope in glycosphingolipids were not decreased in the GnT-III transfectants but were in the ST6Gal I, ST3Gal III, and alpha1,2FT transfectants. These data indicate that ST6Gal I, ST3Gal III, and alpha1,2FT reduced the alpha-galactosyl epitope in both glycoproteins and glycosphingolipids, while GnT-III reduced them only in glycoproteins.     

Differential binding of the lectins Griffonia simplicifolia I and Lycopersicon esculentum to microvascular endothelium: organ-specific localization and partial glycoprotein characterization

The lectins Griffonia simplicifolia I and Lycopersicon esculentum were used to assess the presence of endothelium-specific glycoproteins in the microvasculature of the rat myocardium, diaphragm and superficial cerebral cortex. Organs fixed by intravascular perfusion were processed to obtain semithin (0.5 micron) and thin (less than 0.1 micron) frozen sections that were reacted with biotinylated lectin followed by streptavidin conjugated to Texas Red, for semithin sections, or by streptavidin conjugated to 5-nm colloidal gold particles, for thin sections. Lycopersicon esculentum lectin exclusively labeled the endothelium of all small vessels in all three microvascular beds; it did not bind to components of either the parenchyma or the extracellular matrix. Griffonia simplicifolia I lectin exclusively labeled the endothelium of the entire microvasculature in the myocardium and diaphragm, but marked primarily pericytes in the cerebral microvasculature. It did not label any parenchymal or interstitial organ component. At the electron microscope level, the lectin Griffonia simplicifolia I labeling was associated with the plasmalemma proper and especially with plasmalemmal vesicles and their introits, and Lycopersicon esculentum lectin bound primarily to the luminal plasmalemma in the microvascular beds of the myocardium and diaphragm. In the cerebral cortex, labeling of the microvasculature was clearly different: Griffonia simplicifolia I bound primarily to pericytes and vascular smooth muscle cells whereas Lycopersicon esculentum labeled only the microvascular endothelium. Lysates prepared from the myocardium, diaphragm and cerebral cortex were processed through Griffonia simplicifolia I lectin affinity separation followed by sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) analysis of the fraction obtained. A number of putative endothelium-specific glycoproteins was detected and found to differ qualitatively and quantitatively from organ to organ. The most prominent polypeptide, approximately 97 kDa, was present in substantial amounts in the myocardium and diaphragm, but in considerably lower concentration in the cerebral cortex. The reverse applied for a approximately 55 kDa protein. The preferential distribution of the approximately 97 kDa protein parallels differences in Griffonia simplicifolia I lectin binding by fluorescence and electron microscopy on sections of the corresponding organs. The results provide further evidence for the existence of endothelial glycoproteins specific for different microvascular beds and possibly connected with local functional differentiations.     

Differentiative changes in fucosyltransferase activity in newborn rat epidermal cells.

An enzymatic activity catalyzing the transfer of L-fucose from GDP-L-fucose to a glycoprotein that is associated with the surfaces of the basal cells has been found in the membranous fraction of the cutaneous epidermis from the newborn rat. This fucosyltransferase which is located in the differentiated cells alters the acceptor glycoprotein's lectin-binding specificity from the Isolectin I-B4 of Griffonia simplicifolia (GS I-B4) to the Agglutinin I of Ulex europeus (UEA) and could be responsible for the same change in lectin-binding specificity that occurs as the epidermal basal cell differentiates. Another membraneous fucosyltransferase that can use asialofetuin--but not the GS I-B4-binding glycoprotein--as an acceptor, is also present in the membraneous fraction.     

alpha-D-galactose-bearing glycoproteins on the surface of stimulated murine peritoneal macrophages. Biochemical and immunochemical characterization of purified glycoproteins.

Two glycoproteins were isolated from lysates of thioglycollate-stimulated, murine peritoneal macrophages by affinity chromatography on immobilized Griffonia simplicifolia I lectin and by preparative SDS/PAGE. The glycoproteins were readily labeled on the surface of intact macrophages with 3H and 125I. The labeled glycoproteins migrated as broad bands of molecular mass 92-109 kDa and 115-125 kDa. The mobility of the glycoproteins decreased only slightly after reduction with dithiothreitol, indicating the absence of intersubunit disulfide bridges. The 92-kDa and 115-kDa glycoproteins had pI 5.2-5.4 and pI less than or equal to 4, respectively. Digestion of both glycoproteins with alpha-galactosidase released 23% of their 3H content and abolished their ability to bind to the G. simplicifolia I lectin, showing that they contain terminal alpha-D-galactosyl groups. After reduction with 2-mercaptoethanol, each glycoprotein fraction was sensitive to N-glycanase; the 115-kDa glycoproteins produced a smear with the front at approximately 67 kDa, whereas the 92-kDa glycoprotein gave two bands of 61 kDa and 75 kDa. Unreduced glycoproteins were insensitive to N-glycanase, suggesting the presence of intramolecular disulfide bonds. Although each glycoprotein fraction was sensitive to endoglycosidase H, this enzyme produced only slight changes in molecular mass when compared with N-glycanase. From these results as well as from the specificity of the enzymes involved, it is concluded that each glycoprotein fraction contains complex-type oligosaccharides and a small amount of high-mannose and/or hybrid-type oligosaccharides. While each glycoprotein fraction was bound to Datura stramonium lectin, they failed to react with anti-[i-(Den)] serum and their digestion with endo-beta-galactosidase did not cause a band shift in SDS/PAGE. Taken together, these results suggest the presence of N-acetyllactosamine units which are not arrayed in linear form but occur as single units, bound either to C2 and C6, or to C2 and C4, or both, of outer mannosyl residues on complex-type oligosaccharides. The glycoprotein(s) fraction precipitated with anti-[I (Step)] serum, suggesting the presence of branched lactosaminoglycans. Digestion of both glycoprotein fractions with a mixture of sialidase and O-glycanase did not alter their mobility in SDS/PAGE, suggesting a lack or low content of O-linked trisaccharides and tetrasaccharides. Each glycoprotein fraction was bound specifically to Sambucus nigra and Maackia amurensis immobilized lectins, indicating the presence of sialic acid linked alpha 2,6 to subterminal D-galactose or N-acetylgalactosamine residues, and alpha 2,3 to N-acetyllactosamine residues, respectively.     

Molecular cloning and characterization of an alpha1,3 fucosyltransferase, CEFT-1, from Caenorhabditis elegans

We report on the identification, molecular cloning, and characterization of an alpha1,3 fucosyltransferase (alpha1,3FT) expressed by the nematode, Caenorhabditis elegans . Although C. elegans glycoconjugates do not express the Lewis x antigen Galbeta1-- >4[Fucalpha1-->3]GlcNAcbeta-->R, detergent extracts of adult C.elegans contain an alpha1,3FT that can fucosylate both nonsialylated and sialylated acceptor glycans to generate the Lexand sialyl Lexantigens, as well as the lacdiNAc-containing acceptor GalNAcbeta1-->4GlcNAcbeta1-- >R to generate GalNAcbeta1-->4 [Fucalpha1-->3]GlcNAcbeta1-->R. A search of the C.elegans genome database revealed the existence of a gene with 20-23% overall identity to all five cloned human alpha1,3FTs. The putative cDNA for the C.elegans alpha1,3FT (CEFT-1) was amplified by PCR from a cDNA lambdaZAP library, cloned, and sequenced. COS7 cells transiently transfected with cDNA encoding CEFT-1 express the Lex, but not sLexantigen. The CEFT-1 in the transfected cell extracts can synthesize Lex, but not sialyl Lex, using exogenous acceptors. A second fucosyltransferase activity was detected in extracts of C. elegans that transfers Fuc in alpha1,2 linkage to Gal specifically on type-1 chains. The discovery of alpha-fucosyltransferases in C. elegans opens the possibility of using this well-characterized nematode as a model system for studying the role of fucosylated glycans in the development and survival of C.elegans and possibly other helminths.      

Use of flow cytometry to monitor infection and recombinant human alpha-1,3/4 fucosyltransferase production in baculovirus infected Sf9 cell cultures

This paper describes the setup and the use of a flow cytometric method for monitoring Sf9 insect cell infection by a recombinant baculovirus expressing the human alpha1,3/4 fucosyltransferase Fuc-TIII. Using side scattered light coupled to green fluorescence detection after immunolabeling of the recombinant protein, this method made it possible to monitor baculovirus infection of Sf9 cells grown in batch cultures and infected at different cell densities and multiplicities of infection. The method was able to precisely assess the extent of infection of the insect cells from 60 h postinfection. In asynchronously infected Sf9 cell cultures, the two-step infection process (primary and secondary infection) was well-characterized using this technique. Finally, a reduced sensitivity to baculovirus infection was observed for cells infected at the end of the growth phase compared to the cells infected during exponential growth phase.     

The binding characteristics and utilization of Aleuria aurantia,Lens culinaris and few other lectins in the elucidation of fucosyltransferase activities resembling cloned FT VI and apparently unique to colon cancer cells

Human colon carcinoma cell fucosyltransferase (FT) in contrast to the FTs of several human cancer cell lines, utilized GlcNAcbeta1,4GlcNAcbeta-O-Bn as an acceptor, the product being resistant to alpha1,6-L-Fucosidase and its formation being completely inhibited by LacNAc Type 2 acceptors. Further, this enzyme was twofold active towards the asialo agalacto glycopeptide as compared to the parent asialoglycopeptide. Only 60% of the GlcNAc moieties were released from [14C]fucosylated asialo agalacto triantennary glycopeptide by jack bean beta-N-acetylhexosaminidase. These alpha1,3-L-fucosylating activities on multiterminal GlcNAc residues and chitobiose were further examined by characterizing the products arising from fetuin triantennary and bovine IgG diantennary glycopeptides and their exoglycosidase-modified derivatives using lectin affinity chromatography. Utilization of [14C]fucosylated glycopeptides with cloned FTs indicated that Lens culinaris lectin and Aleuria aurantia lectin (AAL) required, respectively, the diantennary backbone and the chitobiose core alpha1,6-fucosyl residue for binding. The outer core alpha1,3- but not the alpha-1,2-fucosyl residues decreased the binding affinity of AAL. The AAL-binding fraction from [14C]fucosylated asialo fetuin, using colon carcinoma cell extract, contained 60% Endo F/PNGaseF resistant chains. Similarly AAL-binding species from [14C]fucosylated TFA-treated bovine IgG using colon carcinoma cell extract showed significant resistance to endo F/PNGaseF. However, no such resistance was found with the corresponding AAL non- and weak-binding species. Thus colon carcinoma cells have the capacity to fucosylate the chitobiose core in glycoproteins, and this alpha1,3-L-fucosylation is apparently responsible for the AAL binding of glycoproteins. A cloned FT VI was found to be very similar to this enzyme in acceptor substrate specificities. The colon cancer cell FT thus exhibits four catalytic roles, i.e., alpha1,3-L-fucosylation of: (a) Galbeta1,4GlcNAcbeta-; (b) multiterminal GlcNAc units in complex type chain; (c) the inner core chitobiose of glycopeptides and glycoproteins; and (d) the nonreducing terminal chiotobiose unit.     

Lectins as probes to Pneumocystis carinii surface glycocomplexes

The binding characteristics of a panel of commercially available FITC-conjugated lectins to Pneumocystis carinii (Pc) were assessed by fluorescence microscopy and flow cytometry. Rat Pc obtained from infected lung homogenates were incubated with FITC-conjugated lectins in a series of concentrations, counterstained with propidium iodide, and analyzed for percent fluorescence and fluorescence intensity. All organisms bound concanavalin A and Wisteria floribunda agglutinin, 2 representatives of the glucose/mannose-binding group. From the lectin group specific for N-acetylglucosamine, Pc reacted more strongly with wheat germ agglutinin than with Solanum tuberosum agglutinin or Griffonia simplicifolia II lectin. Pneumocystis treated with lectins specific for N-acetyl-D-galactosamine and galactose exhibited much variation; the cells reacted moderately well to soybean agglutinin and less to Bauhinia purpurea, Maclura pomifera and Dolichos biflorus agglutinins and Griffonia simplicifolia I lectin. Arachis hypogaea agglutinin, Viscum album agglutinin and Griffonia simplicifolia I-beta 4 lectin had not effect. The organisms reacted weakly with Ulex europeus I agglutinin which is specific for fucose and did not react with Limax flavus lectin, which is specific for sialic acid. Competitive inhibition studies using relevant carbohydrates were performed to indicate that the positive reactions were specific. These studies should help to elucidate the mechanisms of attachment and pathogenesis of this organism.     

Trafficking and localization studies of recombinant alpha1, 3- fucosyltransferase VI stably expressed in CHO cells

Peripheral alpha1,3-fucosylation of glycans occurs by the action of either one of five different alpha1,3-fucosyltransferases (Fuc-Ts) cloned to date. Fuc-TVI is one of the alpha1,3-fucosyltransferases which is capable to synthesize selectin ligands. The major alpha1, 3- fucosyltransferase activity in human plasma is encoded by the gene for fucosyltransferase VI, which presumably originates from liver cells. While the sequence, chromosomal localization, and kinetic properties of Fuc-TVI are known, immunocytochemical localization and trafficking studies have been impossible because of the lack of specific antibodies. Here we report on the development and characterization of a peptide-specific polyclonal antiserum monospecific to Fuc-TVI and an antiserum to purified soluble recombinant Fuc-TVI crossreactive with Fuc-TIII and Fuc-TV. Both antisera were applied for immunodetection in stably transfected CHO cells expressing the full-length form of this enzyme (CHO clone 61/11). Fuc-TVI was found to be a resident protein of the Golgi apparatus. In addition, more than 30% of cell-associated and released enzyme activity was found in the medium. Maturation and release of Fuc-TVI was analyzed in metabolically labeled CHO 61/11 cells followed by immunoprecipitation. Fuc-TVI occurred in two forms of 47 kDa and 43 kDa bands, while the secreted form was detected as a 43 kDa. These two different intracellular forms arose by posttranslational modification, as shown by pulse-chase experiments. Fuc-TVI was released to the supernatant by proteolytic cleavage as a partially endo-H resistant glycoform.      

A bifunctional diglycosyltransferase forms the Fucalpha1,2Galbeta1,3-disaccharide on Skp1 in the cytoplasm of dictyostelium

Skp1 is a subunit of the Skp1 cullin-1 F-box protein (SCF) family of E3 ubiquitin ligases and of other regulatory complexes in the cytoplasm and nucleus. In Dictyostelium, Skp1 is modified by a pentasaccharide with the type I blood group H antigen (Fucalpha1,2Galbeta1,3GlcNAc-) at its core. Addition of the Fuc is catalyzed by FT85, a 768-amino acid protein whose fucosyltransferase activity maps to the C-terminal half of the protein. A strain whose FT85 gene is interrupted by a genetic insertion produces a truncated, GlcNAc-terminated glycan on Skp1, suggesting that FT85 may also have beta-galactosyltransferase activity. In support of this model, highly purified native and recombinant FT85 are each able to galactosylate Skp1 from FT85 mutant cells. Site-directed mutagenesis of predicted key amino acids in the N-terminal region of FT85 abolishes Skp1 beta-galactosyltransferase activity with minimal effects on the fucosyltransferase. In addition, a recombinant form of the N-terminal region exhibits beta-galactosyltransferase but not fucosyltransferase activity. Kinetic analysis of FT85 suggests that its two glycosyltransferase activities normally modify Skp1 processively but can have partial function individually. In conclusion, FT85 is a bifunctional diglycosyltransferase that appears to be designed to efficiently extend the Skp1 glycan in vivo.     

A non-Golgi alpha 1,2-fucosyltransferase that modifies Skp1 in the cytoplasm of Dictyostelium.

Skp1 is a subunit of the SCF-E3 ubiquitin ligase that targets cell cycle and other regulatory factors for degradation. In Dictyostelium, Skp1 is modified by a pentasaccharide containing the type 1 blood group H trisaccharide at its core. To address how the third sugar, fucose alpha1,2-linked to galactose, is attached, a proteomics strategy was applied to determine the primary structure of FT85, previously shown to copurify with the GDP-Fuc:Skp1 alpha 1,2-fucosyltransferase. Tryptic-generated peptides of FT85 were sequenced de novo using Q-TOF tandem mass spectrometry. Degenerate primers were used to amplify FT85 genomic DNA, which was further extended by a novel linker polymerase chain reaction method to yield an intronless open reading frame of 768 amino acids. Disruption of the FT85 gene by homologous recombination resulted in viable cells, which had altered light scattering properties as revealed by flow cytometry. FT85 was necessary and sufficient for Skp1 fucosylation, based on biochemical analysis of FT85 mutant cells and Escherichia coli that express FT85 recombinantly. FT85 lacks sequence motifs that characterize all other known alpha 1,2-fucosyltransferases and lacks the signal-anchor sequence that targets them to the secretory pathway. The C-terminal region of FT85 harbors motifs found in inverting Family 2 glycosyltransferase domains, and its expression in FT85 mutant cells restores fucosyltransferase activity toward a simple disaccharide substrate. Whereas most prokaryote and eukaryote Family 2 glycosyltransferases are membrane-bound and oriented toward the cytoplasm where they glycosylate lipid-linked or polysaccharide precursors prior to membrane translocation, the soluble, eukaryotic Skp1-fucosyltransferase modifies a protein that resides in the cytoplasm and nucleus.     

Biosynthesis of the carbohydrate antigenic determinants, Globo H, blood group H, and Lewis b: a role for prostate cancer cell alpha1,2-L-fucosyltransferase

Prostate carcinoma LNCaP cells were unique among several human cancer cell lines which include two other prostate cancer cell lines, PC-3 and DU-145, in expressing alpha1,2-L-fucosyltransferase (FT) as an exclusive FT activity. Affinity gel-GDP and Sephacryl S100 HR columns were used for a partial purification of this enzyme from 3.9 x 10(9) LNCaP cells (approximately 200-fold; 40% yield). The K(m) value (2.7 mM) for the LacNAc type 2 acceptor was quite similar to the one reported for the cloned blood group H gene-specified alpha1,2-FT [Chandrasekaran et al. (1996) Biochemistry 35, 8914-8924]. N-Ethylmaleimide was a potent inhibitor (K(i ) 12.5 microM). The enzyme showed four-fold acceptor preference for the LacNAc type 2 unit in comparison to the T-hapten in mucin core 2 structure. Its main features were similar to those of the cloned enzyme: (1) C-6 sulfation of terminal Gal in the LacNAc unit increased the acceptor efficiency, whereas C-6 sialylation abolished acceptor ability; (2) C-6 sulfation of GlcNAc in LacNAc type 2 decreased by 80% the acceptor ability, whereas LacNAc type 1 was unaffected; (3) Lewis x did not serve as an acceptor; (4) the C-4 hydroxyl rather than the C-6 hydroxyl group of the GlcNAc moiety in LacNAc type1 was essential for activity; and (5) the acrylamide copolymer of Galbeta1,3GlcNAcbeta-O-Al was the best acceptor among the acrylamide copolymers. Additionally, highly significant biological features of alpha1,2FT were identified in the present study. The synthesis of Globo H and Lewis b determinants became evident from the fact that Galbeta1,3GalNAcbeta1,3Galalpha-O-Me and Galbeta1,3(Fucalpha1,4)Glc-NAcbeta1,3Galbeta-O-Me served as high-affinity acceptors for this enzyme. Further, D-Fucbeta1,3Gal-NAcbeta1,3Galalpha-O-Me was a very efficient acceptor, indicating that the C-6 hydroxyl group of the terminal Gal moiety in Globo H is not essential for the enzyme activity. Thus, the present study was able to demonstrate three different catalytic roles of LNCaP alpha1,2-FT, namely, the expressions of blood group H, Lewis b from Lewis a, and Globo H.     

Lectin binding studies on murine peritoneal cells: physicochemical characterization of the binding of lectins from Datura stramonium, Evonymus europaea, and Griffonia simplicifolia to murine peritoneal cells

Purified 125I-labeled lectins from Datura stramonium, Evonymus europaea, and Griffonia simplicifolia (I-B4 isolectin) were used to analyze changes in the expression of carbohydrates on the surface of resident (PC) and thioglycollate-stimulated murine (C57B/6J) peritoneal exudate cells (PEC). The lectins from D. stramonium, E. europaea, and G. simplicifolia I-B4 bind specifically to PEC with relatively high affinity (Kd = 5.65 +/- 1.08 X 10(-7) M, 1.08 +/- 0.12 X 10(-8) M, and 1.33 +/- 0.15 X 10(-7) M, respectively). Assuming a single lectin molecule binds to each cell surface saccharide, the number of receptor sites per cell ranged for different cell samples from 22.3 to 50.0 X 10(6), from 3.8 to 4.8 X 10(6), and from 2.0 to 16.8 X 10(6) for D. stramonium, E. europaea, and G. simplicifolia I-B4 lectins, respectively. There were approximately 3- to 7-fold, 16- to 20-fold, and 2- to 20-fold increases in binding capacity for D. stramonium, E. europaea and G. simplicifolia I-B4, respectively, compared to the binding to resident, peritoneal cells. Scatchard plots of the binding of all three lectins to PEC were linear, suggesting that the receptor sites for these lectins are homogeneous and noninteracting. The binding capacity of these lectins to PEC was unchanged after trypsin digestion of cells. The expression of carbohydrates on the surface of PEC was also monitored by an agglutination assay. PEC were agglutinated by all three lectins whereas PC either were not agglutinated or were agglutinated only at high lectin concentrations. On the basis of our knowledge of the carbohydrate binding specificity of the D. stramonium and G. simplicifolia I-B4 lectins, we postulate that, parallel with thioglycolate stimulation, there is an increase in the number of N-acetyllactosamine residues and terminal alpha-D-galactosyl end groups. The blood group B, and H type 1 determinants--DGa1 alpha 1,3[LFuc alpha 1,2]DGa1 beta 1,3(or 4)DGlcNAc and LFuc alpha 1,2DGa1 beta 1,3DG1cNAc, respectively, as well as DGa1 alpha 1,3DGa1 beta 1,3(or 4)DGlcNAc--may be considered to be possible receptors for the E. europaea lectin. These glycoconjugates, present on the surface of peritoneal exudate cells, provide new chemical markers for studying the differentiation of resident peritoneal cells.     

Activity of fucosyltransferases and altered glycosylation in cystic fibrosis airway epithelial cells

Cystic fibrosis (CF) glycoconjugates have a glycosylation phenotype of increased fucosylation and/or decreased sialylation when compared with non-CF. A major increase in fucosyl residues linked alpha 1,3 to antennary GlcNAc was observed when surface membrane glycoproteins of CF airway epithelial cells were compared to those of non-CF airway cells. Importantly, the increase in the fucosyl residues was reversed with transfection of CF cells with wild type CFTR cDNA under conditions which brought about a functional correction of the Cl(-) channel defect in the CF cells. In contrast, examination of fucosyl residues in alpha 1,2 linkage by a specific alpha 1,2 fucosidase showed that cell surface glycoproteins of the non-CF cells had a higher percentage of fucose in alpha 1,2 linkage than the CF cells. Airway epithelial cells in primary culture had a similar reciprocal relationship of alpha 1,2- and alpha 1,3-fucosylation when CF and non-CF surface membrane glycoconjugates were compared. In striking contrast, the enzyme activity and the mRNA of alpha 1,2 fucosyltransferase did not reflect the difference in glycoconjugates observed between the CF and non-CF cells. We hypothesize that mutated CFTR may cause faulty compartmentalization in the Golgi so that the nascent glycoproteins encounter alpha 1,3FucT before either the sialyl- or alpha 1,2 fucosyltransferases. In subsequent compartments, little or no terminal glycosylation can take place since the sialyl- or alpha 1,2 fucosyltransferases are unable to utilize a substrate, which is fucosylated in alpha 1,3 position on antennary GlcNAc. This hypothesis, if proven correct, could account for the CF glycophenotype.     

Isolation of a novel human alpha (1,3)fucosyltransferase gene and molecular comparison to the human Lewis blood group alpha (1,3/1,4)fucosyltransferase gene. Syntenic, homologous, nonallelic genes encoding enzymes with distinct acceptor substrate specificities.

Biochemical and genetic evidence indicates that the human genome may encode four or more distinct GDP-fucose:beta-D-N-acetylglucosaminide 3-alpha-L-fucosyltransferase (alpha(1,3)fucosyltransferase) activities. Genes encoding two of these activities have been previously isolated. These correspond to an alpha(1,3/1,4)fucosyltransferase thought to represent the human Lewis blood group locus and an alpha(1,3)fucosyltransferase expressed in the myeloid lineage. We report here the molecular cloning and expression of a third human alpha(1,3)fucosyltransferase gene, homologous to but distinct from the two previously reported human fucosyltransferase genes. When expressed in transfected mammalian cells, this gene determines expression of a fucosyltransferase capable of using N-acetyllactosamine to form the Lewis x epitope, and alpha(2,3)sialyl-N-acetyllactosamine to construct the sialyl Lewis x moiety. This enzyme shares 91% amino acid sequence identity with the human Lewis blood group alpha(1,3/1,4)fucosyltransferase, yet exhibits only trace amounts of alpha(1,4)fucosyltransferase activity. Polymerase chain reaction analyses were used to demonstrate that the gene is syntenic to the Lewis locus on chromosome 19. These analyses also excluded the possibility that this DNA segment represents an allele of the Lewis locus that encodes alpha(1,3)fucosyltransferase but not alpha(1,4)fucosyltransferase activity. These results are consistent with the hypothesis that this gene encodes the human "plasma type" alpha(1,3)fucosyltransferase, and suggest a molecular basis for a family of human alpha(1,3)fucosyltransferase genes.     

Comparison of the three rat GDP-L-fucose:beta-D-galactoside 2-alpha-L-fucosyltransferases FTA, FTB and FTC.

The complete coding sequences of three rat alpha1,2fucosyltransferase genes were obtained. Sequence analysis revealed that these genes, called FTA, FTB and FTC, were homologous to human FUT1, FUT2 and Sec1, respectively. A distance analysis between all alpha1,2fucosyltransferase sequences available showed that the two domains of the catalytic region evolved differently with little divergence between the FUT2 and Sec1 N-terminal domains, quite distant from that of FUT1. At variance, FUT1 and FUT2 C-terminal domains were less distant while a high evolutionary rate was noted for Sec1 C-terminal domain. Whereas FTA and FTB encode typical glycosyltransferases, FTC lacks the homologous start codon and encodes a protein devoid of intracellular and transmembrane domains. It is located on rat chromosome 1q34. Transfection experiments revealed that unlike FTA and FTB, FTC does not generate enzyme activity. Analysis by flow cytometry showed that H type 2 epitopes were synthesized in Chinese hamster ovary cells transfected by both FTA and FTB cDNA, but only FTB transfectants possessed H type 3 determinants. In REG rat carcinoma cells, both FTA and FTB allowed synthesis of H type 2 and H type 3 at the cell surface. Western blots showed that, in both cell types, FTA was able to synthesize H type 2 epitopes on a larger set of glycoproteins than FTB. Analysis of the kinetic parameters obtained using small oligosaccharides revealed only a slight preference of FTA for type 2 over other types of acceptor substrates, whereas FTB was barely able to fucosylate this substrate.     

Effect of RNA interference on Gal alpha 1,3 Gal expression in PIEC cells

Xenotransplantation from pig to human being is viewed as a potential solution for the acute organ shortage. However, consequent xenorejection induced by Gal alpha 1,3 Gal (Gal, Gal antigen) prevents xenotransplantation from clinical application. Thus, the most attracting attempt to prevent xenorejection is the elimination of Gal. Our study suggested that compared with the human alpha 1,2 fucosyltransferase (FT) gene and porcine antisense alpha 1,3 galactosyltransferase gene, sequence-specific siRNA targeting Gal were capable of suppressing Gal expression markedly, and therefore, significantly inhibiting xenoreactivity and the complement activation with human serum in PIEC cells. We also demonstrated the concordant inhibitory effect of siRNA and human FT gene on Gal and corresponding functions, which implied a practical significance of combined transgenic strategy. The successful application of vector-based dsRNA-GT may extend the list of available modalities in the abrogation of xenorejection in xenotransplantation.     

Biosynthesis of bi-, tri-, and tetraantennary oligosaccharides containing alpha-D-galactosyl residues at their nonreducing termini. Branch specificity of the Ehrlich tumor cell alpha(1,3)-galactosyltransferase

The ability of Ehrlich tumor cell alpha(1,3)-galactosyltransferase to catalyze the incorporation of alpha-D-Gal residues into a specific branch of bi-, tri-, and tetraantennary oligosaccharides has been investigated by acetolysis followed by gel filtration of the fragments on Bio-Gel P-4. Taking advantage of the carbohydrate specificity of the Griffonia simplicifolia I-B4 isolectin, the mono-[14C]alpha-D-Gal derivatives were isolated by affinity chromatography. Analysis of the acetolysis fragments generated by cleavage of the multiantennary substrates indicates that the Ehrlich cell alpha(1,3)-galactosyltransferase acts preferentially on the alpha-D-Man(1,6) arm. This branch is preferred 2.5 times in bi-, 5.6-8.5 times in tri-, and 12.7 times in tetraantennary structures over the alpha-D-Man(1,3) arm. Within the alpha-D-Man(1,6) branch, in turn, there is a 1.3-1.9-fold consistently higher frequency of galactosylation of the beta-D-GlcNAc(1,2) as compared to the beta-D-GlcNAc(1,6) antenna.