Biochemical characterization of plasma-derived tissue factor pathway inhibitor: post-translational modification of free, full-length form with particular reference to the sugar chain.

BACKGROUND: Tissue factor pathway inhibitor (TFPI) is a physiological protease inhibitor that inhibits the initial reactions of the extrinsic blood coagulation pathway. Most TFPI in human plasma is associated with lipoproteins; however, the most functionally active form is thought to be the free, full-length form (f-pTFPI). Cell culture derived TFPI and recombinant TFPI (rTFPI) exhibit variations in their respective anticoagulant activity, which may be caused by post-translational modifications, such as the frequent differences in sugar chain structures among recombinant proteins. Sugar chain structures in rTFPI expressed in Chinese hamster ovary (CHO) cells have been reported previously, but those of plasma TFPI have not been. OBJECTIVES: To purify f-pTFPI and analyze the sugar chain structures. RESULTS AND CONCLUSION: f-pTFPI was purified to homogeneity from blood plasma using a combination of anion-exchange, heparin affinity, immunoaffinity, and reversed-phase chromatographies, resulting in a yield of 76%. f-pTFPI showed a partially phosphorylated glycoprotein comprising a total of 276 amino acids by peptide mapping. The sugar chain structures were analyzed by two-dimensional sugar mapping combined with exoglycosidase digestion of the pyridylamino sugar chains and the following results were obtained. (Sialyl) Galbeta1-3GalNAc was linked to Thr(175), partially to Thr(14) and Ser(174); sialyl complex-type sugar chains to Asn(117) and Asn(167), whereas Asn(228) was not glycosylated. Neuraminidase-resistant acidic sugar chains including sulfated sugar chains were not observed significantly. The protease inhibitory activities of f-pTFPI towards activated factor (F) X and tissue factor-activated FVII complex were identical to those of full-length rTFPI expressed in CHO cells.

Molecular cloning and expression of Caenorhabditis elegans ERp57-homologue with transglutaminase activity.

Formation of cross-linking between proteins via a gamma-glutamyl-epsilon-lysine residue is an important process in many biological phenomena including apoptosis. Formation of this linkage is catalyzed by the enzyme transglutaminase, which is widely distributed from bacteria to the animal kingdom. The simple multi-cellular organism Caenorhabditis elegans also possesses transglutaminase activity associated with apoptosis [Madi, A. et al. (1998) Eur. J. Biochem. 253, 583-590], but no gene with significant homology to vertebrate or bacterial transglutaminases has been found in the C. elegans genome sequence database. On the other hand, protein disulfide isomerases were recently recognized as a new family of transglutaminases [Chandrashekar, R. et al. (1998) Proc. Natl. Acad. Sci. USA 95, 531-536]. To identify the molecule with transglutaminase activity in C. elegans, we isolated from C. elegans a gene homologous to ERp57, which encodes a protein disulfide isomerase, expressed it in recombinant form, and characterized the transglutaminase and protein disulfide isomerase activities of the resultant protein. The C. elegans ERp57 protein had both enzyme activities, and the transglutaminase activity had similar characteristics to the activity in lysate of the whole worm. These results suggested that the ERp57 homologue was one of the substances with transglutaminase activity in C. elegans.

Structural analysis of the sugar chains of human urinary thrombomodulin.

The sugar chains of human urinary thrombomodulin were studied. N- and O-linked sugar chains were simultaneously liberated by hydrazinolysis followed by N-acetylation and were tagged with 2-aminopyridine. Then the structures of the N- and O-linked pyridylamino (PA-) sugar chains were analyzed by two-dimensional sugar mapping combined with exoglycosidase digestion. The major N-linked sugar chains of human urinary thrombomodulin were found to be monosialo- and disialofucosylbiantennary chains, while the major O-linked sugar chain was +/-Siaalpha2-3Galbeta1-3(+/-Siaalpha2-6)GalNAc. Thrombomodulin also contained the reported structure SO4-3GlcAbeta1-3Galbeta1-3(+/-Siaalpha2-6)Galbeta1-4Xyl [H. Wakabayashi, S. Natsuka, T. Mega, N. Otsuki, M. Isaji, M. Naotsuka, S. Koyama, T. Kanamori, K. Sakai, and S. Hase (1999) J. Biol. Chem. 274, 5436-5442]. In addition to these sugar chains, a single Glc was linked to Ser 287.

High molecular weight transglutaminase inhibitor produced by a microorganism (Streptomyces lavendulae Y-200).

Transglutaminases catalyze the cross-linking and amine incorporation of proteins, and are implicated in various biological phenomena such as blood clotting, wound healing, apoptosis, and cell differentiation. Streptomyces lavendulae Y-200, isolated from soil, produced a substance that inhibited transglutaminases. The inhibitory substance was purified from the cultured medium by procedures of acid precipitation, deoxyribonuclease treatment, and gel filtration chromatography. The partially purified sample was dark brown. The inhibitory activity was stable under acidic, alkaline, and high temperature conditions, and resistant to the treatment with proteinases such as trypsin and Pronase. The molecular weight of the inhibitory substance was estimated to be between 10(4) and 10(5) from its permeability through ultrafilter membranes. The acid hydrolysate of the inhibitory substance contained amino acids and sugars. The inhibitory substance inhibited both calcium-dependent and calcium-independent transglutaminases in a competitive manner with a glutamine substrate. The extent of inhibition caused by the calcium-dependent transglutaminase increased with increasing calcium concentration. The results obtained here may help identify a novel regulatory substance of transglutaminase in biological systems.

beta1-4Galactosyltransferase activity of mouse brain as revealed by analysis of brain-specific complex-type N-linked sugar chains.

We previously reported two brain-specific agalactobiantennary N-linked sugar chains with bisecting GlcNAc and alpha1-6Fuc residues, (GlcNAcbeta1-2)(0)(or)(1)Manalpha1-3(GlcNAcbeta1-2M analpha1-6)(GlcNA cbeta1-4)Manbeta1-4GlcNAcbeta1-4(Fucalpha1-6)Glc NAc [Shimizu, H., Ochiai, K., Ikenaka, K., Mikoshiba, K., and Hase, S. (1993) J. Biochem. 114, 334-338]. Here, the reason for the absence of Gal on the sugar chains was analyzed through the detection of other complex type sugar chains. Analysis of N-linked sugar chains revealed the absence of Sia-Gal and Gal on the GlcNAc residues of brain-specific agalactobiantennary N-linked sugar chains. We therefore investigated the substrate specificity of galactosyltransferase activities in brain using pyridylamino derivatives of agalactobiantennary sugar chains with structural variations in the bisecting GlcNAc and alpha1-6Fuc residues as acceptor substrates. While the beta1-4galactosyltransferases in liver and kidney could utilize all four oligosaccharides as substrates, the beta1-4galactosyltransferase(s) in brain could not utilize the agalactobiantennary sugar chain with both bisecting GlcNAc and Fuc residues, but could utilize the other three acceptors. Similar results were obtained using glycopeptides with agalactobiantennary sugar chains and bisecting GlcNAc and alpha1-6Fuc residues as substrates. The beta1-4galactosyltransferase activity of adult mouse brain thus appears to be responsible for producing the brain-specific sugar chains and to be different from beta1-4galactosyltransferase-I. The agalactobiantennary sugar chain with bisecting GlcNAc and alpha1-6Fuc residues acts as an inhibitor against "brain type" beta1-4galactosyltransferase with a K(i) value of 0.29 mM.

A pyridylamination method aimed at automatic oligosaccharide analysis of N-linked sugar chains.

The procedure for preparation of pyridylaminated sugar chains from glycoproteins was improved with a view to its eventual automation. Following on the coupling reaction improvement already reported [N. Kuraya and S. Hase (1992) J. Biochem. 112, 122-126], two further aspects were improved in this study. Instead of sodium bicarbonate-acetic anhydride, volatile reagents were adopted for the re-N-acetylation of hexosamine residues after hydrazinolysis to give rapid removal of excess reagents. Subsequent to the pyridylamination reaction, excess reagents were removed by cation-exchange to isolate the pyridylaminated oligosaccharides in place of gel filtration. These alterations rendered a one-pot reaction possible and resulted in a large reduction in the amount of time needed compared with other methods so far reported. The procedure was successfully applied to the detection of sugar chains from Taka-amylase A and human erythrocyte membranes.

Presence of asparagine-linked N-acetylglucosamine and chitobiose in Pyrus pyrifolia S-RNases associated with gametophytic self-incompatibility.

S-RNases encoded by the S-locus of rosaceous and solanaceous plants discriminate between the S-alleles of pollen in gametophytic self-incompatibility reactions, but it is not clear how. We report the structures of N-glycans attached to each of the N-glycosylation sites of seven S-RNases in Pyrus pyrifolia of the Rosaceae. The structures were identified by chromatographic analysis of pyridylaminated sugar chains prepared from S4-RNase and by liquid chromatography/electrospray ionization-mass spectrometric analysis of the protease digests of reduced and S-carboxymethylated S-RNases. S4-RNase carries various types of sugar chains, including plant-specific ones with beta1-->2-linked xylose and alpha1-->3-linked fucose residues. More than 70% of the total N-glycans of S4-RNase are, however, an N-acetylglucosamine or a chitobiose (GlcNAcbeta1-->4GlcNAc), which has not been found naturally. The N-acetylglucosamine and chitobiose are mainly present at the N-glycosylation sites within the putative recognition sites of the S-RNase, suggesting that these sugar chains may interact with pollen S-product(s).

Molecular cloning and characterization of two zebrafish alpha(1,3)fucosyltransferase genes developmentally regulated in embryogenesis.

Some alpha(1,3)fucosylated oligosaccharides serve as counter receptors to lectin-like adhesion proteins or are expressed with temporal precision during embryogenesis, and alpha(1, 3)fucosyltransferase is a key enzyme in the production of these oligosaccharides. Two alpha(1,3)-fucosyltransferase genes, designated zFT1 and zFT2, were cloned from zebrafish. Sequence comparisons with other genes indicated that zFT1 and zFT2 share about 30% amino acid sequence identity with human alpha(1, 3)fucosyltransferases. Although the alpha(1,3)fucosyltransferases cloned so far can be classified into three types-myeloid, Lewis, and leukocyte-by virtue of their amino acid sequences, phylogenetic analysis indicated that neither zFT1 nor zFT2 belongs to any of these categories. The expression of zFT1 or zFT2 in mammalian cells induces alpha(1,3)fucosyltransferase activity to synthesize the Lewis x structure from pyridylaminated lacto-N-neotetraose; however, lacto-N-tetraose does not serve as a substrate. Reverse transcriptase-polymerase chain reaction analysis revealed that zFT1 is transcribed during a restricted period before hatching, whereas the mRNA for zFT2 was detected only after hatching.

Partial purification and characterization of a novel endo-beta-mannosidase acting on N-linked sugar chains from Lilium longflorum thumb.

An enzyme catalyzing the hydrolysis of the Manbeta1-4GlcNAc linkage of N-linked sugar chains was partially purified and characterized. Endo-beta-mannosidase activity was detected using pyridylaminated (PA-) Manalpha1-6Manbeta1-4GlcNAcbeta1-4GlcNAc as the substrate in a homogenate of lily flowers (Lilium longflorum Thumb). The enzyme was partially purified by ammonium sulfate precipitation, and Q-Sepharose, Superdex 200, hydroxyapatite, Poros PE/M, Mono Q, and Superdex 200 column chromatographies. The optimum pH was 5.0 and the estimated molecular weight of the enzyme was 78,000, as determined by gel filtration. The Km value found for Manalpha1-6Manbeta1-4GlcNAcbeta1-4GlcNAc-PA was 1.4 mM. The enzymatic activity was not influenced by the addition of 10 mM EDTA or 2 mM Ca2+. Experiments on the hydrolysis of several PA-N-linked sugar chains revealed that the enzyme hydrolyzed MannManalpha1-6Manbeta1-4GlcNAcbeta1-4GlcNAc-PA (n = 0-2) into a mixture of MannManalpha1-6Man and GlcNAcbeta1-4GlcNAc-PA, indicating that it is an endoglycosidase in nature. However, the enzyme did not hydrolyze beta1-4mannohexaose or p-nitrophenyl beta-mannopyranoside.

Novel proteoglycan linkage tetrasaccharides of human urinary soluble thrombomodulin, SO4-3GlcAbeta1-3Galbeta1-3(+/-Siaalpha2-6)Galbeta1-4Xyl.

O-linked sugar chains with xylose as a reducing end linked to human urinary soluble thrombomodulin were studied. Sugar chains were liberated by hydrazinolysis followed by N-acetylation and tagged with 2-aminopyridine. Two fractions containing pyridylaminated Xyl as a reducing end were collected. Their structures were determined by partial acid hydrolysis, two-dimensional sugar mapping combined with exoglycosidase digestions, methylation analysis, mass spectrometry, and NMR as SO4-3GlcAbeta1-3Galbeta1-3(+/-Siaalpha2-6)Galbeta1+ ++-4Xyl. These sugar chains could bind to an HNK-1 monoclonal antibody. This is believed to be the first example of a proteoglycan linkage tetrasaccharide with glucuronic acid 3-sulfate and sialic acid.

Systematic analysis of N-linked sugar chains from whole tissue employing partial automation.

A partially automated technique for the isolation and characterization of N-linked sugar chains from glycoproteins of crude tissue samples is established. The N-linked sugar chains from the acetone-extracted tissues are made free by a process of hydrazinolysis and subsequently N-acetylated by GlycoPrep 1000 (Oxford Glycosystems). These free sugar chains are further converted to pyridylamino derivatives by GlycoTag (Takara). Characterization of these sugar chains is achieved by a combination of HPLC columns using a highly sensitive fluorescence detector at femtomole levels. Tissue sample can be successfully pyridylaminated and analyzed to give highly reproducible results with consistent yield, requiring fewer purification steps, minimum skills, and less time. Moreover, fixed tissues can also be analyzed employing this technique, giving a similar sugar chain pattern compared to normal tissue samples. Using this method we show that the pattern of N-linked sugar chains present in human sera or in one small region of brain is strikingly similar among the different individuals. However, the absence of a highlighted peak in one of the samples suggests this method can be extrapolated to identify changes, if any, associated with disorders such as inflammation or cancer. Furthermore, this two-dimensional display of sugar chains would discover the function-specific molecules as we see in proteins.

Development-dependent expression of complex-type sugar chains specific to mouse brain.

We previously detected a fucosylagalactobiantenna with a bisecting GlcNAc residue (BA-2) and one lacking the GlcNAc residue linked to the Manalpha1-3 residue of BA-2 (BA-1), which were enriched specifically in mouse brain [Shimizu, H., Ochiai, K., Ikenaka, K., Mikoshiba, K., and Hase, S. (1993) J. Biochem. 114, 334-338]. Pyridylamino sugar chains were prepared from mouse brains of various ages, and BA-1 and BA-2 were quantified after separation by HPLC. In cerebrum, BA-1 was scarcely expressed in newborn brain but gradually increased in amount during development, while expression of BA-2 reached a maximum 1 week after birth followed by a rapid decrease; in adult mice, the amount of BA-1 was almost the same as that of BA-2. In cerebellum, expression of BA-1 was lower than that of BA-2 at all stages. Glycoproteins with the BA-1 and BA-2 structures were enriched in the membrane fraction, and the glycoproteins solubilized were purified by lectin-affinity chromatography and gel filtration. The results indicated that BA-1 and BA-2 occurred in glycoproteins of more than 20 kDa in cerebellum, but most BA-1 and BA-2 were found in a 80-200 kDa fraction in cerebrum. These results show that the two brain-specific sugar chains are developmentally regulated and linked to the membrane-associated glycoproteins of subcellular organellas.

Structures of the sugar chains of recombinant macrophage colony-stimulating factor produced in Chinese hamster ovary cells.

The structures of the N- and O-linked sugar chains of recombinant human macrophage colony-stimulating factor (rhM-CSF) from Chinese hamster ovary (CHO) cells were studied. rhM-CSF is a homodimeric glycoprotein. Sugar composition analysis revealed that rhM-CSF contained 4.1 mol N-acetylgalactosamine, 10.3 mol N-acetylglucosamine, 5.0 mol mannose, 10.0 mol galactose, 1.4 mol fucose, and 11.8 mol sialic acid per mol of the monomer. The N- and O-linked sugar chains liberated by hydrazinolysis were N-acetylated, and the reducing-end sugar residues were tagged with 2-aminopyridine. The pyridylamino (PA-) sugar chains thus obtained were purified by HPLC. The structures of the PA-sugar chains were analyzed by a combination of reversed-phase and size-fractionation HPLC, and exoglycosidase digestions, from which the structures of the rhM-CSF sugar chains were estimated to be as follows: monosialo biantennary sugar chain (9 mol%), monosialo fucosylbiantennary sugar chain (10 mol%), disialo biantennary sugar chain (30 mol%), disialo fucosylbiantennary sugar chain (28 mol%), disialo triantennary sugar chain (7 mol%), trisialo triantennary sugar chain (11 mol%), and trisialo fucosyltriantennary sugar chain (5 mol%) for the N-linked sugar chains, and asialo (27 mol%), monosialo (51 mol%), and disialo (22 mol%) Galbeta1-3GalNAc for the O-linked sugar chains. Sialic acid residues were linked to the N-linked sugar chains through an alpha2-3 linkage.

Purification and characterization of neutral alpha-mannosidase from hen oviduct: studies on the activation mechanism of Co2+.

Neutral alpha-mannosidase was purified to homogeneity from hen oviduct. The molecular mass of the enzyme was 480 kDa on gel filtration, and the 110-kDa band on SDS-PAGE in the presence of 2-mercaptoethanol indicated that it is composed of four subunits. The activated enzyme hydrolyzed both p-nitrophenyl alpha-D-mannoside and high mannose-type sugar chains. This substrate specificity is almost the same as that reported for the neutral a-mannosidase from Japanese quail oviduct [Oku and Hase (1991) J. Biochem. 110, 982-989]. Manalpha1-6(Manalpha1-3)Manalpha1-6(Manalpha1-3) Manbeta1-4GlcNAc (Km =0.44 mM) was hydrolyzed four times faster than Manalpha1-6(Manalpha1-3)Manalpha1-6(Manalpha1-3) Manbeta1-4GIcNAcbeta1-4GlcNAc, and Manalpha1-6(Manalpha1-2Manalpha1-2Manalpha1-3)++ +Manbeta1-4GlcNAc was obtained as the end product from Man9GlcNAc on digestion with the activated alpha-mannosidase. The enzyme was activated 24-fold on preincubation with Co2+. The activation with other metal ions, like Mn2+, Ca2+, Fe2+, Fe3+, and Sr2+, was less than 5-fold, and Zn2+, Cu2+, and Hg2+ inhibited the enzyme activity. The optimum pHs for both the enzyme activity and activation with Co2+ were around 7. The cobalt ion contents of the purified, EDTA-treated, and Co2+-activated enzymes were 1.5, 0.0, and 3.9, respectively, per molecule. Since the Co2+-activated enzyme gradually lost its activity on incubation with EDTA and the activity was restored promptly on the addition of Co2+, the binding of Co2+ to the enzyme seems to be essential for its activation. The results obtained with protease inhibitors together with those of the SDS-PAGE before and after activation, showed that the proteolytic cleavage reported for the activation of monkey brain alpha-mannosidase seems not to be involved.

Detection of UDP-D-xylose: alpha-D-xyloside alpha 1-->3xylosyltransferase activity in human hepatoma cell line HepG2.

We previously reported the detection of novel O-linked sugar chains classified as being of the glucosyl-O-serine type [Hase et al. (1988) J. Biochem. 104, 867-868]. The sugar chains are a disaccharide (Xyl alpha 1-3Glc) and a trisaccharide (Xyl alpha 1-3Xyl alpha 1-3 Glc) linked to serine residues in epidermal growth factor-like domains of human and bovine blood coagulation factors. The structures of these sugar chains suggested the presence of an alpha 1-->3xylosyltransferase for their biosynthesis. We report here on the detection of alpha 1-->3xylosyltransferase activity which catalyzes the transfer of xylose to Xyl alpha 1-3Glc in the human hepatoma cell line HepG2. We employed pyridylaminated Xyl alpha 1-3Glc as a fluorescent acceptor and UDP-D-Xyl as a donor. The reaction product was purified by reversed-phase HPLC, and the structure of the transfer product isolated was confirmed to be pyridylaminated Xyl alpha 1-3Xyl alpha 1-3Glc by Smith degradation, mass spectrometry, and alpha- and beta-xylosidase digestions. The apparent K(m) value for pyridylaminated Xyl alpha 1-3Glc was 52 mM and for UDP-D-Xyl 0.28 mM. Optimum pH was 7.2. The enzyme was inactivated by addition of EDTA, and its activity was restored by addition of Mn2+ and Mg2+. These results indicate the presence of a novel enzyme which is able to transfer xylose to Xyl alpha 1-3Glc, forming Xyl alpha 1-3Xyl alpha 1-3Glc in human cells.

The fucosyltransferase FucT-VII regulates E-selectin ligand synthesis in human T cells.

Selectin-ligands on T cells contribute to the recruitment of circulating cells into chronic inflammatory lesions in the skin and elsewhere. This report provides the first evidence that a single fucosyltransferase, termed FucT-VII, controls the synthesis of E-selectin ligands in human T-lymphoblasts. The FucT-IV transferase (the ELFT enzyme), in contrast constructs lower avidity E-selectin ligands and requires enzyme levels found only in myeloid cells. Treatment of Jurkat cells with phorbol myristate acetate increased the expression of sialylated Lewis(x)-related sLe(x)related epitopes and induced the synthesis of E-selectin ligands functional at physiologic levels of linear shear-stress. Northern analysis revealed a parallel increase in the steady-state levels FucT-VII mRNA, but there were no increases in the two other leukocyte-associated fucosyltransferases (FucT-IV and VI). The stable transfection of the FucT-VII gene into Jurkat cells induced high levels of the sLe(x)-related epitopes and the synthesis of E-selectin ligands which equal or exceeded the avidity of those on circulating lymphocytes. The growth of T-lymphoblasts under conditions which induced expression of the sLe(x,a) epitopes increased the level of FucT-VII mRNA, the synthesis of sialylated-Lewis(x) structures by cell-free extracts and the synthesis of E-selectin ligands equal in avidity to those on FucT-VII transfectants. In contrast, neither the mRNA levels nor activities of the FucT-IV and VI enzymes increased in association with E-selectin ligand synthesis in T-lymphoblasts. Myeloid cell lines, unlike lymphoblasts, expressed high levels of both the FucT-VII and IV enzymes in conjunction with E-selectin ligands raising the possibility that both enzymes contributed to ligand synthesis. FucT-IV transfected Jurkat cells synthesized low avidity ligands for E-selectin but only in association with CDw65 (VIM-2) carbohydrate epitope. Only blood neutrophils and myeloid cell lines expressed this epitope at the levels associated with E-ligand synthesis in the transfectants. In contrast, native Jurkat cells, blood monocytes, blood lymphocytes, and cultured T-lymphoblasts expressed low levels or none. We conclude that FucT-VII is a principal regulator of E-selectin ligand synthesis in human T-lymphoblasts while both FucT-VII and FucT-IV may direct ligand synthesis in some myeloid cells.

Expression of the alpha(1,3)fucosyltransferase Fuc-TVII in lymphoid aggregate high endothelial venules correlates with expression of L-selectin ligands.

Lymphocyte homing to lymph nodes and Peyer's patches is mediated, in part, by adhesive interactions between L-selectin expressed by lymphocytes and L-selectin ligands displayed at the surface of the cuboidal endothelial cells lining the post-capillary venules within lymphoid aggregates. Candidate terminal oligosaccharide structures thought to be essential for effective L-selectin ligand activity include a sulfated derivative of the sialyl Lewis x tetrasaccharide. Cell type-specific synthesis of this oligosaccharide is presumed to require one or more alpha(1,3)fucosyltransferases, operating upon common 3'-sialylated and/or sulfated N-acetyllactosamine-type precursors. The identity of the alpha(1,3)fucosyltransferase(s) expressed in cells that bear L-selectin ligands has not been defined. We report here the molecular cloning and characterization of a murine alpha(1,3)fucosyltransferase locus whose expression pattern correlates with expression of high affinity ligands for L-selectin. In situ hybridization and immunohistochemical analyses demonstrate that this cDNA and its cognate alpha(1,3)fucosyltransferase are expressed in endothelial cells lining the high endothelial venules of peripheral lymph nodes, mesenteric lymph nodes, and Peyer's patches. These expression patterns correlate precisely with the expression pattern of L-selectin ligands identified with a chimeric L-selectin/IgM immunohistochemical probe and by the high endothelial venule-reactive monoclonal antibody MECA-79. Transcripts corresponding to this cDNA are also detected in isolated bone marrow cells, a source rich in the surface-localized ligands for E- and P-selectins. Sequence and functional analyses indicate that this murine enzyme corresponds to the human Fuc-TVII locus. These observations suggest that Fuc-TVII participates in the generation of alpha(1,3)fucosylated ligands for L-selectin and provide further evidence for a role for this enzyme in E- and P-selectin ligand expression in leukocytes.

Molecular cloning, expression, chromosomal assignment, and tissue-specific expression of a murine alpha-(1,3)-fucosyltransferase locus corresponding to the human ELAM-1 ligand fucosyl transferase.

Terminal Fuc alpha 1-3GlcNAc moieties are displayed by mammalian cell surface glycoconjugates in a tissue-specific manner. These oligosaccharides participate in selectin-dependent leukocyte adhesion and have been implicated in adhesive events during murine embryogenesis. Other functions for these molecules remain to be defined, as do the tissue-specific expression patterns of the corresponding alpha-(1-3)-fucosyltransferase (alpha 1-3FT) genes. This report characterizes a murine alpha 1-3FT that shares 77% amino acid sequence identity with human ELAM ligand fucosyltransferase (ELFT, also termed Fuc-TIV). The corresponding gene maps to mouse chromosome 9 in a region of homology with the Fuc-TIV locus on human chromosome 11q. In vitro, the murine alpha 1-3FT can efficiently fucosylate the trisaccharide Gal alpha 1-3Gal beta 1-4GlcNAc (apparent Km of 0.71 mM) to form an unusual tetrasaccharide (Gal alpha 1-3Gal beta 1-4[Fuc alpha 1-3]GlcNAc) described in periimplantation mouse tissues. The enzyme can also form the Lewis x determinant from Gal beta 1-4GlcNAc (Km = 2.05 mM), and the sialyl Lewis x determinant from NeuNAc alpha 2-3Gal beta 1-4GlcNAc (Km = 1.78mM). However, it does not yield sialyl Lewis x determinants when expressed in a mammalian cell line that maintains sialyl Lewis x precursors. Transcripts from this gene accumulate to low levels in hematopoietic organs, but are unexpectedly abundant in epithelia that line the stomach, small intestine, colon, and epididymus. Epithelial cell-specific expression of this gene suggests function(s) in addition to, and distinct from, its proposed role in selectin ligand synthesis.