Ole Hindsgaul

The α(1.3)fucosyltransferase Fuc-TVII controls leukocyte trafficking through an essential role in L-, E- and P-selectin ligand biosynthesis.

alpha(1,3)Fucosylated oligosaccharides represent components of leukocyte counterreceptors for E- and P-selectins and of L-selectin ligands expressed by lymph node high endothelial venules (HEV). The identity of the alpha(1,3)fucosyltransferase(s) required for their expression has been uncertain, as has a requirement for alpha(1,3)fucosylation in HEV L-selectin ligand activity. We demonstrate here that mice deficient in alpha(1,3) fucosyltransferase Fuc-TVII exhibit a leukocyte adhesion deficiency characterized by absent leukocyte E- and P-selectin ligand activity and deficient HEV L-selectin ligand activity. Selectin ligand deficiency is distinguished by blood leukocytosis, impaired leukocyte extravasation in inflammation, and faulty lymphocyte homing. These observations demonstrate an essential role for Fuc-TVII in E-, P-, and L-selectin ligand biosynthesis and imply that this locus can control leukocyte trafficking in health and disease.

The α(1,3)fucosyltransferases FucT-IV and FucT-VII Exert Collaborative Control over Selectin-Dependent Leukocyte Recruitment and Lymphocyte Homing

E-, P-, and L-selectin counterreceptor activities, leukocyte trafficking, and lymphocyte homing are controlled prominently but incompletely by alpha(1,3)fucosyltransferase FucT-VII-dependent fucosylation. Molecular determinants for FucT-VII-independent leukocyte trafficking are not defined, and evidence for contributions by or requirements for other FucTs in leukocyte recruitment is contradictory and incomplete. We show here that inflammation-dependent leukocyte recruitment retained in FucT-VII deficiency is extinguished in FucT-IV(-/-)/FucT-VII(-/-) mice. Double deficiency yields an extreme leukocytosis characterized by decreased neutrophil turnover and increased neutrophil production. FucT-IV also contributes to HEV-born L-selectin ligands, since lymphocyte homing retained in FucT-VII(-/-) mice is revoked in FucT-IV(-/-)/FucT-VII(-/-) mice. These observations reveal essential FucT-IV-dependent contributions to E-, P-, and L-selectin ligand synthesis and to the control of leukocyte recruitment and lymphocyte homing.

Novel Sulfated Lymphocyte Homing Receptors and Their Control by a Core1 Extension β1,3-N-Acetylglucosaminyltransferase

L-selectin mediates lymphocyte homing by facilitating lymphocyte adhesion to addressins expressed in the high endothelial venules (HEV) of secondary lymphoid organs. Peripheral node addressin recognized by the MECA-79 antibody is apparently part of the L-selectin ligand, but its chemical nature has been undefined. We now identify a sulfated extended core1 mucin-type O-glycan, Gal beta 1-->4(sulfo-->6)GlcNAc beta 1-->3Gal beta 1-->3GalNAc, as the MECA-79 epitope. Molecular cloning of a HEV-expressed core1-beta 1,3-N-acetylglucosaminyltransferase (Core1-beta 3GlcNAcT) enabled the construction of the 6-sulfo sialyl Lewis x on extended core1 O-glycans, recapitulating the potent L-selectin-mediated, shear-dependent adhesion observed with novel L-selectin ligands derived from core2 beta1,6-N-acetylglucosaminyltransferase-I null mice. These results identify Core1-beta 3GlcNAcT and its cognate extended core1 O-glycans as essential participants in the expression of the MECA-79-positive, HEV-specific L-selectin ligands required for lymphocyte homing.

6′-Sulfo Sialyl Lex but Not 6-Sulfo Sialyl Lex Expressed on the Cell Surface Supports L-selectin-mediated Adhesion

In order to determine if a sulfated oligosaccharide on the cell surface can function as an L-selectin ligand, a novel approach for in vitro transfer of oligosaccharides was utilized (Srivastava, G., Kaun, K. J., Hindsgaul, O., and Palcic, M. M. (1992) J. Biol. Chem. 267, 22356-22361). CHO cells were incubated with synthetic 6′-sulfo sialyl Lex, NeuNAcα2→3(sulfate-6)Galβ1→4(Fucα1→3)GlcNAc or 6-sulfo sialyl Lex, NeuNAcα2→3Galβ1→4[(Fucα1→3)sulfate→6GlcNAc] oligosaccharide linked to C-6 of a fucose residue in GDP-fucose and a milk fucosyltransferase. The resultant CHO cells expressing 6′-sulfo sialyl Lex or 6-sulfo sialyl Lex on their cell surface were tested for adhesion to E-selectin and L-selectin chimeric proteins coated on plates. The results indicate that 6′-sulfo sialyl Lex supports L-selectin-mediated adhesion much better than sialyl Lex similarly tagged on the cell surface. In contrast, 6-sulfo sialyl Lex containing a sulfate group on the N-acetylglucosamine residue did not support adhesion with either selectin. These combined results suggest that 6′-sulfo sialyl Lex is a much better ligand than sialyl Lex oligosaccharide for L-selectin.

Poly-N-acetyllactosamine Synthesis in BranchedN-Glycans Is Controlled by Complemental Branch Specificity of i-Extension Enzyme and β1,4-Galactosyltransferase I

Poly-N-acetyllactosamine is a unique carbohydrate that can carry various functional oligosaccharides, such as sialyl Lewis X. It has been shown that the amount of poly-N-acetyllactosamine is increased inN-glycans, when they contain Galβ1→4GlcNAcβ1→6(Galβ1→4GlcNAcβ1→2)Manα1→6 branched structure. To determine how this increased synthesis of poly-N-acetyllactosamines takes place, the branched acceptor was incubated with a mixture of i-extension enzyme (iGnT) and β1,4galactosyltransferase I (β4Gal-TI). First,N-acetyllactosamine repeats were more readily added to the branched acceptor than the summation of poly-N-acetyllactosamines formed individually on each unbranched acceptor. Surprisingly, poly-N-acetyllactosamine was more efficiently formed on Galβ1→4GlcNAcβ1→2Manα→R side chain than in Galβ1→4GlcNAcβ1→6Manα→R, due to preferential action of iGnT on Galβ1→4GlcNAcβ1→2Manα→R side chain. On the other hand, galactosylation was much more efficient on β1,6-linked GlcNAc than β1,2-linked GlcNAc, preferentially forming Galβ1→4GlcNAcβ1→6(GlcNAcβ1→2)Manα1→6Manβ →R. Starting with this preformed acceptor,N-acetyllactosamine repeats were added almost equally to Galβ1→4GlcNAcβ1→6Manα→R and Galβ1→4GlcNAcβ1→2Manα→R side chains. Taken together, these results indicate that the complemental branch specificity of iGnT and β4Gal-TI leads to efficient and equal addition ofN-acetyllactosamine repeats on both side chains of GlcNAcβ1→6(GlcNAcβ1→2)Manα1→6Manβ→R structure, which is consistent with the structures found in nature. The results also suggest that the addition of Galβ1→4GlcNAcβ1→6 side chain on Galβ1→4GlcNAcβ1→2Man→R side chain converts the acceptor to one that is much more favorable for iGnT and β4Gal-TI.

Poly-N-acetyllactosamine Extension inN-Glycans and Core 2- and Core 4-branchedO-Glycans Is Differentially Controlled by i-Extension Enzyme and Different Members of the β1,4-Galactosyltransferase Gene Family

Poly-N-acetyllactosamines are attached to N-glycans, O-glycans, and glycolipids and serve as underlying glycans that provide functional oligosaccharides such as sialyl LewisX. Poly-N-acetyllactosaminyl repeats are synthesized by the alternate addition of β1,3-linked GlcNAc and β1,4-linked Gal by i-extension enzyme (iGnT) and a member of the β1,4-galactosyltransferase (β4Gal-T) gene family. In the present study, we first found that poly-N-acetyllactosamines inN-glycans are most efficiently synthesized by β4Gal-TI and iGnT. We also found that iGnT acts less efficiently on acceptors containing increasing numbers of N-acetyllactosamine repeats, in contrast to β4Gal-TI, which exhibits no significant change. In O-glycan biosynthesis,N-acetyllactosamine extension of core 4 branches was found to be synthesized most efficiently by iGnT and β4Gal-TI, in contrast to core 2 branch synthesis, which requires iGnT and β4Gal-TIV. Poly-N-acetyllactosamine extension of core 4 branches is, however, less efficient than that of N-glycans or core 2 branches. Such inefficiency is apparently due to competition between a donor substrate and acceptor in both galactosylation andN-acetylglucosaminylation, since a core 4-branched acceptor contains both Gal and GlcNAc terminals. These results, taken together, indicate that poly-N-acetyllactosamine synthesis inN-glycans and core 2- and core 4-branchedO-glycans is achieved by iGnT and distinct members of the β4Gal-T gene family. The results also exemplify intricate interactions between acceptors and specific glycosyltransferases, which play important roles in how poly-N-acetyllactosamines are synthesized in different acceptor molecules.

Molecular Cloning and Expression of a Novel Human β-Gal-3-O-sulfotransferase That Acts Preferentially onN-Acetyllactosamine in N- andO-Glycans

A novel cDNA-encoding galactose 3-O-sulfotransferase was cloned by screening the expressed sequence tag data base using the previously cloned cDNA encoding a galactosyl ceramide 3-O-sulfotransferase, which we term Gal3ST-1. The newly isolated cDNA encodes a novel 3-O-sulfotransferase, termed Gal3ST-3, that acts exclusively on N-acetyllactosamine present inN-glycans and core2-branched O-glycans. These conclusions were confirmed by analyzing CD43 chimeric proteins in Chinese hamster ovary cells expressing core2 β1,6-N-acetylglucosaminyltransferase. The acceptor specificity of Gal3ST-3 contrasts with that of the recently cloned galactose 3-O-sulfotransferase (Honke, K., Tsuda, M., Koyota, S., Wada, Y., Iida-Tanaka, N., Ishizuka, I., Nakayama, J., and Taniguchi, N. (2001) J. Biol. Chem. 276, 267–274), which we term Gal3ST-2 in the present study because the latter enzyme can also act on core1 O-glycan and type 1 oligosaccharides, Galβ1→3GlcNAc. Moreover, Gal3ST-3 but not Gal3ST-2 can act on Galβ1→4(sulfo→6)GlcNAc, indicating that disulfated sulfo→3Galβ1→4(sulfo→6) GlcNAc→R may be formed by Gal3ST-3 in combination with GlcNAc 6-O-sulfotransferase. Although both Gal3ST-2 and Gal3ST-3 do not act on galactosyl ceramide, Gal3ST-3 is only moderately more homologous to Gal3ST-2 (40.1%) than to Gal3ST-1 (38.0%) at the amino acid level. Northern blot analysis demonstrated that transcripts for Gal3ST-3 are predominantly expressed in the brain, kidney, and thyroid where the presence of 3′-sulfation ofN-acetyllactosamine has been reported. These results indicate that the newly cloned Gal3ST-3 plays a critical role in 3′-sulfation of N-acetyllactosamine in both O- and N-glycans.

A concise synthesis of the 6-o- and 6′-o-sulfated analogues of the sialyl lewis X tetrasaccharide

The octyl glycoside of the sialyl Lewis X tetrasaccharide and its 6-O-sulfated and 6-O-sulfated analogues were chemically synthesized in a concise manner starting from readily accessible monosaccharide intermediates. The synthesis involved formation of an orthogonally protected tetrasaccharide intermediate from which all three materials were prepared. A selective catalytic hydrogenolysis of four O-benzyl ethers in presence of a 4,6-O-benzylidene group was the key step in the synthetic scheme.

The N-glycolyl form of mouse sialyl Lewis X is recognized by selectins but not by HECA-452 and FH6 antibodies that were raised against human cells

E-, P- and L-selectins critically function in lymphocyte recirculation and recruiting leukocytes to inflammatory sites. MECA-79 antibody inhibits L-selectin-mediated lymphocyte adhesion in several species and does not require sialic acid in its epitope. Many other antibodies, however, recognize human selectin ligands expressing N-acetylneuraminic acid but not mouse selectin ligands expressing N-glycolylneuraminic acid, suggesting that difference in sialic acid in sialyl Lewis X leads to differential reactivity. We found that HECA-452 and FH6 monoclonal antibodies bind Chinese hamster ovary (CHO) cells expressing N-acetylneuraminyl Lewis X oligosaccharide but not its N-glycolyl form. Moreover, synthetic N-acetylneuraminyl Lewis X oligosaccharide but not its N-glycolyl oligosaccharide inhibited HECA-452 and FH6 binding. By contrast, E-, P- and L-selectin bound to CHO cells regardless of whether they express N-acetyl or N-glycolyl form of sialyl Lewis X, showing that selectins have a broader recognition capacity than HECA-452 and FH-6 anti-sialyl Lewis x antibodies.

Efficient synthesis of lactosaminylated core-2 O-glycans.

A series of lactosaminylated oligosaccharides found in mucin type O-glycans was synthesized using a generalized block strategy. The synthesis involved the addition of a protected lactosamine donor to a partially protected T-disaccharide derivative. The nonreducing galactose residues of the deblocked oligosaccharide products could be removed by beta-galactosidase from jack bean to produce the corresponding GlcNAc terminated compounds. A series of tri- to hexasaccharides was thus efficiently produced.