Differential recognition of animal type beta4-galactosylated and alpha3-fucosylated chito-oligosaccharides by two family 18 chitinases from Trichoderma harzianum.

We report the purification of two glycosyl hydrolase family 18 chitinases, Chit33 and Chit42, from the filamentous fungus Trichoderma harzianum and characterization using a panel of different soluble chitinous substrates and inhibitors. We were particularly interested in the potential of these (alpha/beta)(8)-barrel fold enzymes to recognize beta-1,4-galactosylated and alpha-1,3-fucosylated oligosaccharides, which are animal-type saccharides of medical relevance. Three-dimensional structural models of the proteins in complex with chito-oligosaccharides were built to support the interpretation of the hydrolysis data.

Human galectin-1 recognition of poly-N-acetyllactosamine and chimeric polysaccharides.

Human galectin-1 is a dimeric carbohydrate binding protein (Gal-1) (subunit 14.6 kDa) widely expressed by many cells but whose carbohydrate binding specificity is not well understood. Because of conflicting evidence regarding the ability of human Gal-1 to recognize N-acetyllactosamine (LN, Galbeta4GlcNAc) and poly-N-acetyllactosamine sequences (PL, [-3Galbeta4GlcNAcbeta1-]n), we synthesized a number of neoglycoproteins containing galactose, N-acetylgalactosamine, fucose, LN, PL, and chimeric polysaccharides conjugated to bovine serum albumin (BSA).

Purification and cDNA cloning of UDP-GlcNAc:GlcNAcbeta1-3Galbeta1-4Glc(NAc)-R [GlcNAc to Gal]beta1,6N-acetylglucosaminyltransferase from rat small intestine: a major carrier of dIGnT activity in rat small intestine.

A rat intestinal beta1,6N-acetylglucosaminyltransferase (beta1-6GnT) responsible for the formation of the beta1,6-branched poly-N-acetyllactosamine structure has been purified to apparent homogeneity by successive column chromatographic procedures using an assay wherein pyridylaminated lacto- N-triose II (GlcNAcbeta1-3Galbeta1-4Glc-PA) was used as an acceptor substrate and the reaction product was GlcNAcbeta1-3(GlcNAcbeta1-6)Galbeta1-4Glc-PA. The purified enzyme catalyzed the conversion of the polylactosamine acceptor GlcNAcbeta1-3'LacNAc into GlcNAcbeta1-3'(GlcNAcbeta1-6') LacNAc (dIGnT activity), but it could not transfer GlcNAc to LacNAcbeta1-3'LacNAc (cIGnT activity). This enzyme could also convert mucin core 1 and core 3 analogs, Galbeta1-3GalNAcalpha1-O-paranitrophenyl (pNP) and GlcNAcbeta1-3GalNAcalpha1-O-pNP, into Galbeta1-3(GlcNAcbeta1-6) GalNAcalpha1-O-pNP (C2GnT activity) and GlcNAcbeta1-3(GlcNAcbeta1-6)GalNAcalpha1-O-pNP (C4GnT activity), respectively.

Glycosulfopeptides with O-glycans containing sialylated and polyfucosylated polylactosamine bind with low affinity to P-selectin.

P-selectin glycoprotein ligand-1 (PSGL-1), a dimeric mucin on leukocytes, is the best characterized ligand for selectins. P-selectin binds stereospecifically to the extreme N terminus of PSGL-1, which contains three clustered tyrosine sulfates (TyrSO3-) adjacent to a Thr residue with a core 2-based O-glycan expressing sialyl Lewis x (C2-O-sLe(x)). GSP-6, a synthetic glycosulfopeptide modeled after the N terminus of PSGL-1, containing three TyrSO3- residues and a short, monofucosylated C2-O-sLe(x) bound to P-selectin with high affinity (K(d) approximately 650 nm).

Fuc-TIX: a versatile alpha1,3-fucosyltransferase with a distinct acceptor- and site-specificity profile.

alpha1,3-Fucosyltransferases (Fuc-Ts) convert N-acetyllactosamine (LN, Galbeta1-4GlcNAc) to Galbeta1-4(Fucalpha1-3)GlcNAc, the Lewis x (CD15, SSEA-1) epitope, which is involved in various recognition phenomena. We describe details of the acceptor specificity of alpha1,3-fucosyltransferase IX (Fuc-TIX). The unconjugated N- and O-glycan analogs LNbeta1-2Man, LNbeta1-6Manalpha1-OMe, LNbeta1-2Manalpha1-3(LNbeta1-2Manalpha1-6)Manbeta1-4GlcNAc, and Galbeta1-3(LNbeta1-6)GalNAc reacted well in vitro with Fuc-TIX present in lysates of appropriately transfected Namalwa cells.

Several polylactosamine-modifying glycosyltransferases also use internal GalNAcbeta1-4GlcNAc units of synthetic saccharides as acceptors.

The GalNAcbeta1-4GlcNAc determinant (LdN) occurs in some human and bovine glycoconjugates and also in lower vertebrates and invertebrates. It has been found in unsubstituted as well as terminally substituted forms at the distal end of conjugated glycans, but it has not been reported previously at truly internal positions of polylactosamine chains. Here, we describe enzyme-assisted conversion of LdNbeta1-OR oligosaccharides into GlcNAcbeta1-3GalNAcbeta1-4GlcNAcbeta1-OR.

O-glycans on human high endothelial CD34 putatively participating in L-selectin recognition.

Leukocyte traffic into lymph nodes and sites of inflammation is guided by L-selectin. Experiments performed in vitro and with gene-deleted mice suggest that CD34 recognizes L-selectin if decorated by 6-sulfo sialyl Lewis x (sLex) saccharides and the MECA-79 epitope. However, very little is known about glycosylation of human L-selectin ligands.

alpha 2,3-Sialylation of terminal GalNAc beta 1-3Gal determinants by ST3Gal II reveals the multifunctionality of the enzyme. The resulting Neu5Ac alpha 2-3GalNAc linkage is resistant to sialidases from Newcastle disease virus and Streptococcus pneumoniae.

Enzymatic alpha 2,3-sialylation of GalNAc has not been described previously, although some glycoconjugates containing alpha 2,3-sialylated GalNAc residues have been reported. In the present experiments, recombinant soluble alpha 2,3-sialyltransferase ST3Gal II efficiently sialylated the X(2) pentasaccharide GalNAc beta 1-3Gal beta 1-4GlcNAc beta 1-3Gal beta 1-4Glc, globo-N-tetraose GalNAc beta 1-3Gal alpha 1-4Gal beta 1-4Glc, and the disaccharide GalNAc beta 1-3Gal in vitro. The purified products were identified as Neu5Ac alpha 2-3GalNAc beta 1-3Gal beta 1-4GlcNAc beta 1-3Gal beta 1-4Glc, Neu5Ac alpha 2-3GalNAc beta 1-3Gal alpha 1-4Gal beta 1-4Glc, and Neu5Ac alpha 2-3GalNAc beta 1-3Gal, respectively, by matrix-assisted laser desorption/ionization time-of-flight mass spectrometry, enzymatic degradations, and one- and two-dimensional NMR-spectroscopy.

Human alpha3-fucosyltransferases convert chitin oligosaccharides to products containing a GlcNAcbeta1-4(Fucalpha1-3)GlcNAcbeta1-4R determinant at the nonreducing terminus.

Human alpha3-fucosyltransferases (Fuc-Ts) are known to convert N-acetyllactosamine to Galbeta1-4(Fucalpha1-3)GlcNAc (Lewis x antigen); some of them transfer fucose also to GalNAcbeta1-4GlcNAc, generating GalNAcbeta1-4(Fucalpha1-3)GlcNAc determinants. Here, we report that recombinant forms of Fuc-TV and Fuc-TVI as well as Fuc-Ts of human milk converted chitin oligosaccharides of 2-4 GlcNAc units efficiently to products containing a GlcNAcbeta1-4(Fucalpha1-3)GlcNAcbeta1-4R determinant at the nonreducing terminus. The product structures were identified by mass spectrometry and nuclear magnetic resonance experiments; rotating frame nuclear Overhauser spectroscopy data suggested that the fucose and the distal N-acetylglucosamine are stacked in the same way as the fucose and the distal galactose of the Lewis x determinant.

Enzymatic in vitro synthesis of I-branches of mammalian polylactosamines: generation of scaffolds for multiple selectin-binding saccharide determinants.

Polylactosamines are covalent monosaccharide assemblies of the animal kingdom and some bacteria, and are characterized by backbones of interlinked N-acetyllactosamine units (Galbeta1-4GlcNAc, LacNAc). The mammalian LacNAc arrays are linear (blood group i-type) and branched (blood group I-type), and are linked to the core elements of glycolipids as well as O- and N-glycans of glycoproteins and keratan sulfate proteoglycans. Generation of I-branches to linear i-type polylactosamines is initiated by two kinds of beta6GlcNAc transferases.

The acceptor and site specificity of alpha 3-fucosyltransferase V. High reactivity of the proximal and low of the distal galbeta 1-4GlcNAc unit in i-type polylactosamines.

We report here on in vitro acceptor and site specificity of recombinant alpha3-fucosyltransferase V (Fuc-TV) with 40 oligosaccharide acceptors. Galbeta1-4GlcNAc (LN) and GalNAcbeta1-4GlcNAc (LDN) reacted rapidly; Galbeta1-3GlcNAc (LNB) reacted moderately, and GlcNAcbeta1-4GlcNAc (N, N'-diacetyl-chitobiose) reacted slowly yet distinctly. In neutral and terminally alpha3-sialylated polylactosamines of i-type, the reducing end LN unit reacted rapidly and the distal (sialyl)LN group very slowly; the midchain LNs revealed intermediate reactivities.

High endothelial cells synthesize and degrade sLex. Putative implications for L-selectin-dependent recognition.

L-selectin guides lymphocytes into peripheral lymphoid tissues by recognizing glycoprotein ligands decorated with 6-sulfated sialyl Lewis x (sulfo sLex). Here we have used a rat peripheral lymph node high endothelial cell line (Ax) to study in detail the synthesis, expression and degradation of sLex epitope. We show here that Ax cells possess active alpha(1,3)fucosyltransferase Fuc-TVII, the enzyme responsible for the final fucosylation of sialyl-N-acetyllactosamine during sLex synthesis, and express sLex on the cell surface.

Biosynthesis of sialylated and fucosylated selectin ligands of HL-60 cells in vitro. Midchain alpha3-fucose units inhibit terminal alpha6-sialylation but not alpha3-sialylation of polylactosamines.

Polylactosamines Neu5Ac alpha2-3'Lex beta1-3'Lex beta1-3'Lex and Neu5Ac alpha2-3'LNbeta1-3'Lex beta1-3'Lex [Lex, Gal beta1-4(Fuc alpha1-3)GlcNAc; LN, Gal beta1-4GlcNAc] decorate selectin counterreceptors in human HL-60 cells. Here, we show that HL-60 cell lysates catalyze distal alpha3-sialylation of LNbeta1-3'LNbeta1-3'LN and LNbeta1-3'Lex beta1-3'Lex efficiently, outlining two potential sets of biosynthetic pathways leading to the selectin ligands. In one set, alpha3-sialylation precedes internal fucosylation of the polylactosamine backbone, whereas in the other one, internal fucosylation is initiated before alpha3-sialylation.

Isolation and characterization of linear polylactosamines containing one and two site-specifically positioned Lewis x determinants: WGA agarose chromatography in fractionation of mixtures generated by random, partial enzymatic alpha3-fucosylation of pure polylactosamines.

We report that isomeric monofucosylhexasaccharides, Galbeta1-4GlcNAcbeta1-3Galbeta1-4GlcNAcbeta1- 3Galbeta1-4(Fucalpha1-3) GlcNAc, Galbeta1-4GlcNAcbeta1-3Galbeta1-4(Fucalpha1-3) GlcNAcbeta1-3Galbeta1-4 GlcNAc and Galbeta1-4(Fucalpha1-3)GlcNAcbeta1-3Galbeta1- 4GlcNAcbeta1-3Galbeta1-4 GlcNAc, and bifucosylhexasaccharides Galbeta1-4GlcNAcbeta1-3Galbeta1-4(Fucalpha1-3) GlcNAcbeta1-3Galbeta1-4(Fucalpha1-3)GlcNAc, Galbeta1-4(Fucalpha1-3)GlcNAcbeta1-3Galbeta1- 4GlcNAcbeta1-3Galbeta1-4 (Fucalpha1-3)GlcNAc and Galbeta1-4(Fucalpha1-3)GlcNAcbeta1-3Galbeta1-4( Fucalpha1-3)GlcNAcbeta1-3Galbeta1-4GlcNAc can be isolated in pure form from reaction mixtures of the linear hexasaccharide Galbeta1-4GlcNAcbeta1-3Galbeta1-4GlcNAcbeta1- 3Galbeta1-4GlcNAc with GDP-fucose and alpha1,3-fucosyltransferases of human milk. The pure isomers were characterized in several ways;1H-NMR spectroscopy, for instance, revealed distinct resonances associated with the Lewis x group [Galbeta1-4(Fucalpha1-3)GlcNAc] located at the proximal, middle, and distal positions of the polylactosamine chain. Chromatography on immobilized wheat germ agglutinin was crucial in the separation process used; the isomers carrying the fucose at the reducing end GlcNAc possessed particularly low affinities for the lectin.

Enzymatic synthesis of alpha3'sialylated and multiply alpha3fucosylated biantennary polylactosamines. A bivalent [sialyl diLex]-saccharide inhibited lymphocyte-endothelium adhesion organ-selectively.

Multifucosylated sialo-polylactosamines are known to be high affinity ligands for E-selectin. PSGL-1, the physiological ligand of P-selectin, is decorated in HL-60 cells by a sialylated and triply fucosylated polylactosamine that is believed to be of functional importance. Mimicking some of these saccharide structures, we have synthesized enzymatically a bivalent [sialyl diLex]-glycan, Neu5Acalpha2-3'Lexbeta1-3'Lexbeta1-3'(Neu5Acalpha2-3'Lexbeta1-3Lexbe ta1-6')LN [where Neu5Ac is N-acetylneuraminic acid, Lex is the trisaccharide Galbeta1-4(Fucalpha1-3)GlcNAc and LN is the disaccharide Galbeta1-4GlcNAc].

The centrally acting beta1,6N-acetylglucosaminyltransferase (GlcNAc to gal). Functional expression, purification, and acceptor specificity of a human enzyme involved in midchain branching of linear poly-N-acetyllactosamines.

In the present experiments the cDNA coding for a truncated form of the beta1,6N-acetylglucosaminyltransferase responsible for the conversion of linear to branched polylactosamines in human PA1 cells was expressed in Sf9 insect cells. The catalytic ectodomain of the enzyme was fused to glutathione S-transferase, allowing effective one-step purification of the glycosylated 67-74-kDa fusion protein. Typically a yield of 750 microg of the purified protein/liter of suspension culture was obtained.

Purification and characterization of UDP-GlcNAc:Galbeta1-4GlcNAcbeta1-3*Galbeta1-4Glc(NAc)-R(GlcNAc to *Gal) beta1,6N-acetylglucosaminyltransferase from hog small intestine.

A beta1,6N-acetylglucosaminyltransferase (beta1-6GnT) responsible for the formation of the beta1,6-branched poly-N-acetyllactosamine structure has been purified 210,000-fold in 2.4% yield from a homogenate of hog small intestine by successive column chromatographies involving CM-Sepharose FF, Ni2+-chelating Sepharose FF, and UDP-hexanolamine-agarose, using an assay wherein pyridylaminated lacto-N-neotetraose (Galbeta1-4GlcNAcbeta1-3Galbeta1-4Glc-PA) was used as an acceptor substrate, and the reaction product was Galbeta1-4GlcNAcbeta1-3(GlcNAcbeta1-6)Galbeta1-4 Glc-PA. The apparent molecular weight of the purified enzyme was 76,000 under nonreducing conditions.

Biosynthesis of branched polylactosaminoglycans. Embryonal carcinoma cells express midchain beta1,6-N-acetylglucosaminyltransferase activity that generates branches to preformed linear backbones.

Two types of beta1,6-GlcNAc transferases (IGnT6) are involved in in vitro branching of polylactosamines: dIGnT6 (distally acting), transferring to the penultimate galactose residue in acceptors like GlcNAcbeta1-3Galbeta1-4GlcNAcbeta1-R, and cIGnT6 (centrally acting), transferring to the midchain galactoses in acceptors of the type (GlcNAcbeta1-3)Galbeta1-4GlcNAcbeta1-3Galbeta1-+ ++4GlcNAcbeta1-R. The roles of the two transferases in the biosynthesis of branched polylactosamine backbones have not been clearly elucidated. We report here that cIGnT6 activity is expressed in human (PA1) and murine (PC13) embryonal carcinoma (EC) cells, both of which contain branched polylactosamines in large amounts.

Enzymatic synthesis of site-specifically (alpha 1-3)-fucosylated polylactosamines containing either a sialyl Lewis (x), a VIM-2, or a sialylated and internally difucosylated sequence.

By using two different reaction pathways, we generated enzymatically three sialylated and site-specifically alpha 1-3-fucosylated polylactosamines. Two of these are isomeric hexasaccharides Neu5Ac(alpha 2-3)Gal(beta 1-4)GlcNAc(beta 1-3)Gal(beta 1-4)[Fuc(alpha 1-3)] GlcNAc and Neu5Ac(alpha 2-3)Gal(beta 1-4)[Fuc(alpha 1-3)]GlcNAc(beta 1-3)Gal(beta 1-4) GlcNAc, containing epitopes that correspond to VIM-2 and sialyl Lewis (x), respectively. The third one, nonasaccharide Neu5Ac(alpha 2-3)Gal(beta 1-4)GlcNAc(beta 1-3)Gal(beta 1-4)[Fuc(alpha 1-3)] GlcNAc(beta 1-3)Gal(beta 1-4)[Fuc(alpha 1-3)]GlcNAc, is a sialylated and internally difucosylated derivative of a trimeric N-acetyllactosamine.

Complementary acceptor and site specificities of Fuc-TIV and Fuc-TVII allow effective biosynthesis of sialyl-TriLex and related polylactosamines present on glycoprotein counterreceptors of selectins.

The P-selectin counterreceptor PSGL-1 is covalently modified by mono alpha2,3-sialylated, multiply alpha1,3-fucosylated polylactosamines. These glycans are required for the adhesive interactions that allow this adhesion receptor-counterreceptor pair to facilitate leukocyte extravasation. To begin to understand the biosynthesis of these glycans, we have characterized the acceptor and site specificities of the two granulocyte alpha1,3-fucosyltransferases, Fuc-TIV and Fuc-TVII, using recombinant forms of these two enzymes and a panel of synthetic polylactosamine-based acceptors.

Improved enzymatic synthesis of a highly potent oligosaccharide antagonist of L-selectin.

The polylactosamine sLex beta1-3'(sLex beta1-6')LacNAc beta1-3'(sLex beta1-6')LacNAc beta1-3'(sLex beta1-6')LacNAc (7) (where sLex is Neu5Ac alpha2-3Gal beta1-4(Fuc alpha1-3)GlcNAc and LacNAc is Gal beta1-4GlcNAc) is a nanomolar L-selectin antagonist and therefore a potential anti-inflammatory agent (Renkonen et al. (1997) Glycobiology, 7, 453). Here we describe an improved synthesis of 7.

Enzymatic midchain branching of polylactosamine backbones is restricted in a site-specific manner in alpha 1,3-fucosylated chains.

Branched polylactosamines on animal cell surfaces are believed to contribute to multivalent interactions in cell adhesion and cell signalling. Their biosynthesis proceeds via linear precursors that become branched by beta1,6-GlcNAc transferases (IGnT6, GlcNAc to Gal). Previous work has identified the tetrasaccharide Galbeta1-4GlcNAcbeta1-3Galbeta1-4GlcNAc (1) and the hexasaccharide Galbeta1-4GlcNAcbeta1-3Galbeta1-4GlcNAcbeta1- 3Galbeta1-4GlcNAc (4) as acceptors for a rat serum enzyme activity (cIGnT6), which transfers GlcNAcbeta1-6 units to the midchain galactose residues.

The beta 1,6-GlcNAc transferase activity present in hog gastric mucosal microsomes catalyses site-specific branch formation on a long polylactosamine backbone.

We find that the beta 1,6-GlcNAc transferase activity present in hog gastric mucosal microsomes converts the linear pentasaccharide GlcNAc beta 1-3Gal beta 1-4GlcNAc beta 1-3Gal beta 1-4GlcNAc (1) in a site-specific way to the branch-bearing hexasaccharide GlcNAc beta 1-3(GlcNAc beta 1-6)Gal beta 1-4GlcNAc beta 1-3Gal beta 1-4GlcNAc (2). The product is a positional isomer of GlcNAc beta 1-3Gal beta 1-4GlcNAc beta 1-3(GlcNAc beta 1-6)Gal beta 1-4GlcNAc (3), reportedly formed from 1 by another polylactosamine beta 1,6-GlcNAc transferase activity present in human serum (Leppänen et al., Biochemistry, 30 (1991) 9287).

In vitro experimental studies of sialyl Lewis x and sialyl Lewis a on endothelial and carcinoma cells: crucial glycans on selectin ligands.

Extravasation from the blood of malignant tumour cells that form metastasis and leukocytes that go into tissues require contact between selectins and their sialyl Lewis x and sialyl Lewis a (sLe(x) and sLe(a) respectively) decorated ligands. Endothelial cells have been shown to express sLe(x) epitopes in lymph nodes and at sites of inflammation, and this is crucial for the selectin-dependent leukocyte traffic. Besides the ability to synthesize sLe(x) on sialylated N-acetyllactosamine via the action of alpha(1,3)fucosyltransferase(s), endothelial cells can also degrade sLe(x) to Lewis x through the action of alpha(2,3)sialidase(s).

Enzymatic transfer of a beta1,6-linked N-acetylglucosamine to the alpha-galactose of globo-N-tetraose: in vitro synthesis of a novel hybrid pentasaccharide of lacto-globo type.

A novel saccharide was synthesized by incubating globo-N-tetraose, GalNAc beta1-3Gal alpha1-4Gal beta1-4Glc, and UDP[3H]GlcNAc with hog gastric mucosal microsomes, known to contain beta1,6-N-acetylglucosaminyltransferase activity of a broad acceptor specificity. Chromatography and MALDI-TOF mass spectrometry of the product, as well as the amount of incorporated radioactivity indicated that one [3H]GlcNAc residue was transferred to the acceptor saccharide. One- and two-dimensional 1H NMR-spectroscopic analysis of the product and ESI-CID mass spectrometry of the pentasaccharide in permethylated form established its structure as GalNAc beta1-3([3H]GlcNAc beta1-6)Gal alpha1-4Gal beta1-4Glc.