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K L Matta

Publications and source records attributed to K L Matta.

At least 37 records · Page 2Linked to original sources
PubMed1998

Inhibition of L- and P-selectin by a rationally synthesized novel core 2-like branched structure containing GalNAc-Lewisx and Neu5Acalpha2-3Galbeta1-3GalNAc sequences.

The selectins interact in important normal and pathological situations with certain sialylated, fucosylated glycoconjugate ligands containing sialyl Lewisx(Neu5Acalpha2-3Galbeta1-4(Fucalpha1-3)GlcN Ac). Much effort has gone into the synthesis of sialylated and sulfated Lewisxanalogs as competitive ligands for the selectins. Since the natural selectin ligands GlyCAM-1 and PSGL-1 carry sialyl Lewisxas part of a branched Core 2 O-linked structure, we recently synthesized Galbeta1-4(Fucalpha1-3)GlcNAcbeta1-6(SE-3Galbeta1++ +-3)GalNAc1alphaOMe and found it to be a moderately superior ligand for L and P-selectin (Koenig et al. , Glycobiology 7, 79-93, 1997). Other studies have shown that sulfate esters can replace sialic acid in some selectin ligands (Yeun et al. , Biochemistry, 31, 9126-9131, 1992; Imai et al. , Nature, 361, 555, 1993). Based upon these observations, we hypothesized that Neu5Acalpha2-3Galbeta1-3GalNAc might have the capability of interacting with L- and P-selectin. To examine this hypothesis, we synthesized Galbeta1-4(Fucalpha1-3)GlcNAcbeta1-6(Neu5Acalpha2++ +-3Galbeta1-3)-GalNAc alpha1-OB, which was found to be 2- to 3-fold better than sialyl Lexfor P and L selectin, respectively. We also report the synthesis of an unusual structure GalNAcbeta1-4(Fucalpha1- 3)GlcNAcbeta1-OMe (GalNAc-Lewisx-O-methyl glycoside), which also proved to be a better inhibitor of L- and P-selectin than sialyl Lewisx-OMe. Combining this with our knowledge of Core 2 branched structures, we have synthesized a molecule that is 5- to 6-fold better at inhibiting L- and P-selectin than sialyl Lewisx-OMe, By contrast to unbranched structures, substitution of a sulfate ester group for a sialic acid residue in such a molecule resulted in a considerable loss of inhibition ability. Thus, the combination of a sialic acid residue on the primary (beta1-3) arm, and a modified Lexunit on the branched (beta1-6) arm on an O-linked Core 2 structure generated a monovalent synthetic oliogosaccharide inhibitor superior to SLexfor both L- and P-selectin.

Binding, Competitive↗
PubMed1997

Fucosylation of disaccharide precursors of sialyl LewisX inhibit selectin-mediated cell adhesion.

We showed previously that HL-60 and F9 mouse embryonal carcinoma cells will take up and deblock peracetylated Galbeta1-4GlcNAcbeta-O-naphthalenemethanol (Galbeta1-4GlcNAc-NM) and use the disaccharide as a primer of oligosaccharide chains (Sarkar, A. K., Fritz, T. A., Taylor, W. H., and Esko, J. D. (1995) Proc. Natl. Acad. Sci. U. S. A. 92, 3323-3327). We now report that another disaccharide, acetylated GlcNAcbeta1-3Gal-naphthalenemethanol (GlcNAcbeta1-3Gal-NM), has even greater potency and that both compounds will inhibit sialyl LewisX (sLex)-dependent cell adhesion. When fed to U937 cells, acetylated forms of Galbeta1-4GlcNAc-NM and GlcNAcbeta1-3Gal-NM primed oligosaccharides in a dose-dependent manner. Analysis of compounds assembled on Galbeta1-4GlcNAc-NM showed only one product, namely Galbeta1-4(Fucalpha1-3)GlcNAc-NM. In contrast, GlcNAcbeta1-3Gal-NM generated Galbeta1-4GlcNAcbeta1-3Gal-NM, Galbeta1-4(Fucalpha1-3)GlcNAcbeta1-3Gal-NM, NeuAcalpha2-3Galbeta1-4GlcNAcbeta1-3Gal-NM, and NeuAcalpha2-3Galbeta1-4(Fucalpha1-3)GlcNAcbeta1- 3Gal-NM. Both compounds decreased the incorporation of [3H]fucose into cellular glycoconjugates, without affecting the incorporation of [3H]mannosamine, a precursor of sialic acid residues. Moreover, the overall extent of sialylation was not affected based on the reactivity of cells to fluorescein isothiocyanate-conjugated Maackia amurensis lectin. Priming inhibited expression of sLex on cell surface glycoconjugates, which reduced E-selectin-dependent cell adhesion to tumor necrosis factor-alpha-activated human umbilical vein endothelial cells. GlcNAcbeta1-3Gal-NM and Galbeta1-4GlcNAc-NM represent starting points for making enzyme-specific, site-directed inhibitors of glycosyltransferases that could act in living cells.

Acetylation↗
PubMed1997

Selectin inhibition: synthesis and evaluation of novel sialylated, sulfated and fucosylated oligosaccharides, including the major capping group of GlyCAM-1.

Selectins interact with glycoconjugate ligands in important normal and pathological situations. While high affinity recognition of natural ligands is associated with alpha 1-3(4)fucosylated, alpha 2-3sialylated (and/or sulfated) lactosamine sequences, small oligosaccharides that potently inhibit the selectins have not been found. One possibility suggested by other investigators is that high affinity may require unusual sequences not yet tested, for example, the "major capping group" (6'-sulfo-sialyl Le(x)) of the L-selectin ligand GlyCAM-1. To explore this possibility, we synthesized a spectrum of novel synthetic and semisynthetic oligosaccharides related to those on natural ligands. In studying these molecules, we noted that binding of recombinant soluble selectins to immobilized sialyl Le(a) or 3'-sulfo-Le(x) is markedly inhibited by concentrations of chloride above the physiological range. This indicates the ionic nature of the interactions, and shows that buffers typically used in screening assays for inhibitors are not optimal. Using parameters that more closely approximate physiological conditions, we confirmed that alpha 2-3-linked sialic acids, and alpha 1-3(4)fucosylation are important for recognition. Similar results obtained with both types of immobilized targets for the three selectins indicated that the binding sites for sialic acid and sulfate are very close, or identical. While O-sulfate esters mostly improved L- and P-selectin recognition, effects depended upon their position and number. Furthermore, sulfation can also impart some "negative" specificity: the major capping group does not interact with E-selectin. The branched Core 2 sequence seemed to enhance L- and P-selectin binding, however, the best inhibitors still appeared to be sialyl Le(a) and 3'-sulfo-Le(x), with the aglycone group of the latter affecting binding. Of particular note, the "major capping group" of GlyCAM-1 was not an unusually potent nor highly selective inhibitor of L-selectin, even when studying the interaction of L-selectin with native GlyCAM-1 itself.

Carbohydrate Sequence↗
PubMed1997

The enzymatic sulfation of glycoprotein carbohydrate units: blood group T-hapten specific and two other distinct Gal:3-O-sulfotransferases as evident from specificities and kinetics and the influence of sulfate and fucose residues occurring in the carbohydrate chain on C-3 sulfation of terminal Gal.

Enzymatic 3-O-sulfation of terminal beta-Gal residues was investigated by screening sulfotransferase activity present in 37 human tissue specimens toward the following synthesized acceptor moieties: Galbeta1,3GalNAc alpha-O-Al, Galbeta1,4GlcNAcbeta-O-Al, Galbeta1,3GlcNAcbeta-O-Al, and mucin-type Galbeta1,4GlcNAcbeta1,6(Galbeta1,3)GalNAc alpha-O-Bn structures containing a C-3 methyl substituent on either Gal. Two distinct types of Gal: 3-O-sulfotransferases were revealed. One (Group A) was specific for the Galbeta1, 3GalNAc alpha- linkage and the other (Group B) was directed toward the Galbeta1,4GlcNAc branch beta1,6 linked to the blood group T hapten. Enzyme activities found in breast tissues were unique in showing a strict specificity for the T-hapten. Galbeta-O-allyl or benzyl did not serve as acceptors for Group A but were very active with Group B. An examination of activity present in six human sera revealed a specificity of the serum enzyme toward beta1,3 linked Gal, particularly, the T-hapten without beta1,6 branching. Group A was highly active toward T-hapten/acrylamide copolymer, anti-freeze glycoprotein, and fetuin O-glycosidic asialo glycopeptide; less active toward fetuin triantennary asialo glycopeptide; and least active toward bovine IgG diantennary glycopeptide. Group B was moderately and highly active, respectively, with the latter two glycopeptides noted and least active with the first two. Competition experiments performed with Galbeta1,3GalNAc alpha-O-Al and Galbeta1,4GlcNAcbeta1,6(Galbeta1,3)GalNAc alpha-O-Bn having a C-3 substituent (methyl or sulfate) on either Gal reinforced earlier findings on the specificity characteristics of Group A and Group B. Group A displayed a wider range of optimal activity (pH 6.0-7.4), whereas Group B possessed a peak of activity at pH 7.2. Mg2+ stimulated Group A 55% and Group B 150%, whereas Mn+2 stimulated Group B 130% but inhibited Group A 75%. Ca2+ stimulated Group B 100% but inhibited Group A 35%. Group A and Group B enzymes appeared to be of the same molecular size (<100,000 Da) as observed by Sephacryl S-100 HR column chromatography. The following effects upon Gal: 3-O-sulfotransferase activities by fucose, sulfate, and other substituents on the carbohydrate chains were noted. (1) A methyl or GlcNAc substituent on C-6 of GalNAc diminished the ability of Galbeta1,3GalNAc alpha-O-Al to act as an acceptor for Group A. (2) An alpha1,3-fucosyl residue on the beta1,6 branch in the mucin core structure did not affect the activity of Group A toward Gal linked beta1,3 to GalNAc alpha-. (3) Lewis x and Lewis a terminals did not serve as acceptors for either Group A or B enzymes. (4) Elimination of Group B activity on Gal in the beta1,6 branch owing to the presence of a 3-fucosyl or 6-sulfo group on GlcNAc did not hinder any action toward Gal linked beta1,3 to GalNAc alpha. (5) Group A activity on Gal linked beta1,3 to GalNAc remained unaffected by 3'-sulfation of the beta1,6 branch. The reverse was true for Group B. (6) The acceptor activity of the T-hapten was increased somewhat upon C-6 sulfation of GalNAc, whereas, C-6 sialylation resulted in an 85% loss of activity. (7) A novel finding was that Galbeta1,4GlcNAcbeta-O-Al and Galbeta1,3GlcNAcbeta-O-Al, upon C-6 sulfation of the GlcNAc moiety, became 100% inactive and 5- to 7-fold active, respectively, in their ability to serve as acceptors for Group B.

Animals↗
PubMed1996

Inhibition of anti-Gal IgG binding to porcine endothelial cells by synthetic oligosaccharides.

Rejection of pig-to-human or pig-to-primate xenografts is mediated by the natural anti-Gal antibody, which interacts with alpha-galactosyl epitopes (i.e., Gal alpha1-3Gal beta1-4GlcNAc-R) abundantly expressed on porcine cells. The objective of this study was to determine the ability of various synthetic oligosaccharides to inhibit the binding of anti-Gal IgG molecules to porcine endothelial cells in vitro. Such inhibition ultimately may help to reduce or to prevent the in vivo antibody-dependent cell cytotoxicity (ADCC) reaction. In the absence of complement-mediated hyperacute rejection, the ADCC induced by anti-Gal IgG molecules is likely to cause the chronic rejection of xenografts. The synthetic free alpha-galactosyl epitope (Gal alpha1-3Gal beta1-4GlcNAc) was found to be 300-fold more effective than melibiose or alpha-methyl galactoside in inhibiting anti-Gal binding to porcine endothelial cells, and to prevent >90% of the antibody binding at a concentration of 1 mM. The disaccharide Gal alpha1-3Gal was ten-fold less effective than the free alpha-galactosyl epitope. Accordingly, the affinity of the disaccharide to anti-Gal, as measured by equilibrium dialysis, was seven-fold lower than that of the trisaccharide. The effective concentration of oligosaccharides inhibiting anti-Gal is independent of the antibody affinity, but is dependent on the concentration of the antibody. Based on the small difference in affinity between Gal alpha1-3Gal beta1-4GlcNAc and Gal alpha1-3Gal beta1-4GIc, and the large difference in the price of N-acetyllactosamine vs. lactose, it is suggested that lactose may be considered as an appropriate starting material for synthesizing large amounts of a trisaccharide that effectively neutralizes anti-Gal.

Animals↗
PubMed1996

Characterization of the specificities of human blood group H gene-specified alpha 1,2-L-fucosyltransferase toward sulfated/sialylated/fucosylated acceptors: evidence for an inverse relationship between alpha 1,2-L-fucosylation of Gal and alpha 1,6-L-fucosylation of asparagine-linked GlcNAc.

The assembly of complex structures bearing the H determinant was examined by characterizing the specificities of a cloned blood group H gene-specified alpha 1,2-L-fucosyltransferase (FT) toward a variety of sulfated, sialylated, or fucosylated Gal beta 1,3/4GlcNAc beta- or Gal beta 1,3GalNAc alpha-based acceptor structures. (a) As compared to the basic type 2, Gal beta 1,4GlcNAc beta-(K(m) = 1.67 mM), the basic type 1 was 137% active (K(m) = 0.83 mM). (b) On C-6 sulfation of Gal, type 1 became 142.1% active and type 2 became 223.0% active (K(m) = 0.45 mM). (c) On C-6 sulfation of GlcNAc, type 2 showed 33.7% activity. (d) On C-3 or C-4 fucosylation of GlcNAc, both types 1 and 2 lost activity. (e) Type 1 showed 70.8% and 5.8% activity, respectively, on C-6 and C-4 O-methylation of GlcNAc. (f) Type 1 retained 18.8% activity on alpha 2,6-sialylation of GlcNAc. (g) Terminal type 1 or 2 of extended chain had lower activity. (h) With Gal in place of GlcNAc in type 1, the activity became 43.2%. (i) Compounds with terminal alpha 1,3-linked Gal were inactive. (j) Gal beta 1,3GalNAc alpha- (the T-hapten) was approximately 0.4-fold as active as Gal beta 1,4GlcNAc beta-. (k) C-6 sulfation of Gal on the T-hapten did not affect the acceptor activity. (l) C-6 sulfation of GalNAc decreased the activity to 70%, whereas on C-6 sulfation of both Gal and GalNAc the T-hapten lost the acceptor ability. (m) C-6 sialylation of GalNAc also led to inactivity. (n) beta 1,6 branching from GalNAc of the T-hapten by a GlcNAc residue or by units such as Gal beta 1, 4GlcNAc-, Gal beta 1,4(Fuc alpha 1,3)GlcNAc-, or 3-sulfoGal beta 1,4GlcNAc- resulted in 111.9%, 282.8%, 48.3%, and 75.3% activities, respectively. (o) The enhancement of enzyme affinity by a sulfo group on C-6 of Gal was demonstrated by an increase (approximately 5-fold) in the K(m) for Gal beta 1,4GlcNAc beta 1,6(Gal beta 1,3)GalNAc alpha-O-Bn in presence of 6-sulfoGal beta 1,- 4GlcNAc beta-O-Me (3.0 mM). (p) Among the two sites in Gal beta 1, 4GlcNAc beta 1,6(Gal beta 1,3) GalNAc alpha-O-Bn, the enzyme had a higher affinity ( > 3-fold) for the Gal linked to GlcNAc. (q) With respect to Gal beta 1,- 3GlcNAc beta-O-Bn (3.0 mM), fetuin triantennary asialo glycopeptide (2.4 mM), bovine IgG diantennary glycopeptide (2.8 mM), asialo Cowper's gland mucin (0.06 mM), and the acrylamide copolymers (0.125 mM each) containing Gal beta 1,3GlcNAc beta-, Gal beta 1,3(6-sulfo)GlcNAc beta-, Gal beta 1,3GalNAc alpha-, Gal beta 1,3Gal beta-, or Gal alpha 1,3Gal beta- units were 153.6%, 43.0%, 6.2%, 52.5%, 94.9%, 14.7%, 23.6%, and 15.6% active, respectively. (r) Fucosylation by alpha 1,2-L-FT of the galactosyl residue which occurs on the antennary structure of the bovine IgG glycopeptide was adversely affected by the presence of an alpha 1,6-L-fucosyl residue located on the distant glucosaminyl residue that is directly attached to the asparagine of the protein backbone. This became evident from the 4-fold activity of alpha 1,2-L-FT toward bovine IgG glycopeptide after approximately 5% removal of alpha 1,6-linked Fuo.

ABO Blood-Group System↗
PubMed1996

Specificity analysis of three clonal and five non-clonal alpha 1,3-L-fucosyltransferases with sulfated, sialylated, or fucosylated synthetic carbohydrates as acceptors in relation to the assembly of 3'-sialyl-6'-sulfo Lewis x (the L-selectin ligand) and related complex structures.

Unique specificities of the cloned alpha 1,3-L-fucosyltransferases (FTs), FT III (Lewis type), FT IV (myeloid type), and FT V (plasma type), and the alpha 1,3-FTs of Colo 205 (colon carcinoma), HL 60 (myeloid), B142 (lymphoid), EKVX (lung carcinoma), and calf mesenteric lymph nodes (CMLN) were discerned with sulfated, sialylated, and/or fucosylated Gal beta 1,3/4GlcNAc beta-based acceptor moieties. (a) FT V was 1.0-, 20.8-, and 4.6-fold active in forming Lewis x, Lewis y, and 3'-alpha-galactosyl Lewis x, respectively. (b) FT III and FT V formed approximately 4-fold 3'-sulfo Lewis x, as compared to 3'-sialyl Lewis x. (c) FT IV showed great efficiency in forming 3'-sulfo Lewis x (249%) and Lewis x (345%) in mucin-type branched chains. (d) FT III, FT IV, and FT V formed 19%, 62%, and 47% 6-sulfo Lewis x as compared to Lewis x. (e) 6'-Sulfo Lewis x and 3'-sialyl-6'-sulfo Lewis x (GLYCAM ligand) were not synthesized from their immediate precursors by FT III, FT IV, or FT V. (f) FT III, FT IV, and FT V were 311%, 9%, and 188% active, respectively, with 2'-fucosyl lactose but were not active with 2'- fucosyl-6'-sulfo lactose. (g) FT III and FT V were 7.0- and 0.5-fold active in forming Lewis a as compared to Lewis x, whereas, FT IV was inactive. (h) FT III was -2.0-fold more active in forming 3'-alpha-galactosyl Lewis a than Lewis b. (i) FT III synthesized 6-sialyl Lewis a (40% efficiency as compared to Lewis a) from 6-sialyl type 1. (j) FT III did not act on 6'-sulfo or 6'-sialyl type 1 but was 106% and 22% active with 3'-sulfo and 6-sulfo type 1, respectively. (k) The Colo 205 FT activities with type 1 compounds almost paralleled that of FT III except for the low activity (9%) with Gal beta 1,3(NeuAc alpha 2, 6)GlcNAc beta-O-Bn, but with type 2 considerable differences between Colo 205 FT and FT III were noticed. (l) The alpha 1,3-FTs of CMLN, HL60, B142, and EKVX were 1.2-1.7 times active with Fuc alpha 1,2Gal beta 1,4GlcNAc beta- O-pNP and Gal alpha 1,3Gal beta 1,4 GlcNAc beta-O-Bn with respect to Gal beta 1,4GlcNAc beta-O-Al. (m) Both CMLN and HL60 FTs were 2-fold active with 3-sulfoGal beta 1,4GlcNAc in a mucin-type branch structure such as 3-sulfoGal beta 1,4GlcNAc beta 1,6(Gal beta 1,3)GalNAc alpha-O-Bn. (n) The 3'-sulfoLacNAc/acrylamide copolymer, either as an acceptor or as a competitive inhibitor, had the potential to distinguish myeloid type alpha 1,3-FT from the plasma type.

Animals↗
PubMed1996

Chemical synthesis of a hexasaccharide comprising the Lewisx determinant linked beta-(1-->6) to a linear trimannosyl core and the precursor pentasaccharide lacking fucose.

Phenyl 2,3,4-tri-O-acetyl-6-O-chloroacetyl-1-thio-alpha,beta-mannopyranoside (5) was condensed with benzyl O-(2,3,4-tri-O-benzyl-beta-D-mannopyranosyl)-(1-->6)-2,3,4-tri-O-benzyl- alpha-D- mannopyranoside (12) in the presence of NIS-triflic acid to give, after removal of the chloroacetyl group, the key intermediate, benzyl O-(2,3,4-tri-O-acetyl-alpha-D-mannopyranosyl)-(1-->6)-O-(2,3,4-tri-O-ben zyl- beta-D-mannopyranosyl)-(1-->6)-2,3,4-tri-O-benzyl-alpha-D-mannopyranosid e (14). A similar condensation of 6 and 7 with acceptor 14, followed by the removal of protecting groups, afforded 16 and 18, respectively. These compounds are expected to be useful in specificity studies of an antibody raised against a related, synthetic antigen that we are currently investigating.

Antigens, CD↗
PubMed1996

Synthesis of precursors for the dimeric 3-O-SO3Na Lewis X and Lewis A structures.

Stereoselective syntheses of 3-O-SO3Na-beta-Gal-(1-->4)-beta-GlcNAc- (1-->3)-beta-Gal-(1-->4)-GlcNAc-beta-OBn (15) and 3-O-SO3Na-beta-Gal-(1-->3)-beta-GlcNAc-(1-->3)-beta-Gal-(1-->3)- beta-GlcNAc-(1-->3)-beta-Gal-(1-->4)-Glc-beta-OBn (25) were accomplished through the use of two novel glycosyl donors, namely, ethyl O-(2,6-di-O-acetyl-3,4-O-isopropylidene-beta-D- galactopyranosyl)-(1-->4)-3-O-acetyl-2-deoxy-2-phthalimido-1-thio-6-O- trimethylacetyl-beta-D-glucopyranoside (8). and ethyl O-(2,6-di-O-acetyl-3,4-O-isopropylidene-beta-D-galactopyranosyl)- (1-->3)-4-O-acetyl-2-deoxy-2-phthalimido-1-thio-6-O-trimethylace tyl-beta-D-glucopyranoside (18).

CA-19-9 Antigen↗
PubMed1996

Inhibition of human HT-29 colon carcinoma cell adhesion by a 4-fluoro-glucosamine analogue.

Cell surface glycoconjugates play an important role in cellular recognition and adhesion. Modification of these structures in tumour cells could affect tumour cell growth and behaviour, including metastasis. 2-Acetamido-1,3,6-tri-O-acetyl-4-deoxy-4-fluoro-alpha-D-glycopyranose (4-F-GlcNAc) was synthesized as a potential inhibitor and/or modifier of tumour cell glycoconjugates. The effect of this sugar analogue on the adhesive properties of human colon carcinoma HT-29 cells was evaluated. Treatment of HT-29 cells with 4-F-GlcNAc led to reduced cell surface expression of terminal lactosamine, sialy-Le(x) and sialyl-Le(a), as determined by Western blotting and flow cytometry. The aberrant expression of these oligosaccharide structures on the HT-29 cell surface resulted in: (1) decreased E-selectin mediated adhesion of human colon cells to human umbilical cord endothelial cells (HUVEC); (2) impaired adhesion of HT-29 cells to beta-galactoside binding lectin, galectin-1; and (3) reduced ability to form homotypic aggregates. After exposure to 4-F-GlcNAc, lysosomal associated membrane proteins (lamp) 1 and 2, and carcinoembryonic antigen (CEA) detected in HT-29 cells were of lower molecular weight, probably due to impaired glycosylation. These results strongly suggest that modification of tumour cell surface molecules can alter tumour cell adhesion and that tumour cell surface oligosaccharides may be suitable targets for therapeutic exploitation.

Blotting, Western↗
PubMed1995

Synthetic mucin fragments: synthesis of O-sulfo and O-methyl derivatives of allyl O-(beta-D-galactopyranosyl)-(1-->3)-2-acetamido-2-deoxy-alpha-D- galactopyranoside as potential compounds for sulfotransferases.

Allyl 2-acetamido-4,6-O-(4-methoxybenzylidene)-2-deoxy-alpha-D-galact opy ranoside (1) was condensed with either 2,3,4,6-tetra-O-acetyl-alpha-D-galactopyranosyl bromide (2) or 2,3,4-tri-O-benzoyl-6-O-bromoacetyl-alpha-D-galactopyranosyl bromide (14) in the presence of mercuric cyanide. Selective substitution with methyl, sulfo or both at desired positions, followed by the removal of protecting groups, afforded allyl O-(beta-D-galactopyranosyl)-(1-->3)-2-acetamido-2-deoxy-6-O-methyl-alpha -D- galactopyranoside (5), allyl O-(6-O-sulfo-beta-D-galactopyranosyl sodium salt)-(1-->3)-2-acetamido-2-deoxy-6- O-methyl-alpha-D-galactopyranoside (10), allyl O-(beta-D-galactopyranosyl)-(1-->3)-2-acetamido-2-deoxy-6-O-sulfo-alpha- D- galactopyranoside sodium salt (13), allyl O-(6-O-sulfo-beta-D-galactopyranosyl sodium salt)-(1-->3)-2-acetamido-2-deoxy- alpha-D-galactopyranoside (17) and allyl O-(3-O-sulfo-beta-D-galactopyranosyl sodium salt)-(1-->3)-2-acetamido-2-deoxy- alpha-D-galactopyranoside (22). The structures of compounds 5, 10, 13, 17 and 22 were established by 13C NMR and FAB mass spectroscopy.

Acetylgalactosamine↗
PubMed1995

Synthesis of oligosaccharide substrates for N-linked glycoprotein processing enzymes.

The stereoselective syntheses of one pentasaccharide and one tetrasaccharide containing the Glc-alpha-(1-->3)-Man-alpha moiety as their terminal unit, as well as one tetrasaccharide and one trisaccharide containing the Man-alpha-(1-->2)-Man-alpha terminal unit were accomplished through the utilization of two key glycosyl donors, namely, 4-pentenyl 3-O-acetyl-2,4,6-tri-O-benzyl-alpha-D-mannopyranoside and ethyl 2-O-acetyl-3,4,6-tri-O-benzyl-1-thio-alpha-D-mannopyranoside.

Carbohydrate Conformation↗
PubMed1995

Expression of blood group Lewis b determinant from Lewis a: association of this novel alpha (1,2)-L-fucosylating activity with the Lewis type alpha (1,3/4)-L-fucosyltransferase.

Blood group H type 1 [Fuc alpha (1,2)Gal beta (1,3)GlcNAc beta-->] is known as the precursor structure of the blood group determinant, Lewis b [Fuc alpha (1,2)Gal beta (1,3)(Fuc alpha (1,4))GlcNAc beta-->]. Recently, a new biosynthetic route for Lewis b from Lewis a [Gal beta (1,3)(Fuc alpha (1,4))GlcNAc-->] was identified in human gastric carcinoma cells, colon carcinoma Colo 205, and ovarian tumor. The present study demonstrates the association of this new type of alpha (1,2)-L-fucosyltransferase (FT) activity with the Lewis-type alpha (1,3/4)-L-FT as follows: (i) the alpha (1,4)- and novel alpha (1,2)-FT activities of Colo 205 were much less inhibited than the alpha (1,3)-FT activity by N-ethylmaleimide [Ki(microM) = 714.0, 119.0, and 6.5 respectively]. (ii) The alpha (1,4)- and novel alpha (1,2)-FT activities emerged from a Sephacryl S-200 column in identical positions. (iii) A specific inhibitor (copolymer from 3-sulfo-Galbeta(1,3)GlcNAcbeta-O-allyl and acrylamide) of alpha(1,4)-FT activity inhibited both alpha(1,4)- and alpha(1,2)-FT activities in Sephacryl S-200 column effluent to almost the same extent (approximately 80%); (iv) separation of the Lewis-type alpha(1,3/4)-FT from the plasma-type alpha(1,3)-FT by specific elution of the affinity column (bovine IgG glycopep-Sepharose) with lactose and further purification on a Sephacryl S-100 HR column showed that (a) the alpha(1,3)-FT activity was the inherent capacity of the Lewis-type FT (Colo 205 fraction L) since approximately 90% of both the alpha(1,4)- and alpha(1,3)-FT activities is inhibited by the copolymer, (b) the unique ability of catalyzing the alpha(1,2)-L-fucosylation of Gal in Lewis a structure and also the alpha(1,3)-L-fucosylation of Glc in lactose-based structure belonged to the Lewis type enzyme (Colo 205 fraction L), (c) a measurement of the [14C]fucosyl products arising from the two acceptors Galbeta(1,3)(4,6-di-O-Me)GlcNAcbeta-O-Bn and 3-sulfo-Galbeta(1,3)GlcNAcbeta-O-A1 (specific for alpha(1,2) and alpha(1,4), respectively) taken in the same incubation mixture showed mutual inhibition by the acceptors ([Km for the alpha(1,4)-specific acceptor, 3-sulfo-Galbeta(1,3)GlcNAcbeta-O-A], increased from 32 to 50 microM in the presence of 7.5 mM Galbeta(1,3)(4,6-di-O-Me)GlcNAcbeta-O-Bn, whereas Ki for the mutual inhibition of alpha(1,2)-FT activity by the former was 102 microM], and (d) the Lewis-type FT, in contrast to the plasma type FT, was highly effective in fucosylating complex glycopeptides. (iv) A cloned FT (FT III:Lewis type) and the Colo 205 Lewis-type FT (fraction L) showed similar activities toward various acceptors; the enzymatic product resulting from the action of cloned FT on Galbeta(1,3)(Fucalpha(1,4))GlcNAc-beta-O-Bn was identified by FAB mass spectrometry as the difucosyl compound. (v) An examination of six human cell lines indicated that the novel alpha(1,2)-FT activity associates with the alpha(1,4)-FT activity.

Autoradiography↗
PubMed1995

Genetic and enzymatic evidence for Lewis enzyme expression in Lewis-negative cancer patients.

It has been observed that the frequency of individuals with Lewis-negative erythrocytes is significantly higher in cancer patients than in healthy controls. In this study, 20 of the 66 (30.3%) patients with various cancers were typed as Lewis negative from their erythrocytes, while the same frequency in healthy controls was 11.1%. These 20 patients were divided into three groups based on the presence of Lewis blood group antigens and alpha 1-->4-fucosyltransferase in their salivas: group I, 6 patients who had both Lewis antigens and alpha 1-->4-fucosyltransferase activity; group II, 8 patients who had no Lewis antigens but possessed alpha 1-->4-fucosyltransferase activity; group III, 6 patients who had neither Lewis antigens nor alpha 1-->4-fucosyltransferase activity. The genotyping of Le genes by the PCR-RFLP methods, which have been developed and established by us recently, demonstrated that all 14 patients from groups I and II possess Le gene homozygously (Le/Le) or heterozygously (Le/le), whereas all 6 patients from group III were le/le homozygotes. Only the 6 patients from group III were identified as the genuine Lewis-negative individuals. The immunohistochemical staining of the colorectal tumors also showed that the Lewis antigens could be detected on the tumors from groups I and II but not from group III.

Base Sequence↗
PubMed1995

Characterization of sialyloligosaccharide binding by recombinant soluble and native cell-associated CD22. Evidence for a minimal structural recognition motif and the potential importance of multisite binding.

CD22, a B cell-specific receptor of the immunoglobulin superfamily, has been demonstrated to bind to oligosaccharides containing alpha 2-6-linked sialic acid (Sia) residues. Previously, we demonstrated that the minimal structure recognized by this lectin is the trisaccharide Sia alpha 2-6Gal beta 1-4GlcNAc, as found on N-linked, O-linked, or glycolipid structures (Powell, L., and Varki, A. (1994) J. Biol. Chem. 269, 10628-10636). Here we utilize a soluble immunoglobulin fusion construct (CD22Rg) to determine directly by equilibrium dialysis the stoichiometry (2:1) and dissociation constant (32 microM) for Neu5Ac alpha 2-6Gal beta 1-4Glc binding. Inhibition assays performed with over 30 different natural and synthetic sialylated and/or sulfated compounds are utilized to define in greater detail specific structural features involved in oligosaccharide-protein binding. Specifically, the critical features required for binding include the exocyclic hydroxylated side chain of the Sia residue and the alpha 2-6 linkage position to the underlying Gal unit. Surprisingly, alterations of the 2-, 3-, and 4-positions of the latter residue have limited effect on the binding. The nature of the residue to which the Gal is attached may affect binding. Bi(alpha 2-6)-sialylated biantennary oligosaccharides are capable of simultaneously interacting with both lectin sites present on the dimeric CD22Rg fusion construct, giving a marked improvement in binding over monosialylated compounds. Furthermore, data are presented indicating that full-length native CD22, expressed on the surface of Chinese hamster ovary cells, is structurally and functionally a multimeric protein, demonstrating a higher apparent affinity for multiply sialylated compounds over monosialylated compounds. These observations provide a mechanism for strong CD22-dependent cell adhesion despite the relatively low Kd for protein-sugar binding.

Animals↗