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Khushi L Matta

Publications and source records attributed to Khushi L Matta.

16 recordsLinked to original sources
PubMed2006

Synthesis of fluorinated mucin core 2 branched oligosaccharides with the potential of novel substrates and enzyme inhibitors for glycosyltransferases and sulfotransferases.

Syntheses of fluorinated mucin core 2 tri- and tetrasaccharides modified at the C-3 or C-4 position of the pertinent galactose residue are reported. These compounds were used for the study of sialyltransferases and 3-O-sulfotransferases involved in the biosynthesis of O-glycans. Our acceptor substrate specificity studies on three cloned sialyltransferases (Sia-Ts) revealed that a 3- or 4-fluoro substituent in beta1,4Gal resulted in poor acceptors for alpha2,6(N)Sia-T and alpha2,3(N)Sia-T, whereas 4-fluoro-Galbeta1,3GalNAcalpha was a good acceptor for alpha2,3(O)Sia-T. Uniquely, 4-F-Galbeta1,4GlcNAcbeta1,6(Galbeta1,3)GalNAcalpha-OBn was an inhibitor of alpha2,6(N)Sia-T activity but not alpha2,3(N)Sia-T activity. Further we found that the activities of only Gal 3-O-sulfotransferases and not sialyltransferases were adversely affected by a C-3 fluoro substituent at the other Gal terminal of mucin core 2. The strategy of building branched mucin core 2 structures by three glycosidation sequence coupling three classes of glycosyl donors with the reactivity-matching acceptors proved to be successful in syntheses of modified mucin-type core structures of O-glycan. The relative poor yields of the glycosylations using fluorinated galactosyl donors indicated that the fluorine modification dramatically decreased the donor reactivity due to electron-withdrawing effect.

Carbohydrate Conformation↗
PubMed2006

The pattern of glycosyl- and sulfotransferase activities in cancer cell lines: a predictor of individual cancer-associated distinct carbohydrate structures for the structural identification of signature glycans.

Carbohydrate chains of cancer glycoprotein antigens contain major outer changes dictated by tissue-specific regulation of glycosyltransferase genes, the availability of sugar nucleotides, and competition between enzymes for acceptor intermediates during glycan elongation. However, it is evident from recent studies with recombinant mucin probes that the final glycosylation profiles of mucin glycoproteins are mainly determined by the cellular repertoire of glycosyltransferases. Hence, we examined various cancer cell lines for the levels of fucosyl-, beta-galactosyl, beta-N-acetylgalactosaminyl-, sialyl-, and sulfotransferase activities that generate the outer ends of the oligosaccharide chains. We have identified glycosyltransferases activities at the levels that would give rise to O-glycan chains as reported by others in breast cancer cell lines, T47D, ZR75-1, MCF-7, and MDA-MB-231. Most breast cancer cells express Gal-3-O-sulfotransferase specific for T-hapten Gal beta1-->3GalNAc alpha-, whereas the enzyme from colon cancer cells exhibits a vast preference for the Gal beta1,4GlcNAc terminal unit in O-glycans. We also studied ovarian cancer cells SW626 and PA-1 and hepatic cancer cells HepG2. Our studies show that alpha1,2-L-fucosyl-T, alpha(2,3) sialyl-T, and 3-O-Sulfo-T capable of acting on the mucin core 2 tetrasaccharide, Gal beta1,4GlcNAc beta1,6(Gal beta1,3)GalNAc alpha-, can also act on the Globo H antigen backbone, Gal beta1,3GalNAc beta1,3Gal alpha-, suggesting the existence of unique carbohydrate moieties in certain cancer-associated glycolipids. Briefly, our study indicates the following: (i) 3'-Sulfo-T-hapten has an apparent relationship to the tumorigenic potential of breast cancer cells; (ii) the 3'-sulfo Lewis(x), the 3-O-sulfo-Globo unit, and the 3-fucosylchitobiose core could be uniquely associated with colon cancer cells; (iii) synthesis of a polylactosamine chain and T-hapten are favorable in ovarian cancer cells due to negligible sialyltransferase activities; and (iv) a 6'-sialyl LacNAc unit and 3'-sialyl T-hapten appear to be prevalent structures in hepatic cancer cell glycans. Thus, it is apparent that different cancer cells are expressing unique glycan epitopes, which could be novel targets for cancer diagnosis and treatment.

Breast Neoplasms↗
PubMed2006

Molecular characterization of a novel UDP-galactose:fucoside alpha3-galactosyltransferase that modifies Skp1 in the cytoplasm of Dictyostelium.

Skp1 is a nucleocytoplasmic protein that is post-translationally modified by a pentasaccharide, Gal alpha1,Gal alpha1,3Fuc alpha1,2Gal-beta1,3GlcNAc alpha1O-, at a 4-hydroxylated derivative of Pro-143 in the amebazoan Dictyostelium discoideum. An enzymatic activity that catalyzes formation of the Gal alpha1,3Fuc linkage by transfer of Gal from UDP-alphaGal to Fuc alpha1,2Gal beta1,3GlcNAc alpha1O-benzyl, or the corresponding glycoform of Skp1, was described previously in cytosolic extracts of Dictyostelium. A protein GT78 associated with this activity has been purified to chromatographic homogeneity. In-gel tryptic digestion followed by nano-liquid chromatography-mass spectrometry on a quadrupole time-of-flight geometry instrument with data-dependent tandem mass spectrometry acquisition yielded a number of peptide fragmentation spectra, nine of which were manually de novo sequenced and found to map onto a predicted 3-exon gene of unknown function on chromosome 4. GT78 is predicted to comprise 648 amino acids with an N-terminal glycosyltransferase and a C-terminal beta-propeller domain. Overexpression of GT78 with a His6-tag resulted in a 120-fold increase in GalT-activity in cytosolic extracts, and purified His6-GT78 exhibited alpha3GalT-activity toward a synthetic acceptor substrate. Expression of the truncated N-terminal region confirmed the predicted catalytic activity of this domain. Disruption of the GT78 gene led to a loss of enzyme activity in extracts and accumulation of the non-galactosylated isoform of Skp1 in cells. GT78 therefore represents the Skp1 alpha3GalT, and its mechanism conforms to the sequential model of Skp1 glycosylation in the cytoplasm shown for earlier enzymes in the pathway. Informatics studies suggest that related catalytic domains are expressed in the Golgi or cytoplasm of plants, other protozoans, and animals.

Amino Acid Sequence↗
PubMed2006

Quantitative measurement of selectin-ligand interactions: assays to identify a sweet pill in a library of carbohydrates.

Soluble oligosaccharides and glycoproteins can inhibit leukocyte adhesion during a range of vascular ailments including inflammation, thrombosis, and cancer metastasis. The design of such molecules in many cases is based on the structure of naturally occurring selectin ligands. In this case, synthetic selectin-ligand mimetics act as competitive inhibitors of cell adhesion. In an alternate approach, cell-permeable, small-molecule oligosaccharides have been shown to alter the metabolic pathways that lead to the biosynthesis of functional selectin-ligands. The addition of such molecules results in glycoproteins that are defective in their ability to bind selectins. Quantitative in vitro testing of the efficacy of the above inhibition strategies ideally requires the application of assays that mimic the in vivo physiological milieu in terms of the valency of selectin and selectin-ligands, the physiological fluid-flow conditions, and the use of blood cells. Assays that are performed in small volumes are preferable when the quantity of available inhibitor is scarce. Finally, the measurements must account for the rapid on- and off-rates of selectin-mediated binding interactions. This chapter addresses these issues by presenting methods to measure selectin function in enzyme-linked immunosorbent assay and flow cytometry-based static assays, cell-adhesion assays performed under shear flow in cone-plate viscometers, and Biacore surface plasmon resonance measurements of molecular-binding kinetics. Examples are presented where such methods are applied to measure the ability of simple oligosaccharides based on sialyl Lewis-X and complex molecules with the core-2 structure to block selectin function. Such methods may be extended to identify potent selectin antagonists in a library of carbohydrates.

Combinatorial Chemistry Techniques↗
PubMed2005

Analysis of the specificity of sialyltransferases toward mucin core 2, globo, and related structures. identification of the sialylation sequence and the effects of sulfate, fucose, methyl, and fluoro substituents of the carbohydrate chain in the biosynthesis of selectin and siglec ligands, and novel sialylation by cloned alpha2,3(O)sialyltransferase.

Sialic acids are key determinants in many carbohydrates involved in biological recognition. We studied the acceptor specificities of three cloned sialyltransferases (STs) [alpha2,3(N)ST, alpha2,3(O)ST, and alpha2,6(N)ST] and another alpha2,3(O)ST present in prostate cancer cell LNCaP toward mucin core 2 tetrasaccharide [Galbeta1,4GlcNAcbeta1,6(Galbeta1,3)GalNAcalpha-O-Bn] and Globo [Galbeta1,3GalNAcbeta1,3Galalpha-O-Me] structures containing sialyl, fucosyl, sulfo, methyl, or fluoro substituents by identifying the products by electrospray ionization tandem mass spectral analysis and other biochemical methods. The Globo precursor was an efficient acceptor for both alpha2,3(N)ST and alpha2,3(O)ST, whereas only alpha2,3(O)ST used its deoxy analogue (d-Fucbeta1,3GalNAcbeta1,3-Gal-alpha-O-Me); 2-O-MeGalbeta1,3GlcNAc and 4-OMeGalbeta1,4GlcNAc were specific acceptors for alpha2,3(N)ST. Other major findings of this study include: (i) alpha2,3 sialylation of beta1,3Gal in mucin core 2 can proceed even after alpha1,3 fucosylation of beta1,6-linked LacNAc. (ii) Sialylation of beta1,3Gal must precede the sialylation of beta1,4Gal for favorable biosynthesis of mucin core 2 compounds. (iii) alpha2,3 sialylation of the 6-O-sulfoLacNAc moiety in mucin core 2 (e.g., GlyCAM-1) is facilitated when beta1,3Gal has already been alpha2,3 sialylated. (iv) alpha2,6(N)ST was absolutely specific for the beta1,4Gal in mucin core 2. Either alpha1,3 fucosylation or 6-O-sulfation of the GlcNAc moiety reduced the activity. Sialylation of beta1,3Gal in addition to 6-O-sulfation of GlcNAc moiety abolished the activity. (v) Prior alpha2,3 sialylation or 3-O-sulfation of beta1,3Gal would not affect alpha2,6 sialylation of Galbeta1,4GlcNAc of mucin core 2. (vi) A 3- or 4-fluoro substituent in beta1,4Gal resulted in poor acceptors for the cloned alpha2,6(N)ST and alpha2,3(N)ST, whereas 4-fluoro- or 4-OMe-Galbeta1,3GalNAcalpha was a good acceptor for cloned alpha2,3(O)ST. (vii) 4-O-Methylation of beta1,4Gal abolished the acceptor ability toward alpha2,6(N)ST but increased the acceptor efficiency considerably toward alpha2,3(N)ST. (viii) Just like LNCaPalpha1,2-FT and Gal-3-O-sulfotransferase T2, the cloned alpha2,3(N)ST which modifies terminal Gal in Galbeta1,4GlcNAc also efficiently utilizes the terminal beta1,3Gal in the Globo backbone. Utilization of C-3 blocked compounds such as 3-O-sulfo-Galbeta1,3GalNAcbeta1,3Galalpha-OMe as acceptors by cloned alpha2,3(O)ST and analyses of the resulting products by lectin chromatography and mass spectrometry indicate that alpha2,3(O)ST is capable of attaching NeuAc to another position in C-3-substituted beta1,3Gal.

Antigens, Tumor-Associated, Carbohydrate↗
PubMed2005

Affinity and kinetics of sialyl Lewis-X and core-2 based oligosaccharides binding to L- and P-selectin.

Soluble oligosaccharide mimetics of natural selectin ligands act as competitive inhibitors of leukocyte adhesion in models of inflammation. We quantified the binding of simple oligosaccharides based on sialyl Lewis-X (sLe(X)) and complex molecules with the core-2 structure to L- and P-selectin, under both static and fluid flow conditions. Isolated human neutrophils were employed to mimic the physiological valency of selectins and selectin ligands. Surface plasmon resonance studies quantified binding kinetics. We observed the following: (i) The functional group at the anomeric position of carbohydrates plays an important role during selectin recognition, since sLe(X) and sialyl Lewis-a (sLe(a)) were approximately 5-7-fold poorer inhibitors of L-selectin mediated cell adhesion compared to their methyl glycosides. (ii) Despite their homology to physiological glycans, the putative carbohydrate epitopes of GlyCAM-1 and PSGL-1 bound selectins with low affinity comparable to that of sLe(X)-selectin interactions. Thus, besides the carbohydrate portion, the protein core of GlyCAM-1 or the presentation of carbohydrates in clusters on this glycoprotein may contribute to selectin recognition. (iii) A compound Galbeta1,4(Fucalpha1,3)GlcNAcbeta1,6(GalNAcbeta1,3)GalNAcalpha-OMe was identified which blocked L- and P-selectin binding at 30-100-fold lower doses than sLe(X). (iv) Surface plasmon resonance experiments determined that an sLe(X) analogue (TBC1269) competitively inhibited, via steric/allosteric mechanisms, the binding of two anti-P-selectin function blocking antibodies that recognized different epitopes of P-selectin. (v) TBC1269 bound P-selectin via both calcium-dependent and -independent mechanisms, with K(D) of approximately 111.4 microM. The measured on- and off-rates were high (k(off) > 3 s(-)(1), k(on) > 27,000 M(-)(1) s(-)(1)). Similar binding kinetics are expected for sLe(X)-selectin interactions. Taken together, our study provides new insight into the kinetics and mechanisms of carbohydrate interaction with selectins.

Animals↗
PubMed2004

Specificity of a soluble UDP-galactose: fucoside alpha1,3-galactosyltransferase that modifies the cytoplasmic glycoprotein Skp1 in Dictyostelium.

Skp1 is an adaptor-like protein in E3(SCF)-ubiquitin ligases and other multiprotein complexes of the cytoplasm and nucleus. In Dictyostelium, Skp1 is modified by an unusual pentasaccharide containing a Galalpha1-Fuc linkage, whose formation is examined here. A cytosolic extract from Dictyostelium was found to yield, after 2400-fold purification, an activity that could transfer Gal from UDP-Gal to both a Fuc-terminated glycoform of Skp1 and synthetic Fuc conjugates in the presence of Mn(2+) and dithiothreitol. The microsomal fraction was devoid of activity. The linkage formed was Galalpha1,3Fuc based on co-chromatography with only this synthetic isomer conjugate, and sensitivity to alpha1,3/6-galactosidase. Skp1 exhibited an almost 1000-fold lower K(m) and 35-fold higher V(max) compared with a simple alpha-fucoside, but this advantage was abolished by denaturation or alkylation of Cys residues. A comparison of a complete series of synthetic glycosides representing the non-reducing terminal mono-, di-, and trisaccharides of Skp1 revealed, surprisingly, that the disaccharide is most active owing primarily to a V(max) advantage, but still much less active than Skp1 itself because of a K(m) difference. These findings indicate that alpha-GalT1 is a cytoplasmic enzyme whose modification of Skp1 requires proper presentation of the terminal acceptor disaccharide by a folded Skp1 polypeptide, which correlates with previous evidence that the Galalpha1,3Fuc linkage is deficient in expressed mutant Skp1 proteins.

Animals↗
PubMed2004

Determination of linkage position and anomeric configuration in Hex-Fuc disaccharides using electrospray ionization tandem mass spectrometry.

Fixed-energy sequential tandem mass spectrometry (MS(n)) capabilities offered by quadrupole ion trap instruments have been explored in a systematic study of six isomers of Gal-Fucalpha-OBenzyl disaccharides. Under collision-induced dissociation (CID), sodiated molecular species generated in the positive-ion electrospray ionization mode yield simple and predictable mass spectra. Information on interglycosidic linkages and configurations can be deduced from the relative intensities of the selected diagnostic fragments arising from the glycosidic bond cleavages and corroborated by the fragments arising from cross-ring cleavages. As the CID patterns are not dependent on the number of prior tandem mass spectrometric steps, structures can be unambiguously assigned by matching the spectra with a library. The rules governing the fragmentation behavior of this class of oligosaccharides were tested for a representative isomeric disaccharide, Glcbeta1,3Fucalpha-OAllyl. The findings establish a basis for using MS(n) with a quadrupole ion trap instrument to elucidate structures of hexose-fucose subunits from more complicated oligosaccharides. Energy-resolved mass spectra were also acquired by CID tandem triple-quadrupole mass spectrometry. The breakdown behavior of the molecular ions revealed patterns which could differentiate stereoisomers of Gal-Fuc disaccharides over a range of collision energy from 20 to 50 eV.

Disaccharides↗
PubMed2003

Identification of physiologically relevant substrates for cloned Gal: 3-O-sulfotransferases (Gal3STs): distinct high affinity of Gal3ST-2 and LS180 sulfotransferase for the globo H backbone, Gal3ST-3 for N-glycan multiterminal Galbeta1, 4GlcNAcbeta units and 6-sulfoGalbeta1, 4GlcNAcbeta, and Gal3ST-4 for the mucin core-2 trisaccharide.

Sulfated glycoconjugates regulate biological processes such as cell adhesion and cancer metastasis. We examined the acceptor specificities and kinetic properties of three cloned Gal:3-O-sulfotransferases (Gal3STs) ST-2, ST-3, and ST-4 along with a purified Gal3ST from colon carcinoma LS180 cells. Gal3ST-2 was the dominant Gal3ST in LS180. While the mucin core-2 structure Galbeta1,4GlcNAcbeta1,6(3-O-MeGalbeta1,3)GalNAcalpha-O-Bn (where Bn is benzyl) and the disaccharide Galbeta1,4GlcNAc served as high affinity acceptors for Gal3ST-2 and Gal3ST-3, 3-O-MeGalbeta1,4GlcNAcbeta1,-6(Galbeta1,3)GalNAcalpha-O-Bn and Galbeta1,3GalNAcalpha-O-Al (where Al is allyl) were efficient acceptors for Gal3ST-4. The activities of Gal3ST-2 and Gal3ST-3 could be distinguished with the Globo H precursor (Galbeta1,3GalNAcbeta1,3Galalpha-O-Me) and fetuin triantennary asialoglycopeptide. Gal3ST-2 acted efficiently on the former, while Gal3ST-3 showed preference for the latter. Gal3ST-4 also acted on the Globo H precursor but not the glycopeptide. In support of the specificity, Gal3ST-2 activity toward the Galbeta1,4GlcNAcbeta unit on mucin core-2 as well as the Globo H precursor could be inhibited competitively by Galbeta1,4GlcNAcbeta1,6(3-O-sulfoGalbeta1,3)GalNAcalpha-O-Bn but not 3-O-sulfoGalbeta1,-4GlcNAcbeta1,6(Galbeta1,3)GalNAcalpha-O-Bn. Remarkably these sulfotransferases were uniquely specific for sulfated substrates: Gal3ST-3 utilized Galbeta1,4(6-O-sulfo)-GlcNAcbeta-O-Al as acceptor, Gal3ST-2 acted efficiently on Galbeta1,3(6-O-sulfo)GlcNAcbeta-O-Al, and Gal3ST-4 acted efficiently on Galbeta1,3(6-O-sulfo)GalNAcalpha-O-Al. Mg(2+), Mn(2+), and Ca(2+) stimulated the activities of Gal3ST-2, whereas only Mg(2+) augmented Gal3ST-3 activity. Divalent cations did not stimulate Gal3ST-4, although inhibition was noted at high Mn(2+) concentrations. The fine substrate specificities of Gal3STs indicate a distinct physiological role for each enzyme.

Acetylglucosamine↗
PubMed2003

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.

Animals↗
PubMed2003

Complex oligosaccharide investigations: synthesis of an octasaccharide incorporating the dimeric Le(x) structure of PSGL-1.

The synthesis of an octasaccharide containing the dimeric Le(x) oligosaccharide structure found in PSGL-1 carbohydrate chains is reported. Several approaches were investigated employing regioselective and stereoselective glycosylation procedures, and a novel Lewis(x) trisaccharide donor, 7, was prepared and utilized as a key intermediate building block in the scheme developed for the construction of octasaccharide 3. Toward the preparation of 7, investigations into the influence of different protecting groups upon the relative reactivities of disaccharide acceptor moieties, 25 or 26, and the fucosyl donors, 10 and 11, were conducted using similar glycosylating conditions. Dramatic differences were noted between the effects of electron-donating and electron-withdrawing groups upon the reactivity of the acceptor hydroxyl. A similar effect upon the glycosylating capability of the donor molecule was, likewise, observed. The repeat use of donor 7 was instrumental in the synthesis of the desired dimeric octasaccharide structure 3. The structure and purity of 3 and important intermediates were fully characterized by DQF-COSY, TOCSY, ROESY, and ESI mass spectroscopy.

Catalysis↗
PubMed2003

Biologic contribution of P1 promoter-mediated expression of ST6Gal I sialyltransferase.

The synthesis of the common and well-documented Siaalpha 2,6 to Galbeta 1,4GlcNAc structure (Sia6LacNAc) is principally mediated by the sialyltransferase ST6Gal I, which is particularly highly expressed in liver, lactating mammary gland, intestinal epithelia of newborn animals, and B cells. Multiple independent promoters govern the expression of Siat1, the ST6Gal I gene. In liver, elevation of hepatic and serum ST6Gal is part of the acute phase reaction, the hepatic response to systemic trauma, and is governed by the inducible, liver-specific promoter-regulatory region, P1. A constitutive and nontissue-specific promoter, P3, mediates low-level, basal hepatic Siat1 transcription. We generated a mouse specifically unable to use the transcriptional initiation site uniquely used in P1-mediated ST6Gal I expression. These animals, Siat1deltaP1, are viable and display reduced ST6Gal I mRNA in liver with concomitantly reduced sialyltransferase activities in liver and in serum. Siat1deltaP1 animals are unable to elevate hepatic Siat1 mRNA as part of the inflammatory response induced by turpentine. Surprisingly, serum glycoprotein components exhibit normal extent of sialylation, with no noticeable difference in binding to SNA, the alpha2,6-sialyl-specific lectin. Siat1deltaP1 animals also exhibit an outwardly normal B cell response. On intraperitoneal challenge with the pathogen Salmonella typhimurium, a significantly greater accumulation of neutrophils within the peritoneal space was observed in Siat1deltaP1 animals compared to wild-type mice. Siat1deltaP1 mice also exhibit a greater bacterial burden in liver and spleen, accompanied by more pronounced spleno-/hepatomegaly and greater leukocyte infiltration into affected organs than their wild-type counterparts.

Acute-Phase Reaction↗
PubMed2002

A convergent synthesis of core 2 branched sialylated and sulfated oligosaccharides.

A convergent pathway for the syntheses of core 2 oligosaccharide analogues 1 and 2, and a natural form sialylated and sulfated hexasaccharide 3 was developed. Construction of pentasaccharides 24, 27 and hexasaccharide 28 was achieved by complete regioselective glycosylation of the 6-OH in the acceptors 5, 7 and 8, respectively, owing to the much higher reactivity of the primary hydroxyl group over the secondary axial hydroxyl group in these structures. Stereoselective sialylation was accomplished using donor 10 with defined configuration established through X-ray crystallographic analysis. Target oligosaccharides 1-3 were then obtained by the systematic deprotection of intermediates 24, 27 and 29. With these target oligosaccharides 1-3 obtained, biological evaluations of these molecules as enzyme substrates was undertaken and selectin binding studies are planned.

Carbohydrate Sequence↗
PubMed2002

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.

ABO Blood-Group System↗
PubMed2002

High-affinity binding of recombinant human galectin-4 to SO(3)(-)-->3Galbeta1-->3GalNAc pyranoside.

Galectin-4 is a member of galectin family and has two carbohydrate recognition domains. Although galectin-4 has been thought to function in cell adhesion, its precise carbohydrate binding specificity has not yet been clarified. We studied the carbohydrate binding specificity of galectin-4 comparatively with that of galectin-3, using surface plasmon resonance, galectin-3- or -4-Sepharose column chromatography and the inhibition assay of their binding to immobilized asialofetuin. Galectin-3 broadly recognized lactose, type 1, type 2, and core 1. The substitution at the C-2 and C-3 position of beta-galactose in these oligosaccharides with alpha-fucose, alpha-GalNAc, alpha-Neu5Ac, or sulfate increased the binding ability for galectin-3, whereas the substitution at the C-4 or C-6 position diminished the affinity. In contrast, galectin-4 had quite weak affinity to lactose, type 1, and type 2 (K(d) congruent with 8 x 10(-4) M). Galectin-4 showed weak binding ability to core 1 and C-2' or -3'-substituted lactose, type 1, and type 2 with alpha-fucose, alpha-GalNAc, or sulfate (K(d) : 5 x 10(-5) approximately 3 x 10(-4) M). Interestingly, the K(d) value, 3.4 x 10(-6) M, of SO(3)(-)-->3Galbeta1-->3GalNAc-O-Bn to galectin-4 at 25 degrees C was two orders of magnitude lower than that of core 1-O-Bn. 3'-Sialylated core 1 had very weak affinity to galectin-4, suggesting that 3'-O-sulfation of core 1 is critical for the recognition. These results suggest that galectin-4 has a unique carbohydrate binding specificity and interacts with O-linked sulfoglycans.

Antigens, Differentiation↗