Novel interactions of complex carbohydrates with peanut (PNA), Ricinus communis (RCA-I), Sambucus nigra (SNA-I) and wheat germ (WGA) agglutinins as revealed by the binding specificities of these lectins towards mucin core-2 O-linked and N-linked glycans and related structures.

Plant lectins through their multivalent quaternary structures bind intrinsically flexible oligosaccharides. They recognize fine structural differences in carbohydrates and interact with different sequences in mucin core 2 or complex-type N-glycan chain and also in healthy and malignant tissues. They are used in characterizing cellular and extracellular glycoconjugates modified in pathological processes. We study here, the complex carbohydrate-lectin interactions by determining the effects of substituents in mucin core 2 tetrasaccharide Galß1-4GlcNAcß1-6(Galß1-3)GalNAcα-O-R and fetuin glycopeptides on their binding to agarose-immobilized lectins PNA, RCA-I, SNA-I and WGA. Briefly, in mucin core 2 tetrasaccharide (i) structures modified by α2-3/6-Sialyl LacNAc, LewisX and α1-3-Galactosyl LacNAc resulted in regular binding to PNA whereas compounds with 6-sulfo LacNAc displayed no-binding; (ii) strucures bearing α2-6-sialyl 6-sulfo LacNAc, or 6-sialyl LacdiNAc carbohydrates displayed strong binding to SNA-I; (iii) structures with α2-3/6-sialyl, α1-3Gal LacNAc or LewisX were non-binder to RCA-I and compounds with 6-sulfo LacNAc only displayed weak binding; (iv) structures containing LewisX, 6-Sulfo LewisX, α2-3/6-sialyl LacNAc, α2-3/6-sialyl 6-sulfo LacNAc and GalNAc Lewis-a were non-binding to WGA, those with α1-2Fucosyl, α1-3-Galactosyl LacNAc, α2-3-sialyl T-hapten plus 3'/6'sulfo LacNAc displayed weak binding, and compounds with α2-3-sialyl T-hapten, α2.6-Sialyl LacdiNAc, α2-3-sialyl D-Fucß1-3 GalNAc and Fucα-1-2 D-Fucß-1-3GalNAc displaying regular binding and GalNAc LewisX and LacdiNAc plus D-Fuc ß-1-3 GalNAcα resulting in tight binding. RCA-I binds Fetuin triantennary asialoglycopeptide 100 % after α-2-3 and 25 % after α-2-6 sialylation, 30 % after α-1-2 and 100 % after α-1-3 fucosylation, and 50 % after α-1-3 galactosylation. WGA binds 3-but not 6-Fucosyl chitobiose core. Thus, information on the influence of complex carbohydrate chain constituents on lectin binding is apparently essential for the potential application of lectins in glycoconjugate research.

Overexpression of α2,3sialyl T-antigen in breast cancer determined by miniaturized glycosyltransferase assays and confirmed using tissue microarray immunohistochemical analysis.

Glycan structure alterations during cancer regulate disease progression and represent clinical biomarkers. The study determined the degree to which changes in glycosyltransferase activities during cancer can be related to aberrant cell-surface tumor associated carbohydrate structures (TACA). To this end, changes in sialyltransferase (sialylT), fucosyltransferase (fucT) and galactosyltransferase (galT) activity were measured in normal and tumor tissue using a miniaturized enzyme activity assay and synthetic glycoconjugates bearing terminal LacNAc Type-I (Galß1-3GlcNAc), LacNAc Type-II (Galß1-4GlcNAc), and mucin core-1/Type-III (Galß1-3GalNAc) structures. These data were related to TACA using tissue microarrays containing 115 breast and 26 colon cancer specimen. The results show that primary human breast and colon tumors, but not adjacent normal tissue, express elevated ß1,3GalT and α2,3SialylT activity that can form α2,3SialylatedType-IIIglycans (Siaα2-3Galß1-3GalNAc). Prostate tumors did not exhibit such elevated enzymatic activities. α1,3/4FucT activity was higher in breast, but not in colon tissue. The enzymology based prediction of enhanced α2,3sialylated Type-III structures in breast tumors was verified using histochemical analysis of tissue sections and tissue microarrays. Here, the binding of two markers that recognize Galß1-3GalNAc (peanut lectin and mAb A78-G/A7) was elevated in breast tumor, but not in normal control, only upon sialidase treatment. These antigens were also upregulated in colon tumors though to a lesser extent. α2,3sialylatedType-III expression correlated inversely with patient HER2 expression and breast metastatic potential. Overall, enzymology measurements of glycoT activity predict truncated O-glycan structures in tumors. High expression of the α2,3sialylated T-antigen O-glycans occur in breast tumors. A transformation from linear core-1 glycan to other epitopes may accompany metastasis.

Characterization of cancer associated mucin type O-glycans using the exchange sialylation properties of mammalian sialyltransferase ST3Gal-II.

Our previous studies suggest that the α2,3sialylated T-antigen (NeuAcα2,3Galß1,3GalNac-) and associated glycan structures are likely to be elevated during cancer. An easy and reliable strategy to label mucinous glycans that contain such carbohydrates can enable the identification of novel glycoproteins that are cancer associated. To this end, the present study demonstrates that the exchange sialylation property of mammalian ST3Gal-II can facilitate the labeling of mucin glycoproteins in cancer cells, tumor specimens, and glycoproteins in cancer sera. Results show that (i) the radiolabeled mucin glycoproteins of each of the cancer cell lines studied (T47D, MCF7, LS180, LNCaP, SKOV3, HL60, DU4475, and HepG2) is distinct either in terms of the specific glycans presented or their relative distribution. While some cell lines like T47D had only one single sialylated O-glycan, others like LS180 and DU4475 contained a complex mixture of mucinous carbohydrates. (ii) [14C]sialyl labeling of primary tumor cells identified a 25-35 kDa mucin glycoprotein unique to pancreatic tumor. Labeled glycoproteins for other cancers had higher molecular weight. (iii) Studies of [14C] sialylated human sera showed larger mucin glycopeptides and >2-fold larger mucin-type chains in human serum compared to [14C]sialyl labeled glycans of fetuin. Overall, the exchange sialylation property of ST3Gal-II provides an efficient avenue to identify mucinous proteins for applications in glycoproteomics and cancer research.

Fluorinated per-acetylated GalNAc metabolically alters glycan structures on leukocyte PSGL-1 and reduces cell binding to selectins.

Novel strategies to control the binding of adhesion molecules belonging to the selectin family are required for the treatment of inflammatory diseases. We tested the possibility that synthetic monosaccharide analogs can compete with naturally occurring sugars to alter the O-glycan content on human leukocyte cell surface selectin-ligand, P-selectin glycoprotein ligand-1 (PSGL-1). Resulting reduction in the sialyl Lewis-X-bearing epitopes on this ligand may reduce cell adhesion. Consistent with this hypothesis, 50muM per-acetylated 4F-GalNAc added to the growth media of promyelocytic HL-60 cells reduced the expression of the cutaneous lymphocyte associated-antigen (HECA-452 epitope) by 82% within 2 cell doubling cycles. Cell binding to all 3 selectins (L-, E-, and P-selectin) was reduced in vitro. 4F-GalNAc was metabolically incorporated into PSGL-1, and this was accompanied by an approximately 20% reduction in PSGL-1 glycan content. A 70% to 85% reduction in HECA-452 binding epitope and N-acetyl lactosamine content in PSGL-1 was also noted on 4F-GalNAc addition. Intravenous 4F-GalNAc infusion reduced leukocyte migration to the peritoneum in a murine model of thioglycolate-induced peritonitis. Thus, the compound has pharmacologic activity. Overall, the data suggest that 4F-GalNAc may be applied as a metabolic inhibitor to reduce O-linked glycosylation, sialyl Lewis-X formation, and leukocyte adhesion via the selectins.

Scaling down the size and increasing the throughput of glycosyltransferase assays: activity changes on stem cell differentiation.

Glycosyltransferases (glycoTs) catalyze the transfer of monosaccharides from nucleotide-sugars to carbohydrate-, lipid-, and protein-based acceptors. We examined strategies to scale down and increase the throughput of glycoT enzymatic assays because traditional methods require large reaction volumes and complex chromatography. Approaches tested used (i) microarray pin printing, an appropriate method when glycoT activity was high; (ii) microwells and microcentrifuge tubes, a suitable method for studies with cell lysates when enzyme activity was moderate; and (iii) C(18) pipette tips and solvent extraction, a method that enriched reaction product when the extent of reaction was low. In all cases, reverse-phase thin layer chromatography (RP-TLC) coupled with phosphorimaging quantified the reaction rate. Studies with mouse embryonic stem cells (mESCs) demonstrated an increase in overall ß(1,3)galactosyltransferase and α(2,3)sialyltransferase activity and a decrease in α(1,3)fucosyltransferases when these cells differentiate toward cardiomyocytes. Enzymatic and lectin binding data suggest a transition from Lewis(x)-type structures in mESCs to sialylated Galß1,3GalNAc-type glycans on differentiation, with more prominent changes in enzyme activity occurring at later stages when embryoid bodies differentiated toward cardiomyocytes. Overall, simple, rapid, quantitative, and scalable glycoT activity analysis methods are presented. These use a range of natural and synthetic acceptors for the analysis of complex biological specimens that have limited availability.

Mammalian sialyltransferase ST3Gal-II: its exchange sialylation catalytic properties allow labeling of sialyl residues in mucin-type sialylated glycoproteins and specific gangliosides.

While glycosyltransferases are known to display unidirectional enzymatic activity, recent studies suggest that some can also catalyze readily reversible reactions. Recently, we found that mammalian sialyltransferase ST3Gal-II can catalyze the formation of CMP-NeuAc from 5'-CMP in the presence of a donor containing the NeuAcα2,3Galß1,3GalNAc unit [Chandrasekaran, E. V., et al. (2008) Biochemistry 47, 320-330]. This study shows by using [9-(3)H]- or [(14)C]sialyl mucin core 2 compounds that ST3Gal-II exchanges sialyl residues between CMP-NeuAc and the NeuAcα2,3Galß1,3GalNAc unit and also radiolabels sialyl residues in gangliosides GD1a and GT1b, but not GM1. Exchange sialylation proceeds with relative ease, which is evident from the following. (a) Radiolabeleling of fetuin was ~2-fold stronger than that of asialo fetuin when CMP- [9-(3)H]NeuAc was generated in situ from 5'-CMP and [9-(3)H]NeuAcα2,3Galß1,3GalNAcß1,3Galα-O-Me by ST3Gal-II. (b) ST3Gal-II exchanged radiolabels between [(14)C]sialyl fetuin and [9-(3)H]NeuAcα2,3Galß1,3GalNAcß1,3Galα-O-Me by generating CMP-[(14)C]- and -[9-(3)H]NeuAc through 5'-CMP; only 20.3% (14)C and 28.0% (3)H remained with the parent compounds after the sialyl exchange. The [9-(3)H]sialyl-tagged MN glycophorin A, human chorionic gonadotropin ß subunit, GlyCAM-1, CD43, fetuin, porcine Cowper's gland mucin, bovine casein macroglycopeptide, human placental glycoproteins, and haptoglobin were analyzed by using Pronase digestion, mild alkaline borohydride treatment, Biogel P6, lectin agarose, and silica gel thin layer chromatography. Sulfated and sialylated O-glycans were found in GlyCAM-1 and human placental glycoproteins. This technique has the potential to serve as an important tool as it provides a natural tag for the chemical and functional characterization of O-glycan-bearing glycoproteins.

Systems-level studies of glycosyltransferase gene expression and enzyme activity that are associated with the selectin binding function of human leukocytes.

The application of systems biology methods in the emerging field of glycomics requires the collection and integration of glycosyltransferase data at the gene and enzyme level for the purpose of hypothesis generation. We systematically examined the relationship between gene expression, glycosyltransferase activity, glycan expression, and selectin-binding function in different systems, including human neutrophils, undifferentiated HL-60 (human promyelocytic cells), differentiated HL-60, and HL-60 synchronized in specific growth phases. Results demonstrate that 1) the sLe(X) (sialyl-Lewis-X) epitope is expressed in P-selectin glycoprotein ligand-1 (PSGL-1) from neutrophils at higher levels compared with HL-60. This variation may be due to differences in the relative activities of alpha1,3-fucosyltransferases and alpha2,3-sialyltransferases in these two cell types. 2) HL-60 cell differentiation along granulocyte lineage increased the activity of beta1,4GalT and beta1,3GlcNAcT by 1.6- to 3.2-fold. This may contribute to LacNAc chain extension as evidenced by the 1.7-fold increase in DSA-lectin (lectin recognizing LacNAc) binding to cells after differentiation. 3) The activity of enzymes contributing to sLe(X) formation in leukocytes likely varies as ST3[Galbeta1,4GlcNAc] < or = alpha1,3FT[sialyl-LacNAc] < beta1,3GlcNAcT. 4) O-glycan specific glycosyltransferase activity does not undergo periodic variation with cell cycle phases. Overall, gene expression and enzyme activity data combined with knowledge of biochemistry can predict the resulting glycan structures and yield viable experimentally testable hypothesis.

Potential tumor markers for human gastric cancer: an elevation of glycan:sulfotransferases and a concomitant loss of alpha1,2-fucosyltransferase activities.

PURPOSE: Several reports indicate a complexity in glycosyltransferase activities which lead to several tumor associated carbohydrate structures in gastric carcinoma. The present study was aimed to identify the carbohydrate associated transferases which exhibit the most marked and consistent change of activity in gastric tumorigenesis. METHODS: We examined the levels of fucosyl, beta-galactosyl-, beta-N-acetylgalactosaminyl, sialyl- and glycan:sulfotransferase activities, which generate the outer ends of oligosaccharide chains in tumorous and adjacent normal gastric tissues of the same patient in ten gastric carcinoma cases by using well defined specific synthetic acceptors utilized in our several earlier published studies as referenced in the text (e.g. Chandrasekaran et al. in J Biol Chem 279:10032-10041, 2004; Biochemistry 44:15619-15635, 2005; Carbohydr Res 341:983-994, 2006). RESULTS: Among glycosyltransferases only alpha1,2-fucosyltransferase (FT) was unique in showing a remarkable 40-90% decrease of activity in seven cases. Uniquely several fold elevation of Gal3Sulfo-T(2) (1.9 --> 156.7 fold) and Gal3Sulfo-T(4) (2.4 --> 149.0 fold) activities in all ten cases and moderate elevation of GlcNAc6Sulfo-T (1.3 --> 37.5 fold) activities in nine cases were identified. Poorly differentiated Signet ring cell carcinoma expresses mainly Gal3Sulfo-T(2) activity whereas poorly differentiated adenocarcinoma express predominantly Gal3Sulfo-T(4) activity and also GlcNAc6Sulfo-T activity. But, very low level of these sulfotransferase activities were identified in moderately differentiated gastric carcinomas as well as non-epithelial gastric stromal sarcoma. CONCLUSION: Up regulation of glycan:sulfotransferase activities and down regulation of alpha1,2-fucosyltransferase activity are apparently associated with human gastric tumorigenesis.

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.

Glycosaminoglycans of normal and malignant cultured human mammary cells.

Glycosaminoglycans have been characterized from a normal human breast cell line (HBL-100) and two different cell lines from human breast carcinoma (MDA-MB-231 and MCF-7). The glycosaminoglycans were labeled by exposure of cell cultures to [3H]glucosamine and [35S]sulfate and then isolated from both spent media and cells by pronase digestion and cetylpyridinium chloride fractionation. They were further characterized by (a) hexosamine composition, (b) controlled-pore glass exclusion chromatography, (c) reactivity with specific enzymes (hyaluronidase chondroitinase, heparitinase, and heparinase), (d) nitrous acid degradation, and (e) DEAD-Sephadex chromatography. The results indicate that the HBL-100 line synthesizes mainly hyaluronic acid, most of which is secreted into the medium. Chondroitin sulfate and heparan sulfate are the predominant glycosaminoglycans synthesized by the cancer lines; both are found mainly in the spent medium, but the hyaluronic acid synthesized by the MDA-MB-231 line remains cell associated. The cell-associated heparan sulfate had a molecular weight in excess of 13,000 and may contain linkages susceptible to testicular hyaluronidase. The MCF-7 cells produce significantly lower amounts of glycosaminoglycans than do the other two lines.

Structures of the oligosaccharide chains of two forms of alpha 1-acid glycoprotein purified from liver metastases of lung, colon, and breast tumors.

Two forms of alpha 1-acid glycoprotein with common immunological determinants and almost identical amino acid compositions but different amounts of carbohydrate were isolated from liver metastases of primary colon, lung, and breast tumors by extraction with perchloric acid, gel filtration on Sepharose CL-6B and Sephadex G-200, and affinity chromatography on concanavalin A:agarose and Ricinus communis agglutinin l:agarose. Both forms of the antigen yielded single bands which stained for protein and carbohydrate when examined by disc gel electrophoresis and immunodiffusion. The molecular weights of the two forms were 45,000 and 37,000 respectively. The larger form contained about five to six oligosaccharide chains, whereas the smaller form had only three to four chains. The composition and structures of the oligosaccharide chains in the two forms of this glycoprotein were very similar. Each contained di-, tri-, and tetraantennary complex-type oligosaccharide chains. The diantennary oligosaccharide chains caused both forms of alpha 1-acid glycoprotein to be retained by concanavalin A-agarose columns. The lower-molecular-weight form contained fewer chains and correspondingly fewer terminal galactosyl residues. This resulted in the separation of this species from the higher-molecular-weight form on columns containing R. communis agglutinin I. Three types of reduced oligosaccharides were released from the light and heavy forms of alpha 1-acid glycoprotein by treatment with alkaline borohydride or by hydrazinolysis. These chains were isolated by chromatography on concanavalin A:agarose and Bio-Gel P-6 columns. The arrangement and linkage of sugars in the purified oligosaccharides were determined by periodate oxidation, sequential hydrolysis with glycosidases, and methylation analysis. The major oligosaccharide chain, comprising 50 to 55% of the carbohydrate, had a triantennary structure as shown in the structure: (formula; see text) in which NeuNAc is N-acetylneuraminic acid, Gal is galactose, GlcNAc is N-acetylglucosamine, Man is mannose, GlcNAcol is N-acetylglucosaminitol, and Fuc is fucose. Tetraantennary chains comprised about 25 to 30% of the carbohydrate, and the additional outer chain was attached to the alpha 1,6-mannosyl residue through a beta 1,6-linked GlcNAc unit. The remaining 15 to 20% of the oligosaccharide chains had a diantennary structure. The extent of sialylation of these chains varied in samples isolated from tumors of the same histological type from different individuals. However, a relatively constant proportion of the three types of chains was present in different forms of the glycoprotein isolated from liver metastases.

Purification and properties of alpha-D-mannose:beta-1,2-N-acetylglucosaminyl-transferases and alpha-D-mannosidases from human adenocarcinoma.

Two specific N-acetylglucosaminyltransferases, alpha-1,3-mannoside:beta-2-N-acetylglucosaminyltransferase (transferase I) and alpha-1,6-mannoside:beta-2-N-acetylglucosaminyltransferase (transferase II), which catalyze the transfer of N-acetylglucosamine (GlcNAc) from uridine diphospho-GlcNAc to terminal branched alpha-mannosyl (Man) residues, were purified from liver metastases of human colon adenocarcinoma. Transferase I was assayed with Man alpha 1,6(Man alpha 1,3)Man alpha 1,6(Man alpha 1,3)Man beta 1,4GlcNAc beta 1, 4GlcNAc-Asn (Km 0.35 mM), and transferase II was assayed with Man alpha 1,6(GlcNAc beta 1,2Man alpha 1,3)Man beta 1,4GlcNAc beta 1,4-Glc-NAc-Asn (Km 1.0 mM), in which Asn is asparagine. The Km of transferase I for Man alpha 1,6(Man alpha 1,3)Man beta 1,4GlcNAc-beta 1,4)-(Fuc alpha 1,6)GlcNAc-Asn was 1 mM. The specificity of the interaction of transferase I with ovalbumin, ovomucoid, the modified heavy chain of porcine immunoglobulin G and glycopeptides prepared from these glycoproteins was examined by kinetic and structural analysis. The best macromolecular substrates for transferase I were ovalbumin devoid of terminal GlcNAc and some mannose, a solubilized preparation of the heavy chain of porcine immunoglobulin G, devoid of sialic acid, galactose, and terminal GlcNAc, and untreated ovomucoid. The apparent KmS were 45, 19, and 390 microM for ovalbumin, the modified heavy chain of immunoglobulin G, and untreated ovomucoid, respectively. The apparent Km of the enzyme for uridine diphospho-GlcNAc was not significantly influenced by the nature of the glycoprotein acceptor, and it varied between 14 and 20 microM for the different glycoproteins. The structures of the oligosaccharide chains in these glycoproteins which acted as acceptors for the purified enzyme were determined. A major glycopeptide product with the structure Man alpha 1,3(Man alpha 1,6)Man alpha 1,6(14C-GlcNAc beta 1,2Man-alpha 1,3)Man beta 1,4GlcNAc-beta 1,4-GlcNAc-Asn was isolated from both ovalbumin and ovomucoid following incubation with transferase I. The specificity of the enzyme for terminal branched mannosyl residues attached to a beta-linked mannose unit greatly restricts the action of this transferase to this juncture in the synthesis of complex-type oligosaccharide chains of N-asparagine-linked glycoproteins.(ABSTRACT TRUNCATED AT 400 WORDS)

Blood group Tn-active macromolecules from human carcinomas and erythrocytes: characterization of and specific reactivity with mono- and poly-clonal anti-Tn antibodies induced by various immunogens.

In contrast to healthy and noncarcinoma-diseased tissues, greater than 80% of all carcinomas (CAs) tested express immunoreactive O-(2-acetamido-2-deoxy-alpha-D-galacto-pyranosyl)-(1----3)-serine/threon ine [alpha-D-GalpNAc-(1----3)-Ser/Thr] in their glycoproteins. CA cells shed, into the tumor's environment, Tn, which is involved in cancer pathogenesis as adhesion molecule and as autoimmunogen. An increase in density of Tn on primary CA frequently parallels augmented CA aggressiveness. Tn-Active glycoproteins of culture-grown human breast CA DU 4475 cells were isolated from cytoplasm and from spent growth medium, and erythrocyte (RBC) Tn antigen was prepared by (1----3)-beta-D-galactosidase treatment of isolated human O RBC MN glycoprotein-derived Thomsen-Friedenreich (T) antigen. Immunochemical, serological, physical, and chemical analyses showed close resemblance of CA- and RBC-derived Tn antigens. The preponderant carbohydrate in both Tn glycoproteins is the alpha-D-GalpNAc residue, and the antigens have a qualitatively and quantitatively similar amino acid composition. Highly specific rodent monoclonal (Mo) anti-Tn antibodies (Abs) were elicited with Tn RBC and normal O RBC-derived Tn antigen, and compared with CA-anti-Tn MoAbs unwittingly evoked by others. A sensitive enzyme immunoassay (EIA) with Tn antigen as solid phase was developed. In this system, highly purified, "naturally occurring" anti-Tn antibodies, which all humans possess, were more sensitive in quantitating breast CA Tn structures than the anti-Tn MoAbs induced by Tn RBCs, and by RBC- and CA-derived Tn-active antigens. The sensitivity of anti-Tn MoAbs was higher in detecting RBC-Tn.

Isolation, partial characterization, and biological properties of polysaccharides from crude papain.

Two polysaccharides have been isolated from crude papain by precipitation of papain with ammonium sulfate, further precipitation of other proteins with trichloroacetic acid, and chromatography of the supernatant on DEAE-cellulose. The first polysaccharide to be eluted, designated PP-I, contained D-glucuronic acid, D-glucose, D-galactose, L-arabinose, and L-rhamnose, in the approximate molar ratios of 4:1:12:10:4. The other (PP-II), eluted at a higher salt-concentration, contained the same sugars (with about one-third less glucose and more uronic acid) in the approximate molar ratios of 13:1:40:26:12. Reduction of the uronic acid groups of PP-II produced a polysaccharide (PP-II-R) containing the same sugars in the approximate molar ratios of 2:11:37:28:12. Hydrolysis of a mixture of the two polysaccharides yielded an aldobiouronic acid, D-glucosyluronic acid-D-galactose. Neither polysaccharide preparation contained protein. These polysaccharides dramatically affected aggregation and alignment of normal human fibroblasts but had no effect on a mouse embryo fibroblast aneuploid cell-line that does not exhibit contact inhibition of growth or movement. In aggregating cells, these polysaccharides caused the cells to behave as contact-inhibited cells, that is, cell division and nuclear area were decreased.

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.

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.

Human ovarian cancer, lymphoma spleen, and bovine milk GlcNAc:beta1,4Gal/GalNAc transferases: two molecular species in ovarian tumor and induction of GalNAcbeta1,4Glc synthesis by alpha-lactalbumin.

Affinity Gel-UDP was utilized to purify GlcNAc:beta1,4Gal/GalNAc transferases (Ts) from human lymphoma spleen, ovarian tumor, and ovarian cancer sera. Mn(2+) was found to be an absolute requirement for activity. Two molecular species containing both beta1,4Gal/GalNAc-T activities were discernible when the purified ovarian tumor microsomal enzyme was subjected to Sephacryl S-100 HR column chromatography as well as native polyacylamide gel-electrophoresis. Acceptor specificity studies of the affinity-purified lymphoma spleen and ovarian tumor microsomal enzymes and the conventionally purified, as well as the cloned, bovine milk GlcNAc:beta1,4Gal-Ts using a number of synthetic acceptors showed that the beta(1,6)-linked GlcNAc moiety to alpha-GalNAc was the most efficient acceptor. As compared to the purified milk enzyme, the recombinant form exhibited sixfold GlcNAc:beta1,4 GalNAc-T activity and up to eightfold GlcNAc6SO3beta-:beta1,4Gal-T activity. Further, the recombinant enzyme catalyzed the transfer of GalNAc to the terminal beta-linked GlcNAc6SO3 moiety. Alpha-lactalbumin (alpha-LA) inhibited up to 85%, the transfer of Gal to the GlcNAc moiety linked either to Man or GlcNAc. On the contrary, alpha-LA had no significant influence on the transfer of GalNAc to the above acceptors. alpha-LA had no appreciable effect on the recombinant enzyme, except for the transfer of Gal or GalNAc to Glc. Both alpha- and beta-glucosides, as well as alpha-N-acetylglucosaminide, did not serve as acceptors.

Altered eosinophil profile in mice with ST6Gal-1 deficiency: an additional role for ST6Gal-1 generated by the P1 promoter in regulating allergic inflammation.

Cumulative evidence indicates that the sialyltransferase ST6Gal-1 and the sialyl-glycans, which it constructs, are functionally pleiotropic. Expression of the ST6Gal-1 gene is mediated by six distinct promoter/regulatory regions, and we hypothesized that these promoters may be used differentially to produce ST6Gal-1 for different biologic purposes. To examine this hypothesis, we compared a mouse with a complete deficiency in ST6Gal-1 (Siat1 null) with another mouse that we have created previously with a disruption only in the P1 promoter (Siat1DeltaP1). We noted previously greater neutrophilic inflammation associated with ST6Gal-1 deficiency. Here, we report that ST6Gal-1-deficient mice also have significantly elevated eosinophilic responses. Upon i.p. thioglycollate elicitation, eosinophils accounted for over 20% of the total peritoneal inflammatory cell pool in ST6Gal-1-deficient animals, which was threefold greater than in corresponding wild-type animals. A principal feature of allergic respiratory inflammation is pulmonary eosinophilia, we evaluated the role of ST6Gal-1 in allergic lung inflammation. Using OVA and ABPA experimental models of allergic airways, we showed that ST6Gal-1 deficiency led to greater airway inflammation characterized by excessive airway eosinophilia. The severity of airway inflammation was similar between Siat1DeltaP1 and Siat1 null mice, indicating a role for P1-generated ST6Gal-1 in regulating eosinophilic inflammation. Colony-forming assays suggested greater IL-5-dependent eosinophil progenitor numbers in the marrow of ST6Gal-1-deficient animals. Moreover, allergen provocation of wild-type mice led to a significant reduction in P1-mediated ST6Gal-1 mRNA and accompanied decline in circulatory ST6Gal-1 levels. Taken together, the data implicate ST6Gal-1 as a participant in regulating not only Th1 but also Th2 responses, and ST6Gal-1 deficiency can lead to the development of more severe allergic inflammation with excessive eosinophil production.