E. V. Chandrasekaran

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.

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 meth ods in the emerging field of glycomics requires the collection and integration of glycosyltransferase data at the gene and enzyme level for the purpose of hypoth esis generation. We systematically examined the rela tionship between gene expression, glycosyltransferase activity, glycan expression, and selectin‐binding func tion in different systems, including human neutrophils, undifferentiated HL‐60 (human promyelocytic cells), differentiated HL‐60, and HL‐60 synchronized in spe cific growth phases. Results demonstrate that 1) the sLeX (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 α1,3‐fucosyltransferases and α2,3‐sialyltransferases in these two cell types. 2) HL‐60 cell differentiation along granulocyte lineage increased the activity of β1,4GalT and β1,3GlcNAcT 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) bind ing to cells after differentiation. 3) The activity of enzymes contributing to sLeX formation in leukocytes likely varies as ST3[Galβ1,4GlcNAc] ≤ α1,3FT[sialyl‐LacNAc] < β1,3GlcNAcT. 4) O‐glycan specific glycosyltransferase activity does not undergo periodic varia tion 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.— Marathe, D. D., Chandrasekaran, E. V., Lau, J. T. Y., Matta, K. L., Neelamegham, S. Systems‐level studies of glycosyltransferase gene expression and enzyme activity that are associated with the selectin binding function of human leukocytes. FASEB J. 22, 4154–4167 (2008)

Use of sialylated or sulfated derivatives and acrylamide copolymers of Galβ1,3GalNAcα- and GalNAcα- to determine the specificities of blood group T- and Tn-specific lectins and the copolymers to measure anti-T and anti-Tn antibody levels in cancer patients

Sialylated or sulfated derivatives and acrylamide copolymers of blood group T-(Galβ1,3GalNAcα-) and Tn-(GalNAcα) haptens were studied for their interaction with the lectins of peanut (PNA),Agaricus bisporus-(ABA),Helix pomatia-(HPA) andVicia villosa B4-(VVA), using asialo Cowpers gland mucin (ACGM), which contains both T and Tn epitopes, as the coating substrate in enzyme linked lectin assay. Both T and Tn copolymers (∼40 haptens) showed high affinity and strict specificity; although the T-copolymer at 0.05–0.07 µm concentration caused 50% inhibition of interaction of either PNA or ABA with ACGM, there was little inhibition of the HPA and VVA interactions at over 100 times that concentration. The Tn-copolymer at 0.02–0.05 µm inhibited HPA or VVA interaction with ACGM by 50% but gave virtually no inhibition of PNA and ABA binding. Sialyl, sulfate or methyl group substitution on C-6 of GalNAc of the T-haptene did not prevent interaction with PNA but almost abolished interaction with ABA. In contrast, sialyl or sulfate group on C-6 and sulfate on C-3 of Gal in Galβ1,3GalNAcα- inhibited almost completely the interaction of PNA with ACGM but had only a slight effect on the interaction of ABA; C-6 substitution with either sialic acid or sulfate on GalNAcα- almost abolished the interaction of both HPA and VVA with ACGM. Preliminary studies revealed a significant depression in the serum level of anti-T (two to three-fold decrease) and anti-Tn (∼ two-fold decrease) antibodies in breast cancer compared with normal control subjects when the acrylamide T- and Tn-copolymers were used as coating substrates in enzyme linked immunoassays.

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 (Siat1ΔP1). 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 Siat1ΔP1 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.

Characterization of distinct Gal : 3-O-sulfotransferase activities in human tumor epithelial cell lines and of calf lymph node GlcNAc : 6-O-sulfotransferase activity

We found earlier in human breast and colon tumors, an augmented level of Gal : 3-O-sulfotransferase activities showing, respectively, an acceptor preference to blood group T-hapten (Group A enzymes) or Galβ1,4GlcNAc (Group B enzymes) on the mucin Core 2 structure [Chandrasekaran EV, Jain RK, Vig R, and Matta KL (1997) Glycobiology 7: 753–68]. The present study reports these enzyme activities in human tumor cell lines and normal tissues. The human colon tumor epithelial cell lines, akin to their parent tumors, express Group B enzyme activity. The acceptor specificity and kinetic properties, such as divalent metal ion activation and pH dependent activity profile, of the colon cancer line LS180 enzyme activity are identical to those of colon tissue specimens. Consistent with breast tumor specimens, the Group A enzyme activity is present in human breast tumor epithelial cell lines, with some exceptions. The Gal : 3-O-sulfotransferases show specific binding to Aleuria aurantia lectin, suggesting the presence of asparagine linked carbohydrate chains containing an inner core α1,6-fucosyl residue on these enzymes. Calf lymph nodes contain GlcNAc : 6-O-sulfotransferase as well as Group A Gal : 3-O-sulfotransferase activities, which differ in pH dependent profiles, pH optima (7.6 and 7.0, respectively) and the influence of Mn2+.

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.

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 β(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 Galβ1,3GalNAc-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.

Synthesis of Gal-β-(1→4)-GlcNAc-β-(1→6)-[Gal-β- (1→3)]-GalNAc-α-OBn oligosaccharides bearing O-methyl or O-sulfo groups at C-3 of the Gal residue: specific acceptors for Gal: 3-O-sulfotransferases

Our recent studies have revealed the existence of two distinct Gal: 3-O-sulfotransferases capable of acting on the C-3 position of galactose in a Core 2 branched structure, e.g., Galβ1→4GlcNAcβ1→6(Galβ1→3)GalNacα1→OBenzyl as acceptor to give 3-O-sulfoGalβ1→4GlcNAcβ1→3(Galβ1→3)GalNAcα1→OB 20 and Galβ1→4GlcNAcβ1→6(3-O-sulfoGalβ1→3)GalNAcα1→OB 23. We herein report the synthesis of these two compounds and also that of other modified analogs that are highly specific acceptors for the two sulfotransferases. Appropriately protected 1-thio-glycosides 7, 8, and 10 were employed as glycosyl donors for the synthesis of our target compounds.

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.

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 (Galβ1-3GlcNAc), LacNAc Type-II (Galβ1-4GlcNAc), and mucin core-1/Type-III (Galβ1-3GalNAc) 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 β1,3GalT and α2,3SialylT activity that can form α2,3SialylatedType-IIIglycans (Siaα2-3Galβ1-3GalNAc). 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 Galβ1-3GalNAc (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.