Jun Xue

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.

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 (NeuAcα2,3Galβ1,3GalNac-) 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.

A Concise Synthesis of N-Trichloroethoxycarbonyl Lactosamine Trichloroacetimidate Donor and its Application in the Synthesis of Gal(β1-4)GlcNAc(β1-3)L-Fuc(α-OAll)

A large-scale practical approach is described for converting D-glucosamine into lactosamine donor. 2-Naphthylmethyl (NAP) functionality is utilized as anomeric protecting group of N-trichloroethoxycarboonyl (N-Troc) glucosamine, which can avoid an unexpected intermolecular aglycon transfer observed in TMSOTf-mediated glycosylation when using corresponding thioglycoside as glycosyl acceptor. N-Troc protected glucosamine 9 shows enhanced reactivity as glycosyl acceptor, which was confirmed by systemic comparison with various N-protected glucosamine derivatives in TMSOTf-mediated glycosylation. The lactosamine imidate 14 exhibits valuable glycosyl donor property for β-selective glycosylation which furnished the desired trisaccharide Gal(β1-4)GlcNAc(β1-3)L-Fuc(α-OAll) in high yield. This sequence is part of O-linked chains of human clotting factor IX and mammalian Notch 1, and it was also found to be required for fringe to inhibit Jaggedl-dependent Notch activation.