Publications

The density, molecular isoform, and posttranslational modifications of CD44 can markedly influence growth and metastatic behavior of tumors. Many CD44 functions, including some involving tumors, have been attributed to its ability to recognize hyaluronan (HA). However, only certain CD44-bearing cells bind soluble or immobilized HA. We now show that CD44 made by wild-type Chinese hamster ovary (CHO-K1) cells and a ligand-binding subclone differ with respect to N-linked glycosylation. While both bear CD44 with highly branched, complex-type glycoforms, CD44 expressed by the wild type was more extensively sialylated. CHO-K1 cells which failed to recognize HA when grown in culture gained this ability when grown as a solid tumor and reverted to a non-HA-binding state when returned to culture. The ability of CHO-K1 cells to recognize HA was also reversibly induced when glucose concentrations in the medium were reduced. Glucose restriction influenced CD44-mediated HA binding by many but not all, of a series of murine tumors. Glucose concentrations and glycosylation inhibitors only partially influenced CD44 receptor function on resting murine B lymphocytes. These observations suggest that glucose levels or other local environmental conditions may markedly influence glycosylation pathways used by some tumor cells, resulting in dramatic alteration of CD44-mediated functions.

It has been assumed that membrane-bound glycosyltransferases function within the Golgi apparatus to glycosylate glycoproteins. We now report, however, that a truncated, soluble recombinant form of the murine alpha1,3-galactosyltransferase expressed in human 293 cells is highly efficient and comparable to the full-length enzyme in alpha-galactosylating both newly synthesized membrane-associated and secreted glycoproteins. Although the soluble enzyme was secreted by cells as expected, we also found that the full-length, membrane-associated form was secreted. Unexpectedly, both secreted forms are cleaved identically at two primary sites within the stem region by endogenous protease(s) at the indicated positions in the sequence 73KDWW (downward arrow) FPS (downward arrow) WFKNG. These results demonstrate that the soluble alpha1,3-galactosyltransferase is functional within the cell and that specific proteolysis occurs in the stem region. The widespread occurrence of different soluble glycosyltransferases secreted by cells suggests that normal glycoconjugate biosynthesis may involve cooperation between membrane-bound and soluble enzymes.

Galectin-1 is a beta-galactoside-binding protein secreted by animal cells, and it exists in a monomer-dimer equilibrium (Kd approximately 7 microM). The function(s) of galectin-1 is(are) not yet defined, but dimerization and divalency are presumably important. Crystal structures of the mammalian galectin-1 dimer predict N- and C-terminal interactions at the subunit interface. To examine the mechanism of dimer formation and possibly generate active monomeric galectin-I, mutations were made in the N- and C-termini of recombinant hamster galectin-1. N-Gal-1 contains disruptions of three hydrophobic amino acids at the N-terminus; V5D-Gal-1 contains a single mutation of Val5 to Asp; N/C-Gal-1 contains multiple changes in hydrophobic amino acids at both the N- and C-termini. All mutants behave as monomers in size-exclusion HPLC and native gel electrophoresis. N-Gal-1 and V5D-Gal-1 bind weakly to lactosyl-Sepharose, but N/C-Gal-1 is nonfunctional. In equilibrium dialysis, N-Gal-1 and V5D-Gal-1 bind N-acetyllactosamine with a Kd approximately 90 microM, which is similar to that of native lectin. At high concentrations, V5D-Gal-1 and N-Gal-1 dimerize and can be covalently cross-linked with disuccinimidyl suberate. The Kd values of the monomer-dimer equilibrium for V5D-Gal-1 and N-Gal-1 are estimated to be approximately 60 microM and approximately 250 microM, respectively. The cross-linked dimers of V5D and N-Gal-1 were isolated and were similar to native lectin in both hemagglutinating activity and high-affinity binding to lactosyl-Sepharose. Thus, specific mutations in galectin-1 can alter monomer-dimer equilibrium without affecting carbohydrate-binding activity. The availability of active monomers and functional covalent dimers of galectin-1 should aid in future studies aimed at understanding the biological function(s) of the lectin and the role of divalency.

P-selectin glycoprotein ligand-1 (PSGL-1) is a disulfide-bonded homodimeric mucin-like glycoprotein on leukocytes that interacts with both P- and E-selectin. In this report we describe the structures of the Ser/Thr-linked O-glycans of PSGL-1 synthesized by HL-60 cells metabolically radiolabeled with 3H-sugar precursors. In control studies, the O-glycans on CD43 (leukosialin), a mucin-like glycoprotein also expressed by HL-60 cells, were analyzed and compared to those of PSGL-1. O-Glycans were released from Ser/Thr residues by mild base/borohydride treatment of purified glycoproteins, and glycan structures were determined by a combination of techniques. In contrast to expectations, PSGL-1 is not heavily fucosylated; a majority of the O-glycans are disialylated or neutral forms of the core-2 tetrasaccharide Galbeta1-->4GlcNAcbeta1-->6(Galbeta1-->3)GalNAcOH++ +. A minority of the O-glycans are alpha-1,3-fucosylated that occur as two major species containing the sialyl Lewis x antigen; one species is a disialylated, monofucosylated glycan, and the other is a monosialylated, trifucosylated glycan having a polylactosamine backbone. CD43 lacks the fucosylated glycans found on PSGL-1 and is enriched for the nonfucosylated, disialylated core-2 hexasaccharide. These results demonstrate that PSGL-1 contains unique fucosylated O-glycans that are predicted to be critical for high affinity interactions between PSGL-1 and selectins.

Schistosomiasis is a helminthic parasitic disease that results in a wide-ranging pathology in the approximately 200 million infected people worldwide. Much of the immunity to the parasite is directed against carbohydrate determinants in both glycoproteins and glycolipids from the adult worms and their eggs. Circulating glycoproteins derived from the parasite may be diagnostic for the disease. Recent studies of the structures of schistosome-derived glycoconjugates reveal that they exhibit several interesting and novel motifs. Many schistosome glycans are rich in fucose and devoid of sialic acid. It is surprising that some of the fucosylated schistosome glycans contain the Lewis x (Le(x)) antigen that is also found on human leukocytes and other tissues. These Le(x)-containing glycans elicit autoantibodies, and the glycans may affect lymphocyte functions. This review highlights recent progress in schistosome research in terms of structure, function, and biosynthesis of glycoconjugates. It is hoped that the deeper understanding being gained about glycoconjugates will foster innovative new strategies for lessening the mortality and morbidity caused by these parasites.

We now report a solid-phase assay for CMPNeuAc: Galbeta1-3/4GlcNAc-R alpha-2,3-sialyltransferase (alpha2,3ST) that is nonradioactive and allows specific identification of the sialylated product. An acceptor glycoprotein, desialylated fetuin, is immobilized on a microtiter plate. The transfer of sialic acid from CMPNeuAc generates the product NeuAc alpha2-3Gal beta1-4GlcNAc-R that is specifically bound by biotinylated Maackia amurensis leukoagglutinin (MAL). The binding of biotinylated MAL is measured with either an absorbance-based reagent (streptavidin conjugated to alkaline phosphatase) or a light-based reagent (streptavidin conjugated to the bioluminescent protein aequorin, Aqualite). The rat liver alpha2,3ST was used to optimize the assay. The formation of product is linear with respect to time and dependence on the amounts of CMPNeuAc, enzyme, and acceptor coated on the plates. As little as 0.2 microU of enzyme can be measured using the streptavidin-aequorin reagent. The assay is useful with crude tissue extracts, as demonstrated by the determination of the alpha2,3ST activity in human serum and in microsomes of HL-60 and Chinese hamster ovary cells.

P-selectin glycoprotein ligand-1 (PSGL-1), a sialomucin on human leukocytes, mediates rolling of leukocytes on P-selectin expressed by activated platelets or endothelial cells under shear forces. PSGL-1 requires both tyrosine sulfate and O-linked glycans to bind P-selectin. Electron microscopy of rotary-shadowed PSGL-1 purified from human neutrophils indicated that it is a highly extended molecule with an extracellular domain that is -50 nm long. Both individual PSGL-1 molecules and rosettes composed of several molecules presumably attached at their transmembrane segments were observed. The extracellular domain of PSGL-1 has 318 residues, including a signal peptide from residues 1-18 and a propeptide from residues 19-41. Using bacterially expressed fusion proteins and synthetic peptides derived from the extracellular domain, we mapped the epitopes for two IgG anti-PSGL-1 monoclonal antibodies, PL1 and PL2. PL2 bound to a region within residues 188-235 that is located in a series of decameric consensus repeats. PL1, which blocks binding of PSGL-1 to P-selectin, recognized an epitope spanning residues 49-62. This sequence overlaps the tyrosine sulfation sites at residues 46, 48, and 51 that have been implicated in binding of PSGL-1 to P-selectin. Our results demonstrate that PSGL-1 is a long, extended molecule and suggest that the P-selectin binding site is located near the N terminus, well above the membrane. This location may facilitate interactions of PSGL-1 with P-selectin under shear stress.

The Lewis x antigen (Le(x); Gal beta 1-4[Fuc alpha 1-3]GlcNac beta1-R), which is present on the surfaces of human cells, is also synthesized by the human helminthic parasite Schistosoma mansoni. We now report that IgM and IgG antibodies to Le(x) antigens are present in the sera of humans and rhesus monkeys infected with S. mansoni, whereas these antibodies are completely absent in uninfected individuals. The sera from infected humans and monkeys mediate specific complement-dependent cytolysis of human promyelocytic leukemic HL-60 cells, which bear surface Le(x) antigen. Furthermore, the major activity in sera from infected individuals toward HL-60 cells is due to anti-Le(x) reactivity.