The selectins have attracted intense interest because of their carbohydrate-recognition properties and their pivotal roles in leukocyte trafficking. Future studies will center on the mechanisms for regulating the expression of the selectins and their ligands, the molecular details of selectin binding to glycoprotein ligands and small carbohydrates, and the biophysical principles that selectins employ to mediate attachment and rolling of leukocytes under flow.
The functions of sulphated monosaccharides within glycosaminoglycans (GAGs) and glycoproteins are being studied intensely, but progress is hindered by an inability to selectively desulphate glycoconjugates. We recently identified an N-acetylglucosamine-6-sulphate sulphatase (NG6SS) from bovine kidney that can remove sulphate from N-acetylglucosamine-6-sulphate (GlcNAc-6-SO4) within oligosaccharides and glycoproteins. However, the potential 'endosulphatase' activity of the NG6SS toward GAGs is not known. To test for this possibility, [3H]glucosamine-, [3H]galactose- and 35SO4-labelled keratan sulphate (KS) were separately prepared by metabolic radiolabelling of bovine cornea. NG6SS quantitatively removed sulphate from KS without release of sugar fragments. The enzyme had a Km of 4.7 mM toward free GlcNAc-6-SO4, but its Km for commercially available bovine corneal KS was found to be 9.1 microM. Analyses of both KS and heparan sulphate after treatment with NG6SS demonstrated significant loss of sulphate from GlcNAc-6-SO4 in both GAGs. These findings may be relevant for future studies aimed at defining the function(s) of GlcNAc-6-SO4 residues in GAGs and understanding the catabolism of GAGs, especially in regard to sulphatidoses, such as Sanfilippo D syndrome in humans, which involves a deficiency of NG6SS activity.
In the accompanying study (Cho, M., and Cummings, R. D. (1995) J. Biol. Chem. 270, 5198-5206), we reported that Chinese hamster ovary (CHO) cells synthesize galectin-1. We have now used several approaches to define the subcellular location and biosynthesis of galectin-1 in these cells. Galectin-1 was present on the cell surface, as assessed by immunofluorescent staining with monospecific antibody to the protein. Quantitation of the surface-localized galectin-1 was achieved by metabolically radiolabeling cells with [35S]Met/Cys and measuring the amount of lectin (i) sensitive to trypsin, (ii) accessible to biotinylating reagents, and (iii) accessible to the haptenic disaccharide lactose. By all three procedures, approximately 1/2 of the radiolabeled galectin-1 associated with cells was shown to be on the cell surface with the remainder intracellular. The kinetics of externalization of galectin-1 was monitored by pulse-chase radiolabeling, and it was shown that cells secrete the protein with a t1/2 approximately 20 h. The cell surface form of galectin-1 in CHO cells was active and bound to surface glycoconjugates, but lectin accumulating in the culture media was inactive. Lectin synthesized by mutant Lec8 CHO cells, which are unable to galactosylate glycoproteins was not found on the surface and quantitatively accumulated in the media in an inactive form. Taken together, our results demonstrate that galectin-1 is quantitatively externalized by CHO cells and can associate with surface glycoconjugates where the lectin activity is stabilized.
We report our studies on the characterization of an approximately 14-kDa lectin, termed galectin-1 that we have found to be expressed by Chinese hamster ovary (CHO) cells. cDNA for galectin-1 from CHO cells was prepared and sequenced, and a recombinant form (rGal-1) was expressed in Escherichia coli. A mutated form of the protein that fully retained activity was also constructed (termed C2SrGal-1) in which Cys-2 was changed to Ser-2. rGal-1 was stable in the presence of reducing agent, but it quickly lost all activity in the absence of reducing agent. In contrast, glycoprotein ligands, such as basement membrane laminin, stabilized the activity of rGal-1 in the absence of reducing agent (t1/2 = 2 weeks). C2SrGal-1 was stable in the presence or absence of either ligand or reducing agent. Unexpectedly, galectin-1 was found to exist in a reversible and active monomer-dimer equilibrium with a Kd approximately 7 microM and an equilibration time of t1/2 approximately 10 h. Addition of haptenic sugars did not affect this equilibrium. Galectin-1 isolated from the cytosol of CHO cells was found to exist as monomers and dimers. These studies demonstrate that galectin-1 binding to a biological ligand stabilizes its activity and that the monomer/dimer state of the protein is regulated by lectin concentration.
Neutrophils roll on P-selectin expressed by activated platelets or endothelial cells under the shear stresses in the microcirculation. P-selectin glycoprotein ligand-1 (PSGL-1) is a high affinity ligand for P-selectin on myeloid cells. However, it has not been demonstrated that PSGL-1 contributes to the rolling of neutrophils on P-selectin. We developed two IgG mAbs, PL1 and PL2, that appear to recognize protein-dependent epitopes on human PSGL-1. The mAbs bound to PSGL-1 on all leukocytes as well as on heterologous cells transfected with PSGL-1 cDNA. PL1, but not PL2, blocked binding of 125-I-PSGL-1 to immobilized P-selectin, binding of fluid-phase P-selectin to myeloid and lymphoid leukocytes, adhesion of neutrophils to immobilized P-selectin under static conditions, and rolling of neutrophils on P-selectin-expressing CHO cells under a range of shear stresses. PSGL-1 was localized to microvilli on neutrophils, a topography that may facilitate its adhesive function. These data indicate that (a) PSGL-1 accounts for the high affinity binding sites for P-selectin on leukocytes, and (b) PSGL-1 must interact with P-selectin in order for neutrophils to roll on P-selectin at physiological shear stresses.
The formation of tri- and tetraantennary complex-type N-linked oligosaccharides in animal glycoproteins is partly regulated by UDP-N-acetylglucosamine:beta-6-D-mannoside beta-1,6-N-acetylglucosaminyltransferase (EC 2.4.1.155) (GlcNAc-T V), which generates 2,6-branched mannose. In Chinese hamster ovary (CHO) cells we found that 2,6-branched mannosyl structures are preferentially contained on lysosome-associated membrane proteins (LAMPs) and are generally low or absent in other cellular glycoproteins (Do, K.-Y. and Cummings, R.D. (1993) J. Biol. Chem. 268, 22028-22035). To determine the mechanism by which GlcNAc-T V appears to preferentially recognize glycoproteins, we examined the activity of purified GlcNAc-T V toward a variety of glycoprotein acceptors. Because GlcNAc-T V requires as acceptors oligosaccharides lacking outer galactosyl and sialyl residues, we utilized two classes of acceptor preparations. The first class of acceptor was enzymatically desialylated (DS) and degalactosylated (DG) preparations of bovine fetuin, human transferrin, and human fibrinogen. The second class was glycoproteins in extracts of the mutant CHO cell line, Lec8 CHO, which cannot add galactose or sialic acid to N-linked oligosaccharides. GlcNAc-T V was highly active toward DSDG-fetuin, -transferrin, and -fibrinogen (Km values ranged between 30 and 74 microM), and the catalytic efficiencies (Vmax/Km) of the enzyme toward different acceptors were comparable. In the case of fetuin, each of its three sites for attachment of N-linked oligosaccharides were shown to be utilized equally well by GlcNAc-T V. Notably, the enzyme exhibited a 2-3-fold higher rate of transfer toward DSDG-transferrin when it was further denatured by reduction and S-carboxymethylation. When extracts of Lec8 CHO cells were used as acceptors, GlcNAc-T V preferentially transferred to LAMPs, and only low level transfer was observed to other cell-derived glycoproteins, thus demonstrating specificity of GlcNAc-T V toward native glycoprotein acceptors. When the cell-derived glycoproteins were denatured by reduction and S-carboxymethylation prior to use as acceptors for Glc-NAc-T V, significant transfer occurred to other glycoproteins. These results demonstrate that the mechanism of glycoprotein-specific branching by GlcNAc-T V is determined primarily by its accessibility to available bi/triantennary oligosaccharides on glycoproteins and not by its recognition of peptide determinants or conformation-specific determinants.
We previously demonstrated that P-selectin binds with high affinity to a trace, homodimeric glycoprotein ligand on human myeloid cells. The ligand carries the sialyl Lewis x (sLe(x)) epitope, a limited number of N-linked glycans, and clustered, sialylated O-linked glycans. In this study we demonstrate that the polypeptide component of this ligand is identical to that of P-selectin glycoprotein ligand-1 (PSGL-1), a molecule recently identified by expression cloning from a human myeloid cell cDNA library. We have examined the effects of glycosidases on purified, radioiodinated PSGL-1 from human neutrophils to further characterize the structure and function of the attached oligosaccharides. We found that PSGL-1 had poly-N-acetyllactosamine, only some of which could be removed with endo-beta-galactosidase. The majority of the Le(x) and sLe(x) structures were on endo-beta-galactosidase-sensitive chains. Peptide:N-glycosidase F (PNGaseF) treatment removed at least two of the three possible N-linked oligosaccharides from PSGL-1. Expression of Le(x) and sLe(x) was not detectably altered by PNGaseF digestion, indicating that these structures were primarily on O-linked poly-N-acetyllactosamine. Endo-beta-galactosidase-treated PSGL-1 retained the ability to bind to P-selectin, suggesting that some of the oligosaccharides recognized by P-selectin were either on enzyme-resistant poly-N-acetyllactosamine or on chains which lack poly-N-acetyllactosamine. PNGaseF treatment did not affect the ability of PSGL-1 to bind to P-selectin, demonstrating that the oligosaccharides required for P-selectin recognition are O-linked. PSGL-1 also bound to E-selectin, but with at least 50-fold lower affinity than to P-selectin. These data suggest that PSGL-1 from human neutrophils displays complex, sialylated, and fucosylated O-linked poly-N-acetyllactosamine that promote high affinity binding to P-selectin, but not to E-selectin.
N-Acetylglucosamine-6-sulfate sulfatase (NG6SS) is an enzyme that catalyzes the hydrolysis of sulfate esters from the C-6 hydroxyl of N-acetylglucosamine. We report our purification and characterization of the enzyme and the discovery that it can remove sulfate from internally sulfated GlcNAc on glycopeptides and glycoproteins. The enzyme was purified from bovine kidney over 200,000-fold using a combination of ion-exchange and size-exclusion chromatography. NG6SS is soluble and occurs as a single subunit with apparent solution molecular weight of 60.2 kDa on gel filtration chromatography and approximately 52.5 and 57.8 kDa on reducing and nonreducing SDS/PAGE, respectively. The enzyme is highly basic and exhibits a broad pH range with an optimum at pH 6.5 and a temperature optimum of 37 degrees C. Among the mono- and disaccharide sulfates tested, only GlcNAc-6-SO4 is an effective substrate with a Km of 4.7 mM, and either free sulfate or phosphate inhibits the activity. Unexpectedly, we found that the enzyme displays endosulfatase activity and quantitatively releases 35SO4 from 35SO4-labeled glycopeptides and intact glycoproteins isolated from human Molt-3 cells, which we have previously shown to synthesize glycoproteins containing GlcNAc-6-SO4 residues within the sequence Gal beta 1-4[SO-3-6]-GlcNAc beta 1-R of complex-type N-linked oligosaccharides. The N-terminal sequence of the bovine NG6SS was homologous to a human-liver-derived N-acetylglucosamine-6-sulfatase. The endosulfatase activity of bovine kidney NG6SS may be important in its potential role in the degradation of sulfated glycans and may make this enzyme a valuable reagent to study the biological functions of sulfated glycoproteins.
