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R D Cummings

Publications and source records attributed to R D Cummings.

At least 55 records · Page 3Linked to original sources
PubMed1996

Characterization of monomeric forms of galectin-1 generated by site-directed mutagenesis.

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.

Amino Acid Sequence↗
PubMed1996

Structures of the O-glycans on P-selectin glycoprotein ligand-1 from HL-60 cells.

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.

Antigens, CD↗
PubMed1996

Absorbance- and light-based solid-phase assays for CMPNeuAc:Galbeta1-4GlcNAc-R alpha-2,3-sialyltransferase.

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.

Aequorin↗
PubMed1996

Visualization of P-selectin glycoprotein ligand-1 as a highly extended molecule and mapping of protein epitopes for monoclonal antibodies.

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.

Amino Acid Sequence↗
PubMed1996

Transcriptional regulation of alpha1,3-galactosyltransferase in embryonal carcinoma cells by retinoic acid. Masking of Lewis X antigens by alpha-galactosylation.

Treatment of mouse teratocarcinoma F9 cells with all-trans-retinoic acid (RA) causes a 9-fold increase in steady-state levels of mRNA for UDP-Gal:beta-D-Gal alpha1,3-galactosyltransferase (alpha1,3GT) beginning at 36 h. Enzyme activity rises in a similar fashion, which also parallels the induction of laminin and type IV collagen. Nuclear run-on assays indicate that this increase in alpha1,3GT in RA-treated F9 cells, like that of type IV collagen, is transcriptionally regulated. Differentiation also results in increased secretion of soluble alpha1,3GT activity into the growth media. The major alpha-galactosylated glycoprotein present in the media of RA-treated F9 cells, but not of untreated cells, was identified as laminin. Differentiation of F9 cells is accompanied by an increase in alpha-galactosylation of membrane glycoproteins and a decrease in expression of the stage-specific embryonic antigen, SSEA-1 (also known as the Lewis X antigen or LeX), which has the structure Galbeta1-4(Fucalpha1-3)GlcNAcbeta1-R. However, flow cytometric analyses with specific antibodies and lectins, following treatment of cells with alpha-galactosidase, demonstrate that differentiated cells contain LeX antigens that are masked by alpha-galactosylation. Thus, RA induces alpha1,3GT at the transcriptional level, resulting in major alterations in the surface phenotype of the cells and masking of LeX antigens.

Animals↗
PubMed1996

Post-translational modifications of recombinant P-selectin glycoprotein ligand-1 required for binding to P- and E-selectin.

P-selectin glycoprotein ligand-1 (PSGL-1) is a mucin-like ligand for P- and E-selectin on human leukocytes. PSGL-1 requires sialylated, fucosylated O-linked glycans and tyrosine sulfate to bind P-selectin. Less is known about the determinants that PSGL-1 requires to bind E-selectin. To further define the modifications required for PSGL-1 to bind P- and E-selectin, we transfected Chinese hamster ovary (CHO) cells with cDNAs for PSGL-1 and specific glycosyltransferases. CHO cells synthesize only core 1 O-linked glycans (Galbeta1-3GalNAcalpha1-Se r/Thr); they lack core 2 O-linked glycans (Galbeta1-3(Galbeta1-4GlcNAcbeta1-6)GalNAcalpha1 -Ser/Thr) because they do not express the core 2 beta1 6-N-acetylglucosaminyltransferase (C2GnT). CHO cells also lack alpha1 3 fucosyltransferase activity. PSGL-1 expressed on transfected CHO cells bound P- and E-selectin only when it was co-expressed with both C2GnT and an alpha1 3 fucosyltransferase (Fuc-TIII, Fuc-TIV, or Fuc-TVII). Chromatography of beta-eliminated O-linked glycans from PSGL-1 co-expressed with C2GnT confirmed synthesis of core 2 structures. Tyrosine residues on PSGL-1 expressed in CHO cells were shown to be sulfated. Phenylalanine replacement of three tyrosines within a consensus sequence for tyrosine sulfation abolished binding to P-selectin but not to E-selectin. These results demonstrate that PSGL-1 requires core 2 O-linked glycans that are sialylated and fucosylated to bind P- and E-selectin. PSGL-1 also requires tyrosine sulfate to bind P-selectin but not E-selectin.

Animals↗
PubMed1996

Schistosoma mansoni: characterization of an alpha 1-3 fucosyltransferase in adult parasites.

We report that extracts of Schistosoma mansoni contain a GDPFuc:Gal beta 1-4GlcNAc (Fuc to GlcNAc) alpha 1-3 fucosyltransferase (alpha 1,3 FT) capable of synthesizing the antigenic determinant known as Lewis x (Le(x), Gal beta 1-4[Fuc alpha 1-3]GlcNAc beta 1-R). When the acceptor lacto-N-neotetraose (LNnT, Gal beta 1-4GlcNAc beta 1-3Gal beta 1-4Glc) was incubated with extracts of S. mansoni in the presence of GDPFuc and Mn2+, Fuc was transferred to generate the pentasaccharide lacto-N-fucopentaose III (LNFPIII, Gal beta 1-4[Fuc alpha 1-3]GlcNAc beta 1-3Gal Beta 1-4Glc). The enzyme did not transfer efficiently to the isomeric oligosaccharide lacto-N-tetraose (LNT, Gal beta 1-3GlcNAc beta 1-3Gal beta 1-4Glc). The activity of the schistosome alpha 1,3 FT toward LNnT was dependent upon time, protein and GDPFuc. Interestingly, the schistosome alpha 1,3 FT was also able to transfer Fuc to a sialic acid-containing trisaccharide NeuAc alpha 2-3 Gal beta 1-4 GlcNAc to produce the tetrasaccharide sialyl Lewis x (2,3 sLe(x), NeuAc alpha 2-3 Gal beta 1-4[Fuc 1-3]GlcNAc), although the rate of reaction with the sialylated acceptor was <5% of the rate obtained toward nonsialylated acceptor. The schistosome alpha 1,3 FT was relatively resistant to inhibition by N-ethylmaleimide. The enzymatic properties of the schistosome alpha 1,3 FT resemble those of the human myeloid fucosyltransferase FTIV and not those of other known human fucosyltransferase.

Animals↗
PubMed1996

Schistosoma mansoni infection in humans and primates induces cytolytic antibodies to surface Le(x) determinants on myeloid cells.

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.

Animals↗
PubMed1996

Glycobiology of schistosomiasis.

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.

Animals↗
PubMed1995

Tyrosine sulfation of P-selectin glycoprotein ligand-1 is required for high affinity binding to P-selectin.

P-selectin glycoprotein ligand-1 (PSGL-1) is a mucin-like glycoprotein on leukocytes that is a high affinity ligand for P-selectin. Previous studies have shown that sialylation and fucosylation of PSGL-1 are required for its binding to P-selectin, but other post-translational modifications of PSGL-1 may also be important. We demonstrate that PSGL-1 synthesized in human HL-60 cells can be metabolically labeled with [35S]sulfate that is incorporated primarily into tyrosine sulfate. Treatment of PSGL-1 with a bacterial arylsulfatase releases sulfate from tyrosine, resulting in a concordant decrease in binding to P-selectin. These studies demonstrate that tyrosine sulfate on PSGL-1 functions in conjunction with sialylated and fucosylated glycans to mediate high affinity binding to P-selectin.

Arylsulfatases↗
PubMed1995

Isolation and characterization of human milk bile salt-activated lipase C-tail fragment.

Glycosylation positions and oligosaccharide characteristics in the proline-rich, mucin-like, C-terminal region (C-tail) of human milk bile salt-activated lipase (BAL) were studied in order to assess the possible physiological functions of this region. A large-scale purification method has been devised to purify the C-tail fragment from human milk BAL. Chymotryptic, tryptic, and cyanogen bromide cleavages of partially purified BAL and subsequent molecular sieve chromatography yielded 20-30 mg of C-tail fragment from 1 L of human milk. The N-terminal sequence and amino acid composition of the purified C-tail fragment establish that it is derived from residues 528-712 of the enzyme. The O-glycosylated carbohydrates of the C-tail fragment contain fucose, galactose, glucosamine, galactosamine, and neuraminic acid in a molar ratio of 1:3:2:1:0.3, respectively. beta-Elimination reaction revealed that nine threonine residues and less than one serine residue were glycosylated. Edman degradation of C-tail fragment and its pronase subfragment suggest a number of glycosylation sites which are flanked by a consensus motif of PVPP. We suggest that this motif may serve as a signal for O-glycosylation in the C-tail region of BAL. Immunochemical studies indicated that the oligosaccharide chains in the C-tail region of BAL contain Lewis x and Lewis a antigens and, less prominently, sialyl Lewis x and sialyl Lewis a antigens. C-tail fragment was also found to bind jacalin lectin. These observations suggest the possibility that the C-tail region may contribute to adhesive activity in the physiological function of BAL.

Amino Acid Sequence↗
PubMed1995

Alpha-lactalbumin induces bovine milk beta 1,4-galactosyltransferase to utilize UDP-GalNAc.

We now report that alpha-lactalbumin (alpha-LA) has a novel effect on bovine milk UDP-Gal:GlcNAc-beta 1,4-galactosyltransferase (beta 1,4-GT) and induces the enzyme to efficiently utilize UDP-GalNAc as a donor. In the presence of alpha-LA the enzyme transfers GalNAc to free GlcNAc to produce GalNAc beta 1-4GlcNAc at a rate 55% of that compared to the rate when UDP-Gal is the donor in the absence of alpha-LA. The stimulation by alpha-LA is dependent on the concentrations of alpha-LA, acceptor, and sugar nucleotide. Interestingly, beta 1,4-GT is unable to transfer Gal-NAc to Glc with or without alpha-LA. alpha-LA also stimulates the transfer of GalNAc from UDP-GalNAc to various chitin oligomers, although the degree of stimulation decreases as the acceptor size increases. Thus, bovine milk beta 1,4-GT has an inherent ability to utilize two different sugar nucleotides and the sugar nucleotide preference is regulatable by alpha-LA.

Animals↗
PubMed1995

Leukocyte trafficking mediated by selectin-carbohydrate interactions.

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.

Animals↗
PubMed1995

Galectin-1, a beta-galactoside-binding lectin in Chinese hamster ovary cells. I. Physical and chemical characterization.

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.

Amino Acid Sequence↗
PubMed1995

Galectin-1, a beta-galactoside-binding lectin in Chinese hamster ovary cells. II. Localization and biosynthesis.

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.

Animals↗
PubMed1995

P-selectin glycoprotein ligand-1 mediates rolling of human neutrophils on P-selectin.

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.

Amino Acid Sequence↗
PubMed1995

Sulphate release from keratan sulphate and heparan sulphate by bovine N-acetylglucosamine-6-sulphate sulphatase.

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.

Animals↗