Publications by Year: 2008

2008
Stowell SR, Arthur CM, Mehta P, Slanina KA, Blixt O, Leffler H, Smith DF, Cummings RD. Galectin-1, -2, and -3 exhibit differential recognition of sialylated glycans and blood group antigens. J Biol Chem. 2008;283 (15) :10109-23.Abstract
Human galectins have functionally divergent roles, although most of the members of the galectin family bind weakly to the simple disaccharide lactose (Galbeta1-4Glc). To assess the specificity of galectin-glycan interactions in more detail, we explored the binding of several important galectins (Gal-1, Gal-2, and Gal-3) using a dose-response approach toward a glycan microarray containing hundreds of structurally diverse glycans, and we compared these results to binding determinants on cells. All three galectins exhibited differences in glycan binding characteristics. On both the microarray and on cells, Gal-2 and Gal-3 exhibited higher binding than Gal-1 to fucose-containing A and B blood group antigens. Gal-2 exhibited significantly reduced binding to all sialylated glycans, whereas Gal-1 bound alpha2-3- but not alpha2-6-sialylated glycans, and Gal-3 bound to some glycans terminating in either alpha2-3- or alpha2-6-sialic acid. The effects of sialylation on Gal-1, Gal-2, and Gal-3 binding to cells also reflected differences in cellular sensitivity to Gal-1-, Gal-2-, and Gal-3-induced phosphatidylserine exposure. Each galectin exhibited higher binding for glycans with poly-N-acetyllactosamine (poly(LacNAc)) sequences (Galbeta1-4GlcNAc)(n) when compared with N-acetyllactosamine (LacNAc) glycans (Galbeta1-4GlcNAc). However, only Gal-3 bound internal LacNAc within poly(LacNAc). These results demonstrate that each of these galectins mechanistically differ in their binding to glycans on the microarrays and that these differences are reflected in the determinants required for cell binding and signaling. The specific glycan recognition by each galectin underscores the basis for differences in their biological activities.
Zheng Q, van Die I, Cummings RD. A novel alpha1,2-fucosyltransferase (CE2FT-2) in Caenorhabditis elegans generates H-type 3 glycan structures. Glycobiology. 2008;18 (4) :290-302.Abstract
The alpha1,2-fucosyltransferase family (alpha1,2FT) is the largest family of glycosyltransferases in the genome of the free-living nematode Caenorhabditis elegans, and early evidence suggests that each member may have a unique activity. Here we describe a C. elegans gene (designated CE2FT-2) encoding an alpha1,2FT that has the potential to generate the sequence Fucalpha1-2Galbeta1-3GalNAcalpha-R, which is the H-type 3 blood group structure. The CE2FT-2 cDNA encodes a putative transmembrane protein that shows approximately 42% amino acid identity to a previously cloned C. elegans alpha1,2FT (termed CE2FT-1), but has a very low identity ( approximately 16-20%) to alpha1,2FT sequences in humans, rabbits, and mice. A recombinant form of CE2FT-2 expressed in human 293T cells has a high alpha1,2FT activity toward Galbeta1-3GalNAcalpha-O-pNP, but unexpectedly, the enzyme is inactive toward the acceptor Galbeta-O-phenyl. Thus, CE2FT-2 differs from all other alpha1,2FTs previously described from animals that all utilize Galbeta-O-phenyl. CE2FT-2 is expressed at all stages of worm development, but remarkably, promoter analysis of the CE2FT-2 gene using green fluorescent protein reporter constructs indicates that the CE2FT-2 is expressed exclusively in pharyngeal cells of the worm from embryo to an adult stage. Because pharyngeal cells are known to secrete their glycoconjugates to the nematode surface, these results may indicate that products of CE2FT-2 contribute to interactions of the nematode with its environment or are used as ligands for bacterial attachment. These findings, along with those on other alpha1,2FTs in C. elegans, suggest that each alpha1,2FT in this organism may have a unique acceptor specificity, expression pattern, and biological function.
Song X, Xia B, Lasanajak Y, Smith DF, Cummings RD. Quantifiable fluorescent glycan microarrays. Glycoconj J. 2008;25 (1) :15-25.Abstract
A glycan microarray was developed by using 2,6-diaminopyridine (DAP) as a fluorescent linker and printing of the glycan-DAP conjugates (GDAPs) on epoxy-activated glass slides. Importantly, all coupled GDAPs showed a detectable level of concentration-dependent GDAP fluorescence under blue laser excitation (495 nm) that can be used for both grid location and on-slide quantification. A glycan array including a large number of GDAP's derived from natural and commercially available free glycans was constructed and glycan interactions with various plant lectins were investigated. In addition, binding parameters of lectins to glycans were obtained by varying both the amount of GDAPs on the array and the lectin concentration in analyses. These data demonstrate the general utility of GDAP microarrays for functional glycomic analyses and for determining binding parameters of glycan binding proteins (GBPs).
Klopocki AG, Yago T, Mehta P, Yang J, Wu T, Leppänen A, Bovin NV, Cummings RD, Zhu C, McEver RP. Replacing a lectin domain residue in L-selectin enhances binding to P-selectin glycoprotein ligand-1 but not to 6-sulfo-sialyl Lewis x. J Biol Chem. 2008;283 (17) :11493-500.Abstract
Selectin-ligand interactions (bonds) mediate leukocyte rolling on vascular surfaces. The molecular basis for differential ligand recognition by selectins is poorly understood. Here, we show that substituting one residue (A108H) in the lectin domain of L-selectin increased its force-free affinity for a glycosulfopeptide binding site (2-GSP-6) on P-selectin glycoprotein ligand-1 (PSGL-1) but not for a sulfated-glycan binding site (6-sulfo-sialyl Lewis x) on peripheral node addressin. The increased affinity of L-selectinA108H for 2-GSP-6 was due to a faster on-rate and to a slower off-rate that increased bond lifetimes in the absence of force. Rather than first prolonging (catching) and then shortening (slipping) bond lifetimes, increasing force monotonically shortened lifetimes of L-selectinA108H bonds with 2-GSP-6. When compared with microspheres bearing L-selectin, L-selectinA108H microspheres rolled more slowly and regularly on 2-GSP-6 at low flow rates. A reciprocal substitution in P-selectin (H108A) caused faster microsphere rolling on 2-GSP-6. These results distinguish molecular mechanisms for L-selectin to bind to PSGL-1 and peripheral node addressin and explain in part the shorter lifetimes of PSGL-1 bonds with L-selectin than P-selectin.
Farrand S, Hotze E, Friese P, Hollingshead SK, Smith DF, Cummings RD, Dale GL, Tweten RK. Characterization of a streptococcal cholesterol-dependent cytolysin with a lewis y and b specific lectin domain. Biochemistry. 2008;47 (27) :7097-107.Abstract
The cholesterol-dependent cytolysins (CDCs) are a large family of pore-forming toxins that often exhibit distinct structural changes that modify their pore-forming activity. A soluble platelet aggregation factor from Streptococcus mitis (Sm-hPAF) was characterized and shown to be a functional CDC with an amino-terminal fucose-binding lectin domain. Sm-hPAF, or lectinolysin (LLY) as renamed herein, is most closely related to CDCs from Streptococcus intermedius (ILY) and Streptococcus pneumoniae (pneumolysin or PLY). The LLY gene was identified in strains of S. mitis, S. pneumoniae, and Streptococcus pseudopneumoniae. LLY induces pore-dependent changes in the light scattering properties of the platelets that mimic those induced by platelet aggregation but does not induce platelet aggregation. LLY monomers form the typical large homooligomeric membrane pore complex observed for the CDCs. The pore-forming activity of LLY on platelets is modulated by the amino-terminal lectin domain, a structure that is not present in other CDCs. Glycan microarray analysis showed the lectin domain is specific for difucosylated glycans within Lewis b (Le (b)) and Lewis y (Le (y)) antigens. The glycan-binding site is occluded in the soluble monomer of LLY but is apparently exposed after cell binding, since it significantly increases LLY pore-forming activity in a glycan-dependent manner. Hence, LLY represents a new class of CDC whose pore-forming mechanism is modulated by a glycan-binding domain.
Stowell SR, Qian Y, Karmakar S, Koyama NS, Dias-Baruffi M, Leffler H, McEver RP, Cummings RD. Differential roles of galectin-1 and galectin-3 in regulating leukocyte viability and cytokine secretion. J Immunol. 2008;180 (5) :3091-102.Abstract
Galectin-1 (Gal-1) and galectin-3 (Gal-3) exhibit profound but unique immunomodulatory activities in animals but their molecular mechanisms are incompletely understood. Early studies suggested that Gal-1 inhibits leukocyte function by inducing apoptotic cell death and removal, but recent studies show that some galectins induce exposure of the common death signal phosphatidylserine (PS) independently of apoptosis. In this study, we report that Gal-3, but not Gal-1, induces both PS exposure and apoptosis in primary activated human T cells, whereas both Gal-1 and Gal-3 induce PS exposure in neutrophils in the absence of cell death. Gal-1 and Gal-3 bind differently to the surfaces of T cells and only Gal-3 mobilizes intracellular Ca2+ in these cells, although Gal-1 and Gal-3 bind their respective T cell ligands with similar affinities. Although Gal-1 does not alter T cell viability, it induces IL-10 production and attenuates IFN-gamma production in activated T cells, suggesting a mechanism for Gal-1-mediated immunosuppression in vivo. These studies demonstrate that Gal-1 and Gal-3 induce differential responses in T cells and neutrophils, and identify the first factor, Gal-3, capable of inducing PS exposure with or without accompanying apoptosis in different leukocytes, thus providing a possible mechanism for galectin-mediated immunomodulation in vivo.
Stowell SR, Arthur CM, Slanina KA, Horton JR, Smith DF, Cummings RD. Dimeric Galectin-8 induces phosphatidylserine exposure in leukocytes through polylactosamine recognition by the C-terminal domain. J Biol Chem. 2008;283 (29) :20547-59.Abstract
Human galectins have distinct and overlapping biological roles in immunological homeostasis. However, the underlying differences among galectins in glycan binding specificity regulating these functions are unclear. Galectin-8 (Gal-8), a tandem repeat galectin, has two distinct carbohydrate recognition domains (CRDs) that may cross-link cell surface counter receptors. Here we report that each Gal-8 CRD has differential glycan binding specificity and that cell signaling activity resides in the C-terminal CRD. Full-length Gal-8 and recombinant individual domains (Gal-8N and Gal-8C) bound to human HL60 cells, but only full-length Gal-8 signaled phosphatidylserine (PS) exposure in cells, which occurred independently of apoptosis. Although desialylation of cells did not alter Gal-8 binding, it enhanced cellular sensitivity to Gal-8-induced PS exposure. By contrast, HL60 cell desialylation increased binding by Gal-8C but reduced Gal-8N binding. Enzymatic reduction in surface poly-N-acetyllactosamine (polyLacNAc) glycans in HL60 cells reduced cell surface binding by Gal-8C but did not alter Gal-8N binding. Cross-linking and light scattering studies showed that Gal-8 is dimeric, and studies on individual subunits indicate that dimerization occurs through the Gal-8N domain. Mutations of individual domains within full-length Gal-8 showed that signaling activity toward HL60 cells resides in the C-terminal domain. In glycan microarray analyses, each CRD of Gal-8 showed different binding, with Gal-8N recognizing sulfated and sialylated glycans and Gal-8C recognizing blood group antigens and polyLacNAc glycans. These results demonstrate that Gal-8 dimerization promotes functional bivalency of each CRD, which allows Gal-8 to signal PS exposure in leukocytes entirely through C-terminal domain recognition of polyLacNAc glycans.
Karmakar S, Stowell SR, Cummings RD, McEver RP. Galectin-1 signaling in leukocytes requires expression of complex-type N-glycans. Glycobiology. 2008;18 (10) :770-8.Abstract
Dimeric galectin-1 (dGal-1) is a homodimeric lectin with multiple proposed functions. Although dGal-1 binds to diverse glycans, it is unclear whether dGal-1 preferentially binds to specific subsets of glycans on cell surfaces to transmit signals. To explore this question, we selectively inhibited major glycan biosynthetic pathways in human HL60, Molt-4, and Jurkat cells. Inhibition of N-glycan processing blocked surface binding of dGal-1 and prevented dGal-1-induced Ca(2+) mobilization and phosphatidylserine exposure. By contrast, inhibition of O-glycan or glycosphingolipid biosynthesis did not affect dGal-1 binding or dGal-1-induced Ca(2+) mobilization and phosphatidylserine exposure. These results demonstrate that dGal-1 preferentially binds to and signals through glycoproteins containing complex-type N-glycans in at least some leukocyte subsets.
Zupancic ML, Frieman M, Smith D, Alvarez RA, Cummings RD, Cormack BP. Glycan microarray analysis of Candida glabrata adhesin ligand specificity. Mol Microbiol. 2008;68 (3) :547-59.Abstract
The Candida glabrata genome encodes at least 23 members of the EPA (epithelial adhesin) family responsible for mediating adherence to host cells. To better understand the mechanism by which the Epa proteins contribute to pathogenesis, we have used glycan microarray analysis to characterize their carbohydrate-binding specificities. Using Saccharomyces cerevisiae strains surface-expressing the N-terminal ligand-binding domain of the Epa proteins, we found that the three Epa family members functionally identified as adhesins in Candida glabrata (Epa1, Epa6 and Epa7) bind to ligands containing a terminal galactose residue. However, the specificity of the three proteins for glycans within this class varies, with Epa6 having a broader specificity range than Epa1 or Epa7. This result is intriguing given the close homology between Epa6 and Epa7, which are 92% identical at the amino acid level. We have mapped a five-amino-acid region within the N-terminal ligand-binding domain that accounts for the difference in specificity of Epa6 and Epa7 and show that these residues contribute to adherence to both epithelial and endothelial cell lines in vitro.
Ju T, Lanneau GS, Gautam T, Wang Y, Xia B, Stowell SR, Willard MT, Wang W, Xia JY, Zuna RE, et al. Human tumor antigens Tn and sialyl Tn arise from mutations in Cosmc. Cancer Res. 2008;68 (6) :1636-46.Abstract
Neoplastic lesions typically express specific carbohydrate antigens on glycolipids, mucins, and other glycoproteins. Such antigens are often under epigenetic control and are subject to reversion and loss upon therapeutic selective pressure. We report here that two of the most common tumor-associated carbohydrate antigens, Tn and sialyl Tn (STn), result from somatic mutations in the gene Cosmc that encodes a molecular chaperone required for formation of the active T-synthase. Diverse neoplastic lesions, including colon cancer and melanoma-derived cells lines, expressed both Tn and STn antigen due to loss-of-function mutations in Cosmc. In addition, two human cervical cancer specimens that showed expression of the Tn/STn antigens were also found to have mutations in Cosmc and loss of heterozygosity for the cross-linked Cosmc locus. This is the first example of somatic mutations in multiple types of cancers that cause global alterations in cell surface carbohydrate antigen expression.
Kawar ZS, Johnson TK, Natunen S, Lowe JB, Cummings RD. PSGL-1 from the murine leukocytic cell line WEHI-3 is enriched for core 2-based O-glycans with sialyl Lewis x antigen. Glycobiology. 2008;18 (6) :441-6.Abstract
Leukocyte trafficking involves specific recognition between P-selectin and L-selectin and PSGL-1 containing core 2-based O-glycans expressing sialyl Lewis x (SLe(x)) antigen. However, the structural identity of the glycan component(s) displayed by murine neutrophil PSGL-1 that contributes to its P-selectin counter-receptor activity has been uncertain, since these cells express little if any SLe(x) antigen, and because there have been no direct studies to examine murine PSGL-1 glycosylation. To address this uncertainty, we studied PSGL-1 glycosylation in the murine cell line WEHI-3 using metabolic-radiolabeling with (3)H-monosaccharide precursors to detect low-abundance O-glycan structures. We report that PSGL-1 from WEHI-3 cells expresses a di-sialylated core 2 O-glycan containing the SLe(x) antigen. This fucosylated O-glycan is scarce on PSGL-1 and essentially undetectable in total leukocyte glycoproteins from WEHI-3 cells. These results demonstrate that WEHI-3 cells selectively fucosylate PSGL-1 to generate functionally important core 2-based O-glycans containing the SLe(x) antigen.
Ju T, Aryal RP, Stowell CJ, Cummings RD. Regulation of protein O-glycosylation by the endoplasmic reticulum-localized molecular chaperone Cosmc. J Cell Biol. 2008;182 (3) :531-42.Abstract
Regulatory pathways for protein glycosylation are poorly understood, but expression of branchpoint enzymes is critical. A key branchpoint enzyme is the T-synthase, which directs synthesis of the common core 1 O-glycan structure (T-antigen), the precursor structure for most mucin-type O-glycans in a wide variety of glycoproteins. Formation of active T-synthase, which resides in the Golgi apparatus, requires a unique molecular chaperone, Cosmc, encoded on Xq24. Cosmc is the only molecular chaperone known to be lost through somatic acquired mutations in cells. We show that Cosmc is an endoplasmic reticulum (ER)-localized adenosine triphosphate binding chaperone that binds directly to human T-synthase. Cosmc prevents the aggregation and ubiquitin-mediated degradation of the T-synthase. These results demonstrate that Cosmc is a molecular chaperone in the ER required for this branchpoint glycosyltransferase function and show that expression of the disease-related Tn antigen can result from deregulation or loss of Cosmc function.
Varki AP, Baum LG, Bellis SL, Cummings RD, Esko JD, Hart GW, Linhardt RJ, Lowe JB, McEver RP, Srivastava A, et al. Working group report: the roles of glycans in hemostasis, inflammation and vascular biology. Glycobiology. 2008;18 (10) :747-9.
Cerri DG, Rodrigues LC, Stowell SR, Araujo DD, Coelho MC, Oliveira SR, Bizario JCS, Cummings RD, Dias-Baruffi M, Costa MCR. Degeneration of dystrophic or injured skeletal muscles induces high expression of Galectin-1. Glycobiology. 2008;18 (11) :842-50.Abstract
Muscle degenerative diseases such as Duchenne Muscular Dystrophy are incurable and treatment options are still restrained. Understanding the mechanisms and factors responsible for muscle degeneration and regeneration will facilitate the development of novel therapeutics. Several recent studies have demonstrated that Galectin-1 (Gal-1), a carbohydrate-binding protein, induces myoblast differentiation and fusion in vitro, suggesting a potential role for this mammalian lectin in muscle regenerative processes in vivo. However, the expression and localization of Gal-1 in vivo during muscle injury and repair are unclear. We report the expression and localization of Gal-1 during degenerative-regenerative processes in vivo using two models of muscular dystrophy and muscle injury. Gal-1 expression increased significantly during muscle degeneration in the murine mdx and in the canine Golden Retriever Muscular Dystrophy animal models. Compulsory exercise of mdx mouse, which intensifies degeneration, also resulted in sustained Gal-1 levels. Furthermore, muscle injury of wild-type C57BL/6 mice, induced by BaCl(2) treatment, also resulted in a marked increase in Gal-1 levels. Increased Gal-1 levels appeared to localize both inside and outside the muscle fibers with significant extracellular Gal-1 colocalized with infiltrating CD45(+) leukocytes. By contrast, regenerating muscle tissue showed a marked decrease in Gal-1 to baseline levels. These results demonstrate significant regulation of Gal-1 expression in vivo and suggest a potential role for Gal-1 in muscle homeostasis and repair.
Fu J, Gerhardt H, McDaniel MJ, Xia B, Liu X, Ivanciu L, Ny A, Hermans K, Silasi-Mansat R, McGee S, et al. Endothelial cell O-glycan deficiency causes blood/lymphatic misconnections and consequent fatty liver disease in mice. J Clin Invest. 2008;118 (11) :3725-37.Abstract
Mucin-type O-glycans (O-glycans) are highly expressed in vascular ECs. However, it is not known whether they are important for vascular development. To investigate the roles of EC O-glycans, we generated mice lacking T-synthase, a glycosyltransferase encoded by the gene C1galt1 that is critical for the biosynthesis of core 1-derived O-glycans, in ECs and hematopoietic cells (termed here EHC T-syn(-/-) mice). EHC T-syn(-/-) mice exhibited embryonic and neonatal lethality associated with disorganized and blood-filled lymphatic vessels. Bone marrow transplantation and EC C1galt1 transgene rescue demonstrated that lymphangiogenesis specifically requires EC O-glycans, and intestinal lymphatic microvessels in EHC T-syn(-/-) mice expressed a mosaic of blood and lymphatic EC markers. The level of O-glycoprotein podoplanin was significantly reduced in EHC T-syn(-/-) lymphatics, and podoplanin-deficient mice developed blood-filled lymphatics resembling EHC T-syn(-/-) defects. In addition, postnatal inactivation of C1galt1 caused blood/lymphatic vessel misconnections that were similar to the vascular defects in the EHC T-syn(-/-) mice. One consequence of eliminating T-synthase in ECs and hematopoietic cells was that the EHC T-syn(-/-) pups developed fatty liver disease, because of direct chylomicron deposition via misconnected portal vein and intestinal lymphatic systems. Our studies therefore demonstrate that EC O-glycans control the separation of blood and lymphatic vessels during embryonic and postnatal development, in part by regulating podoplanin expression.
Lawrence R, Olson SK, Steele RE, Wang L, Warrior R, Cummings RD, Esko JD. Evolutionary differences in glycosaminoglycan fine structure detected by quantitative glycan reductive isotope labeling. J Biol Chem. 2008;283 (48) :33674-84.Abstract
To facilitate qualitative and quantitative analysis of glycosaminoglycans, we tagged the reducing end of lyase-generated disaccharides with aniline-containing stable isotopes (12C6 and 13C6). Because different isotope tags have no effect on chromatographic retention times but can be discriminated by a mass detector, differentially isotope-tagged samples can be compared simultaneously by liquid chromatography/mass spectrometry and quantified by admixture with known amounts of standards. The technique is adaptable to all types of glycosaminoglycans, and its sensitivity is only limited by the type of mass spectrometer available. We validated the method using commercial heparin and keratan sulfate as well as heparan sulfate isolated from mutant and wild-type Chinese hamster ovary cells, and select tissues from mutant and wild-type mice. This new method provides more robust, reliable, and sensitive means of quantitative evaluation of glycosaminoglycan disaccharide compositions than existing techniques allowing us to compare the chondroitin and heparan sulfate compositions of Hydra vulgaris, Drosophila melanogaster, Caenorhabditis elegans, and mammalian cells. Our results demonstrate significant differences in glycosaminoglycan structure among these organisms that might represent evolutionarily distinct functional motifs.