Publications by Year: 2017

Seeberger PH, Cummings RD. Glycans in Biotechnology and the Pharmaceutical Industry. In: Essentials of Glycobiology. 3rd edition. ; 2017. Publisher's VersionAbstract
Several classes of successful commercial products are based on isolated or synthetic glycans. This chapter summarizes the use of glycans as vaccines and therapeutics. Applications of glycan mimics as drugs are also discussed.
Cummings RD, Darvill AG, Etzler ME, Hahn MG. Glycan-Recognizing Probes as Tools. In: ; 2017. Publisher's VersionAbstract
Antibodies, lectins, microbial adhesins, viral agglutinins, and other proteins with carbohydrate-binding modules, collectively termed glycan-recognizing probes (GRPs), are widely used in glycan analysis because their specificities enable them to discriminate among a diverse variety of glycan structures. The native multivalency of many of these molecules promotes high-affinity avidity binding to the glycans and cell surfaces containing those glycans. This chapter describes the variety of commonly used GRPs, the types of analyses to which they may be applied, and cautionary principles that affect their optimal use.
Wilson IBH, Cummings RD, Aebi M. Nematoda. In: Essentials of Glycobiology. 3rd edition. ; 2017. Publisher's VersionAbstract
This chapter focuses on the nematode (roundworm) Caenorhabditis elegans as an example of the phylum Nematoda. C. elegans provides a powerful genetic system for studying glycans during embryological development and in primitive organ systems.
Cummings RD, Etzler ME, Surolia A. L-Type Lectins. In: Essentials of Glycobiology. 3rd edition. ; 2017. Publisher's VersionAbstract
The L-type lectins occur in the seeds of leguminous plants, and they have structural motifs that are present in a variety of glycan-binding proteins (GBPs) from other eukaryotic organisms. The structures of many of these lectins have been characterized, and many L-type lectins are used in a wide range of biomedical and analytical procedures. This chapter discusses the structure–function relationships of these lectins and the various biological roles they have in different organisms.
Parodi A, Cummings RD, Aebi M. Glycans in Glycoprotein Quality Control. In: Essentials of Glycobiology. 3rd edition. ; 2017. Publisher's VersionAbstract
N-Glycans affect glycoprotein folding because of their hydrophilic nature. In the endoplasmic reticulum (ER), the processing of N-glycans yields a series of truncated N-glycans that serve as checkpoints that dictate the life or death of many newly made membrane and secreted proteins. Other glycan modifications also may affect glycoprotein folding in the ER. This chapter describes glycan-mediated quality-control processes in the ER and Golgi apparatus and what happens to glycoproteins that fail their “final folding examination.”
Brazil JC, Sumagin R, Stowell SR, Goo L, Louis NA, Cummings RD, Parkos CA. Expression of Lewis-a glycans on polymorphonuclear leukocytes augments function by increasing transmigration. J Leukoc Biol. 2017;102 (3) :753-762. Publisher's VersionAbstract
PMN-expressed fucosylated glycans from the Lewis glycan family, including Lewis-x (Lex) and sialyl Lewis-x (sLex), have previously been implicated in the regulation of important PMN functions, including selectin-mediated trafficking across vascular endothelium. Although glycans, such as Lex and sLex, which are based on the type 2 sequence (Galβ1-4GlcNAc-R), are abundant on PMNs, the presence of type 1 Galβ1-3GlcNAc-R glycans required for PMN expression of the closely related stereoisomer of Lex, termed Lewis-A (Lea), has not, to our knowledge, been reported. Here, we show that Lea is abundantly expressed by human PMNs and functionally regulates PMN migration. Using mAbs whose precise epitopes were determined using glycan array technology, Lea function was probed using Lea-selective mAbs and lectins, revealing increased PMN transmigration across model intestinal epithelia, which was independent of epithelial-expressed LeaAnalyses of glycan synthetic machinery in PMNs revealed expression of β1-3 galactosyltransferase and α1-4 fucosyltransferase, which are required for Lea synthesis. Specificity of functional effects observed after ligation of Lea was confirmed by failure of anti-Lea mAbs to enhance migration using PMNs from individuals deficient in α1-4 fucosylation. These results demonstrate that Lea is expressed on human PMNs, and its specific engagement enhances PMN migration responses. We propose that PMN Lea represents a new target for modulating inflammation and regulating intestinal, innate immunity.
Schneider C, Wicki S, Graeter S, Timcheva TM, Keller CW, Quast I, Leontyev D, Djoumerska-Alexieva IK, Kasermann F, Jakob SM, et al. IVIG regulates the survival of human but not mouse neutrophils. Sci Rep. 2017;7 :1296. Publisher's VersionAbstract
Intravenous immunoglobulin (IVIG) are purified IgG preparations made from the pooled plasma from thousands of healthy donors and are being tested in preclinical mouse models. Inherent challenges, however, are the pluripotency of IVIG and its xenogeneicity in animals. IVIG can alter the viability of human neutrophils via agonistic antibodies to Fas and Siglec-9. In this study, we compared the effects of IVIG on human and mouse neutrophils using different death assays. Different commercial IVIG preparations similarly induced cytokine-dependent death in human neutrophils, whereas they had no effects on the survival of either peripheral blood or bone marrow neutrophils from C57BL/6 or BALB/c mice. F(ab’)2 but not Fc fragments of IVIG induced death of human neutrophils, whereas neither of these IVIG fragments, nor agonistic monoclonal antibodies to human Fas or Siglec-9 affected the viability of mouse neutrophils. Pooled mouse IgG, which exhibited a different immunoprofile compared to IVIG, also had no effect on mouse cells. Together, these observations demonstrate that effects of IVIG on neutrophil survival are not adequately reflected in current mouse models, despite the key role of these cells in human inflammatory and autoimmune diseases.
Laan LC, Williams AR, Stavenhagen K, Giera M, Kooij G, Vlasakov L, Kalay H, Kringel H, Nejsum P, Thamsborg SM, et al. The whipworm (Trichuris suis) secretes prostaglandin E2 to suppress proinflammatory properties in human dendritic cells. FASEB J. 2017;31 (2) :719-731. Publisher's VersionAbstract
Clinical trials have shown that administration of the nematode Trichuris suis can be beneficial in treating various immune disorders. To provide insight into the mechanisms by which this worm suppresses inflammatory responses, an active component was purified from T. suis soluble products (TsSPs) that suppress­­­­ TNF and IL-12 secretion from LPS-activated human dendritic cells (DCs). Analysis by liquid chromatography tandem mass spectrometry identified this compound as prostaglandin (PG)E2. The purified compound showed similar properties compared with TsSPs and commercial PGE2 in modulating LPS-induced expression of many cytokines and chemokines and in modulating Rab7B and P2RX7 expression in human DCs. Furthermore, the TsSP-induced reduction of TNF secretion from DCs is reversed by receptor antagonists for EP2 and EP4, indicating PGE2 action. T. suis secretes extremely high amounts of PGE2 (45–90 ng/mg protein) within their excretory/secretory products but few related lipid mediators as established by metabololipidomic analysis. Culture of T. suis with several cyclooxygenase (COX) inhibitors that inhibit mammalian prostaglandin synthesis affected the worm’s motility but did not inhibit PGE2 secretion, suggesting that the worms can synthesize PGE2 via a COX-independent pathway. We conclude that T. suis secretes PGE2 to suppress proinflammatory responses in human DCs, thereby modulating the host’s immune response.—Laan, L. C., Williams, A. R., Stavenhagen, K., Giera, M., Kooij, G., Vlasakov, I., Kalay, H., Kringel, H., Nejsum, P., Thamsborg, S. M., Wuhrer, M., Dijkstra, C. D., Cummings, R. D., van Die, I. The whipworm (Trichuris suis) secretes prostaglandin E2 to suppress proinflammatory properties in human dendritic cells
Hanes MS, Moreman KW, Cummings RD. Biochemical characterization of functional domains of the chaperone Cosmc. PLoS One. 2017;12 (6). Publisher's VersionAbstract
Cosmc is an endoplasmic reticulum chaperone necessary for normal protein O-GalNAc glycosylation through regulation of T-synthase, its single client. Loss-of-function of Cosmc results in expression of the Tn antigen, which is associated with multiple human diseases including cancer. Despite intense interest in dysregulated expression of the Tn antigen, little is known about the structure and function of Cosmc, including domain organization, secondary structure, oligomerization, and co-factors. Limited proteolysis experiments show that Cosmc contains a structured N-terminal domain (CosmcΔ256), and biochemical characterization of CosmcΔ256 reveals wild type chaperone activity. Interestingly, CosmcE152K, which shows loss of function in vivo, exhibits wild type-like activity in vitro. Cosmc and CosmcE152K heterogeneously oligomerize and form monomeric, dimeric, trimeric, and tetrameric species, while CosmcΔ256 is predominantly monomeric as characterized by chemical crosslinking and blue native page electrophoresis. Additionally, Cosmc selectively binds divalent cations in thermal shift assays and metal binding is abrogated by the CosmcΔ256 truncation, and perturbed by the E152K mutation. Therefore, the N-terminal domain of Cosmc mediates T-synthase binding and chaperone function, whereas the C-terminal domain is necessary for oligomerization and metal binding. Our results provide new structure-function insight to Cosmc, indicate that Cosmc behaves as a modular protein and suggests points of modulation or regulation of in vivo chaperone function.
Tiemeyer M, Aoki K, Paulson J, Cummings RD, York WS, Karlsson NG, Lisacek F, Packer NH, Campbell MP, Aoki NP, et al. GlyTouCan: an accessible glycan structure repository. Glycobiology. 2017;27 (10) :915-919. Publisher's VersionAbstract
Rapid and continued growth in the generation of glycomic data has revealed the need for enhanced development of basic infrastructure for presenting and interpreting these datasets in a manner that engages the broader biomedical research community. Early in their growth, the genomic and proteomic fields implemented mechanisms for assigning unique gene and protein identifiers that were essential for organizing data presentation and for enhancing bioinformatic approaches to extracting knowledge. Similar unique identifiers are currently absent from glycomic data. In order to facilitate continued growth and expanded accessibility of glycomic data, the authors strongly encourage the glycomics community to coordinate the submission of their glycan structures to the GlyTouCan Repository and to make use of GlyTouCan identifiers in their communications and publications. The authors also deeply encourage journals to recommend a submission workflow in which submitted publications utilize GlyTouCan identifiers as a standard reference for explicitly describing glycan structures cited in manuscripts.
Zhong AH, Gordon Jiang Z, Cummings RD, Robson SC. Various N-glycoforms differentially upregulate E-NTPDase activity of the NTPDase3/CD39L3 ecto-enzymatic domain. Purinergic Signal. 2017;Sep 27 :1-9. Publisher's VersionAbstract
The GDA1/CD39 ecto-nucleoside triphosphate diphosphosphohydrolase (E-NTPDase) superfamily is a group of eight heavily glycosylated ecto-enzymes that hydrolyze extracellular nucleosides di- and tri-phosphates in the presence of divalent cations, to generate the monophosphate derivatives. This catalytic process differentially regulates a complex array of purinergic signaling responses. NTPDase3/CD39L3is dominantly expressed in pancreatic islet cells, where it may regulate insulin secretion, and has seven N-linked glycosylation sites with four close to five highly conserved domains called “apyrase conserved regions” (ACRs). In a manner similar to CD39, NTPDase3/CD39L3 uses ATP as its preferential substrate and also possesses significant activities toward other triphosphate and diphosphate nucleosides. To understand the mechanism of the ecto-NTPDase activity and substrate specificity, potentially impacted by N-glycans, we have generated soluble enzymatic domains of NTPDase3/CD39L3 in human embryotic kidney cells with four different glycan modifications. These include mannose5–9 glycans with kifunesine treatment, single GlcNAc-Asn by treatment with EndoH, de-glycosylated form by treatment with PNGaseF, and wild-type glycans. Our functional data indicate that the non-glycosylated NTPDase3/CD39L3 ecto-enzymatic domain retains activity, but that N-glycan attachments, such as the GlcNAc-Asn, substantially upregulate specific NTPDase activity by 2–20 fold. Both the Vmax and the Km on di- or tri-phosphate nucleosides are substantially and differentially altered by the glycan attachments. Structural modeling analysis based on putative structures derived from bacterial-originated CD39 domain proteins suggests that N-glycan modifications at Asn149 next to ACR2 and/or Asn454, N-terminal to ACR5 have critical roles in regulating the catalytic pocket of NTPDase3/CD39L3. Our data provide both new insights into the enzymatic mechanisms of NTPDase family members and further evidence that N-glycans directly modulate functional ectonucleotidase activities.
Byrd-Leotis L, Cummings RD, Steinhauer DA. The Interplay between the Host Receptor and Influenza Virus Hemagglutinin and Neuraminidase. Int J Mol Sci. 2017;18 (1541). Publisher's VersionAbstract
The hemagglutinin (HA) and neuraminidase (NA) glycoproteins of influenza A virus are responsible for the surface interactions of the virion with the host. Entry of the virus is mediated by functions of the HA: binding to cellular receptors and facilitating fusion of the virion membrane with the endosomal membrane. The HA structure contains receptor binding sites in the globular membrane distal head domains of the trimer, and the fusion machinery resides in the stem region. These sites have specific characteristics associated with subtype and host, and the differences often define species barriers. For example, avian viruses preferentially recognize α2,3-Sialic acid terminating glycans as receptors and mammalian viruses recognize α2,6-Sialic acid. The neuraminidase, or the receptor-destroying protein, cleaves the sialic acid from cellular membrane constituents and viral glycoproteins allowing for egress of nascent virions. A functional balance of activity has been demonstrated between the two glycoproteins, resulting in an optimum level of HA affinity and NA enzymatic cleavage to allow for productive infection. As more is understood about both HA and NA, the relevance for functional balance between HA and NA continues to expand, with potential implications for interspecies transmission, host adaptation, and pathogenicity.
Taniguchi T, Woodward AM, Magnelli P, McColgan NM, Lehoux S, Jacobo SMP, Mauris J, Argüeso P. N-Glycosylation affects the stability and barrier function of the MUC16 mucin. J Biol Chem. 2017;292 (June 30) :11079-11090. Publisher's VersionAbstract
Transmembrane mucins are highly O-glycosylated glycoproteins that coat the apical glycocalyx on mucosal surfaces and represent the first line of cellular defense against infection and injury. Relatively low levels of N-glycans are found on transmembrane mucins, and their structure and function remain poorly characterized. We previously reported that carbohydrate-dependent interactions of transmembrane mucins with galectin-3 contribute to maintenance of the epithelial barrier at the ocular surface. Now, using MALDI-TOF mass spectrometry, we report that transmembrane mucin N-glycans in differentiated human corneal epithelial cells contain primarily complex-type structures with N-acetyllactosamine, a preferred galectin ligand. In N-glycosylation inhibition experiments, we find that treatment with tunicamycin and siRNA-mediated knockdown of the Golgi N-acetylglucosaminyltransferase I gene (MGAT1) induce partial loss of both total and cell-surface levels of the largest mucin, MUC16, and a concomitant reduction in glycocalyx barrier function. Moreover, we identified a distinct role for N-glycans in promoting MUC16's binding affinity toward galectin-3 and in causing retention of the lectin on the epithelial cell surface. Taken together, these studies define a role for N-linked oligosaccharides in supporting the stability and function of transmembrane mucins on mucosal surfaces.