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Structure and biosynthesis of glycoprotein carbohydrates
No full textIn eukaryotes, the majority of cell surface and secreted proteins are covalently modified with carbohydrates. This type of posttranslational modification, glycosylation, is inherently complex exhibiting extensive chemical and conformational heterogeneity. Despite this complexity, there are common structural and biosynthetic principles. Here, we present an introduction to the different hierarchies of carbohydrate structure from stereochemistry of monosaccharides and their linkages to the structural diversity of complex mammalianglycosylation. These structures are discussed in the context of the glycan biosynthetic pathways, with an emphasis on N-linked glycosylation, and we outline the nomenclature with which these complex structures can be described. We describe the biosynthesis and crystal structures of antibodies and their glycans to illustrate the conformational properties of glycoprotein carbohydrates and how therapeutic antibodies are being developed by modulating these glycans.</p
Chapter 10 - Antibody glycosylation
No full textThe antibody Fc region is posttranslationally modified by N-linked glycosylation. In immunoglobulin G (IgG), the processing of the glycans is restricted by the presence of extensive interaction with the protein surface. The resulting set of antibody glycoforms exhibit a range of effector functions. In this chapter, we outline the impact of glycosylation on the immune function of antibodies and discuss the implications for monoclonal antibody and intravenous immunoglobulin therapies.</p
Dissecting the molecular mechanism of IVIg therapy : the interaction between serum IgG and DC-SIGN is independent of antibody glycoform or Fc domain
No full textIntravenous immunoglobulin (IVIg) therapy is used to treat a wide range of autoimmune conditions and consists of pooled immunoglobulin G (IgG) from healthy donors. The immunosuppressive effects of IVIg are, in part, attributed to terminal α2,6-linked sialic acid residues on the N-linked glycans of the IgG Fc (fragment crystallizable) domain. This α2,6-sialylated Fc (sFc) has been reported to bind to the carbohydrate recognition domain (CRD) of the cell-surface lectin DC-SIGN (dendritic cell-specific intercellular adhesion molecule-3-grabbing non-integrin) and its murine orthologue SIGN-R1 (specific intracellular adhesion molecule-grabbing non-integrin R1) and, via this interaction, to signal the downstream expression of immunosuppressive cytokines and receptors. Consistent with this model, the antiinflammatory effect of IVIg treatment is abolished in a murine knock-out of SIGN-R1 and can be restored by a knock-in with human DC-SIGN. In contrast, however, existing glycan array and X-ray crystallographic studies indicate that the CRDs of both SIGN-R1 and DC-SIGN bind to a restricted set of primarily oligomannose-type glycans that does not include the glycans found on sFc. We attempted to reconcile these immunological and biophysical observations. We first generated hypersialylated, desialylated, deglycosylated and untreated serum IgG and found that the affinity for the complete extracellular region of the DC-SIGN tetramer was similar for all antibody glycoforms. Moreover, the binding could be attributed to cross-reactive, polyclonal Fab (fragment antigen-binding) specificities in serum as neither recombinant Fc nor sFc bound to DC-SIGN. In addition, serum IgG exhibited no competition against known ligands of the DC-SIGN CRD. These findings lead us to suggest that IVIg therapy does not involve binding of IgG Fc to DC-SIGN and that alternative cell-surface lectins are required for the antiinflammatory activity of sFc
MALDI-MS/MS with traveling wave ion mobility for the structural analysis of N-Linked glycans
Full text linkThe preference for singly charged ion formation by MALDI makes it a better choice than electrospray ionization for profiling mixtures of N-glycans. For structural analysis, fragmentation of negative ions often yields more informative spectra than fragmentation of positive ones but such ions are more difficult to produce from neutral glycans under MALDI conditions. This work investigates conditions for the formation of both positive and negative ions by MALDI from N-linked glycans released from glycoproteins and their subsequent MS/MS and ion mobility behaviour. 2,4,6-Trihydroxyacetophenone (THAP) doped with ammonium nitrate was found to give optimal ion yields in negative ion mode. Ammonium chloride or phosphate also yielded prominent adducts but anionic carbohydrates such as sulfated N-glycans tended to ionize preferentially. Carbohydrates adducted with all three adducts (phosphate, chloride, and nitrate) produced good negative ion CID spectra but those adducted with iodide and sulfate did not yield fragment ions although they gave stronger signals. Fragmentation paralleled that seen following electrospray ionization providing superior spectra than could be obtained by PSD on MALDI-TOF instruments or with ion traps. In addition, ion mobility drift times of the adducted glycans and the ability of this technique to separate isomers also mirrored those obtained following ESI sample introduction. Ion mobility also allowed profiles to be obtained from samples whose MALDI spectra showed no evidence of such ions allowing the technique to be used in conditions where sample amounts were limiting. The method was applied to N-glycans released from the recombinant human immunodeficiency virus glycoprotein, gp120
Emerging principles for the therapeutic exploitation of glycosylation.
No full textGlycosylation plays a key role in a wide range of biological processes. Specific modification to a glycan's structure can directly modulate its biological function. Glycans are not only essential to glycoprotein folding, cellular homeostasis, and immune regulation but are involved in multiple disease conditions. An increased molecular and structural understanding of the mechanistic role that glycans play in these pathological processes has driven the development of therapeutics and illuminated novel targets for drug design. This knowledge has enabled the treatment of metabolic disorders and the development of antivirals and shaped cancer and viral vaccine strategies. Furthermore, an understanding of glycosylation has led to the development of specific drug glycoforms, for example, monoclonal antibodies, with enhanced potency
Going Beyond Counting First Authors in Author Co-citation Analysis
Full text linkThe present study examines one of the fundamental aspects of author co-citation analysis (ACA) - the way co-citation
counts are defined. Co-citation counting provides the data on which all subsequent statistical analyses and mappings
are based, and we compare ACA results based on two different types of co-citation counting - the traditional type that
only counts the first one among a cited work's authors on the one hand and a non-traditional type that takes into
account the first 5 authors of a cited work on the other hand. Results indicate that the picture produced through this non-traditional author co-citation counting contains more coherent author groups and is therefore considerably clearer. However, this picture represents fewer specialties in the research field being studied than that produced through the traditional first-author co-citation counting when the same number of top-ranked authors is selected and analyzed. Reasons for these effects are discussed
Variations on the Author
Full text link“Variations on the Author” discusses two of Eduardo Coutinho’s recent films (Um Dia na Vida, from 2010, and Últimas Conversas, posthumously released in 2015) and their contribution to the general question of documentary authorship. The director’s filmography is characterized by a consistent yet self-effacing form of authorial self-inscription: Coutinho often features as an interviewer that rather than express opinions propels discourses; an interviewer that is good at listening. This mode of self-inscription characterizes him as an author who is not expressive but who is nonetheless markedly present on the screen. In Um Dia na Vida, however, Coutinho is completely absent form the image, while Últimas Conversas, on the contrary, includes a confessional prologue that moves the director from the margins to the center of his films. This article examines the ways in which these works stand out in the filmography of a director who offers new insights into the notion of cinematic authorship
Appropriate Similarity Measures for Author Cocitation Analysis
Full text linkWe provide a number of new insights into the methodological discussion about author cocitation analysis. We first argue that the use of the Pearson correlation for measuring the similarity between authors’ cocitation profiles is not very satisfactory. We then discuss what kind of similarity measures may be used as an alternative to the Pearson correlation. We consider three similarity measures in particular. One is the well-known cosine. The other two similarity measures have not been used before in the bibliometric literature. Finally, we show by means of an example that our findings have a high practical relevance.information science;Pearson correlation;cosine;similarity measure;author cocitation analysis
Selective deactivation of serum IgG: a general strategy for the enhancement of monoclonal antibody receptor interactions.
No full textSerum IgG is a potent inhibitor of monoclonal antibody (mAb) binding to the cell-surface Fcγ receptors (FcγRs), which mediate cytotoxic and phagocytic effector functions. Here, we show that this competition can be eliminated, selectively, by the introduction to serum of (i) an enzyme that displaces Fc from FcγRs and (ii) a modification present in the therapeutic mAb that renders it resistant to that enzyme. Specifically, we show that (i) EndoS (endoglycosidase S) cleaves only complex-type glycans of the type found on IgG but (ii) is inactive against an engineered IgG Fc with oligomannose-type glycans. EndoS thus reduces FcγR binding of serum IgG, but not that of engineered mAb. Introduction of both the engineered mAb and endoglycosidase in serum leads to a dramatic increase in FcγR binding compared to the introduction of mAb in serum alone. Antibody receptor refocusing is a general technique for boosting the effector signal of therapeutic antibodies
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