Impact of glycosylation on the effect of cytokines. A special focus on oncology

The clinical use of cytokines is largely confined to the field of cancer treatment. Industrial procedures for the preparation of cytokines may or may not affect the glycosylation status of several cytokines. The glycosylation of cytokines may be of importance for the accomplishment of their biological functions, including their biological stability, their interaction with specific receptors and their pharmacokinetic behavior. The purpose of this review is thus to describe and discuss the role of cytokine glycosylation, based on experimental data, and with the clinical perspective on oncology. There is no general rule governing the effects induced by the presence of glycosylation on cytokine activity. Among different cytokines concerned by glycosylation status, data concerning G-CSF are sufficient to translate experimental findings into clinical evidence. For this cytokine, glycosylation confers advantages for both stability and biological activity. Although at weight equivalence, the glycosylated form confers greater biological activity than the non-glycosylated form, clinical studies have demonstrated that efficacy remains the same when glycosylated and non-glycosylated G-CSF are prescribed at bioequivalent doses.     

Protein glycosylation in diverse cell systems: implications for modification and analysis of recombinant proteins

A major challenge for the biotechnology industry is to engineer the glycosylation pathways of expression systems to synthesize recombinant proteins with human glycosylation. Inappropriate glycosylation can result in reduced activity, limited half-life in circulation and unwanted immunogenicity. In this review, the complexities of glycosylation in human cells are explained and compared with glycosylation in bacteria, yeasts, fungi, insects, plants and nonhuman mammalian species. Key advances in the engineering of the glycosylation of expression systems are highlighted. Advances in the challenging and technically complex field of glycan analysis are also described. The emergence of a new generation of expression systems with sophisticated engineering for humanized glycosylation of glycoproteins appears to be on the horizon.     

Lectin-based three-color flow cytometric approach for studying cell surface glycosylation changes that occur during apoptosis.

BACKGROUND: Changes in cell surface glycosylation that accompany apoptosis are thought to be involved in the recognition and removal of apoptotic cells by phagocytes, but in most instances these changes are ill defined. To improve our understanding of this phenomenon, we designed a trivariate flow cytometry procedure that allows direct comparison of cell surface glycosylation in apoptotic and viable cells. METHODS: The annexin V/propidium iodide assay has been adapted for cell surface glycosylation analysis by combining the use of these two reagents with biotinylated lectins, and this has been used to investigate camptothecin-induced apoptosis in U-937 cells. RESULTS: Although numerous lectins are potent inducers of apoptosis, we found that it is possible to determine lectin concentrations that produce interpretable data without inducing significant cytotoxicity even when using apoptogenic lectins. That apoptosis is associated with a marked decrease in cell surface sialylation was confirmed by using the sialic acid-specific lectins Maackia amurensis agglutinin and Sambucus nigra agglutinin. These observations were corroborated by lectin blotting analysis with the same lectins. CONCLUSIONS: Species- and cell-dependent altered glycosylation patterns are likely to be associated with different modes of apoptosis. The easy and versatile method described in this report should be useful for exploring this field.     

The biosynthesis of flavonoid glycosides and their importance in chemosystematics

The present article reviews flavonoid O-glycosyltransferases with respect to the historical background, isolation and purification methods, properties of the enzymes involved (especially substrate specificities) and genetic control. The possible biosynthetic pathway leading to the formation of C-glycosides is also discussed. The second part of the article is an attempt to indicate the importance of glycosylation patterns in the field of chemosystematics, especially on the intra- and infra-specific levels. The position and nature of glycosylation are first discussed, and this is followed by examples indicating the importance of glycosylation patterns.     

Effect of altered glycosylation on the structure of the I-like domain of beta1 integrin: a molecular dynamics study

Glycosylation plays an important role in the regulation of integrin function. Molecular mechanisms underlying the effects of altered glycosylation on beta1 integrin structure and function are still largely unknown. In this study, we used a molecular modeling approach to study the effects of altered glycosylation, with alpha2-6 sialic acid and without alpha2-6 sialic acid, on the structure of the I-like domain of the beta1 integrin. Our results demonstrated that altered glycosylation affected the interactions between oligosaccharides and the I-like domain, which in turn changed the accessibility of the specificity-determining loop for ligand binding. Altered glycosylation caused significant conformational changes for most of the key functional regions of the I-like domain of beta1 integrin, including the metal ion-dependent adhesion site that contains a DLSYS motif, and other critical residues for ligand binding (Asn-224, Glu-229, Asp-233, Asp-267, and Asp-295). In addition, altered glycosylation caused significant movement of the alpha1 and alpha7 helices, which are important for the activation of beta1 integrin. The results from this study offered molecular mechanisms for the experimental observations that variant glycosylation regulates integrin function.     

Structure-function relationship in glycosylated alpha-chymotrypsin as probed by IMAC and IMACE.

Chemical glycosylation of bovine alpha-chymotrypsin, by a glucosamine adduct on the carboxyl group, results in the modification of its catalytic activity. The structural alterations of alpha-chymotrypsin resulting from its glycosylation are studied by immobilized metal-ion affinity chromatography (IMAC) and immobilized metal-ion affinity capillary electrophoresis (IMACE). The chemical glycosylation of alpha-chymotrypsin generates two distinct subpopulations of the protein: one which totally loses the initial affinity for IDA-Cu(II) and another which exhibits an increased affinity for the metal chelate ligand.     

Engineering glycoproteins for use as pharmaceuticals

The fact that glycosylation is not a significant process in prokaryotes means that many of the proteins produced by genetically engineered bacteria are not identical to their eukaryotic counterparts. Although glycosylation affects the physical, chemical and biological nature of proteins, its pharmacological value in potential protein pharmaceuticals is not easy to predict. However, the development of mammalian cell culture methods for expressing recombinant DNA-derived glycoproteins will permit further studies in the field.     

Different Binding Affinities of Pb2+ and Cu2+ to Glycosylation Variants of Human Serum Transferrin Interfere with the Detection of Carbohydrate-Deficient Transferrin

Carbohydrate-deficient transferrin (CDT) is a specific biomarker of alcohol abuse, and for diagnosis of chronic alcohol, abuse is often determined using isoelectric focusing (IEF) and chromatographic techniques. To allow this method to be used for the diagnosis of alcohol abuse, inferences of various physical and chemical factors with the detection of CDT have been investigated. However, few reports have focused thus far on whether different metal ions have different binding affinities to CDT and HTf variants or further interfere in the detection of CDT. Here, in order to figure out whether and how metal ions such as Pb(2+) and Cu(2+) bind to holo-human serum transferrin (holo-HTf) and further interfere in CDT detection, the binding characteristics and the binding parameters of holo-HTf with metal ions such as Pb(2+) and Cu(2+) were investigated using UV-visible spectroscopy, Fluorescence spectroscopy, and ICP-MS. Moreover, whether the metal ions such as Pb(2+) and Cu(2+) will reduce the diagnostic accuracy of CDT in clinic was investigated using IEF. The present study demonstrates that Pb(2+) and Cu(2+) have different binding affinities to holo-HTf variants and produce different changes in the relative amounts of each glycosylation isoforms of HTf. Accordingly, the glycosylation chains of HTf will affect the binding affinities of glycosylation isoforms with Pb(2+) and Cu(2+), causing further interferences in CDT detection.     

Regulation of integrin αIIbβ3 ligand binding and signaling by the metal ion binding sites in the β I domain

The ability of αIIbβ3 to bind ligands and undergo outside-in signaling is regulated by three divalent cation binding sites in the β I domain. Specifically, the metal ion-dependent adhesion site (MIDAS) and the synergistic metal binding site (SyMBS) are thought to be required for ligand binding due to their synergy between Ca(2+) and Mg(2+). The adjacent to MIDAS (ADMIDAS) is an important ligand binding regulatory site that also acts as a critical link between the β I and hybrid domains for signaling. Mutations in this site have provided conflicting results for ligand binding and adhesion in different integrins. We have mutated the β3 SyMBS and ADMIDAS. The SyMBS mutant abolished ligand binding and outside-in signaling, but when an activating glycosylation mutation in the αIIb Calf 2 domain was introduced, the ligand binding affinity and signaling were restored. Thus, the SyMBS is important but not absolutely required for integrin bidirectional signaling. The ADMIDAS mutants showed reduced ligand binding affinity and abolished outside-in signaling, and the activating glycosylation mutation could fully restore integrin signaling of the ADMIDAS mutant. We propose that the ADMIDAS ion stabilizes the low-affinity state when the integrin headpiece is in the closed conformation, whereas it stabilizes the high-affinity state when the headpiece is in the open conformation with the swung-out hybrid domain.     

Pactolus I-domain: functional switching of the Rossmann fold

Murine Pactolus is a neutrophil-specific single chain glycoprotein that plays a role as an apoptosis marker for macrophages. The extracellular region of the protein shows strong sequence similarities to integrin beta-subunits. Critical sequence modifications differentiate its function when compared to the integrin family. We show experimentally that Pactolus I-domain does not bind divalent metal ions, indicating that ligand binding is not mediated through a metal ion-dependent adhesion site (MIDAS). NMR data was used to map secondary structure and the strand pairing within the beta-sheet to confirm an overall Rossmann fold topology. Homology modeling enhanced by the NMR data was used to determine the overall structure, with two key loop insertions/deletions (insertion 2 and SDL) that distinguish the Pactolus I-domain from the integrin alpha I-domain and beta I-domains. NMR peak exchange broadening is observed due to dimerization, correlating to the beta I-domain and beta propeller heterodimerization region within the integrin headpiece. Two unique N-linked glycosylation sites (Asn151 and Asn230) within this region disrupt dimerization and may account for why Pactolus is not found to associate with an alpha-subunit. These changes in quaternary structure, ligand binding loops, glycosylation, and metal sites illustrate how evolution has rapidly and effectively altered key aspects of the integrin beta-subunit to derive a protein of novel function on an existing protein scaffold.     

Mass spectrometry for congenital disorders of glycosylation, CDG

Congenital disorders of glycosylation (CDG) constitute a group of diseases affecting N-linked glycosylation pathways. The classical type of CDG, now called CDG-I, results from deficiencies in the early glycosylation pathway for biosynthesis of lipid-linked oligosaccharide and its transfer to proteins in endoplasmic reticulum, while the CDG-II diseases are caused by defects in the subsequent processing steps. Mass spectrometry (MS) produced a milestone in CDG research, by localizing the CDG-I defect to the early glycosylation pathway in 1992. Currently, MS of transferrin, either by electrospray ionization or matrix-assisted laser desorption/ionization, plays the central role in laboratory screening of CDG-I. On the other hand, the glycopeptide analysis recently developed for site-specific glycans of glycoproteins allows detailed glycan analysis in a high throughput manner and will solve problems in CDG-II diagnosis. These techniques will facilitate studying CDG, a field now expanding to O-linked glycosylation and to acquired as well as inherited conditions that can affect protein glycosylation.     

Role of unusual O-glycans in intercellular signaling

In the last two decades, our knowledge of the role of glycans in development and signal transduction has expanded enormously. While most work has focused on the importance of N-linked or mucin-type O-linked glycosylation, recent work has highlighted the importance of several more unusual forms of glycosylation that are the focus of this review. In particular, the ability of O-fucose glycans on the epidermal growth factor-like (EGF) repeats of Notch to modulate signaling places glycosylation alongside phosphorylation as a means to modulate protein-protein interactions and their resultant downstream signals. The recent discovery that O-glucose modification of Notch EGF repeats is also required for Notch function has further expanded the range of glycosylation events capable of modulating Notch signaling. The prominent role of Notch during development and in later cell-fate decisions underscores the importance of these modifications in human biology. The role of glycans in intercellular signaling events is only beginning to be understood and appears ready to expand into new areas with the discovery that thrombospondin type 1 repeats are also modified with O-fucose glycans. Finally, a rare form of glycosylation called C-mannosylation modifies tryptophans in some signaling competent molecules and may be a further layer of complexity in the field. We will review each of these areas focusing on the glycan structures produced, the consequence of their presence, and the enzymes responsible.     

蓝色花形成分子机理研究进展

花色是观赏植物的重要特征, 在自然界中蓝色花占比很少, 很多观赏植物都缺少蓝色种质。因此, 研究蓝色花形成的分子机理对于蓝色花定向育种具有重要意义。研究表明, 花色的形成主要是通过花青苷积累, 花青素通过糖基化形成花青苷, 再通过酰基、甲基化修饰及金属离子络合反应, 在特定的液泡pH环境中呈现出稳定的蓝色。该文从花青苷合成与代谢途径入手, 对蓝色花形成关键基因功能、花青苷各位点酰化的影响、金属离子的作用、液泡pH值相关基因研究及蓝色花分子育种等方面进行综述。     

Lectin microarray profiling of metastatic breast cancers

Altered protein glycosylation compared with the disease-free state is a universal feature of cancer cells. It has long been established that distinct glycan structures are associated with specific forms of cancer, but far less is known about the complete array of glycans associated with certain tumors. The cancer glycome has great potential as a source of biomarkers, but progress in this field has been hindered by a lack of available techniques for the elucidation of disease-associated glycosylation. In the present study, lectin microarrays consisting of 45 lectins with different binding preferences covering N- and O-linked glycans were coupled with evanescent-field activated fluorescent detection in the glycomic analysis of primary breast tumors and the serum and urine of patients with metastatic breast cancer. A single 50 μm section of a primary breast tumor or <1 μL of breast cancer patient serum or urine was sufficient to detect glycosylation alterations associated with metastatic breast cancer, as inferred from lectin-binding patterns. The high-throughput, sensitive and relatively simple nature of the simultaneous analysis of N- and O-linked glycosylation following minimal sample preparation and without the need for protein deglycosylation makes the lectin microarray analysis described a valuable tool for discovery phase glycomic profiling.     

Mechanism of Bacterial Oligosaccharyltransferase: IN VITRO QUANTIFICATION OF SEQUON BINDING AND CATALYSIS*

N-Linked glycosylation is an essential post-translational protein modification in the eukaryotic cell. The initial transfer of an oligosaccharide from a lipid carrier onto asparagine residues within a consensus sequon is catalyzed by oligosaccharyltransferase (OST). The first X-ray structure of a complete bacterial OST enzyme, Campylobacter lari PglB, was recently determined. To understand the mechanism of PglB, we have quantified sequon binding and glycosylation turnover in vitro using purified enzyme and fluorescently labeled, synthetic peptide substrates. Using fluorescence anisotropy, we determined a dissociation constant of 1.0 μm and a strict requirement for divalent metal ions for consensus (DQNAT) sequon binding. Using in-gel fluorescence detection, we quantified exceedingly low glycosylation rates that remained undetected using in vivo assays. We found that an alanine in the −2 sequon position, converting the bacterial sequon to a eukaryotic one, resulted in strongly lowered sequon binding, with in vitro turnover reduced 50,000-fold. A threonine is preferred over serine in the +2 sequon position, reflected by a 4-fold higher affinity and a 1.2-fold higher glycosylation rate. The interaction of the +2 sequon position with PglB is modulated by isoleucine 572. Our study demonstrates an intricate interplay of peptide and metal binding as the first step of protein N-glycosylation.     

Optimisation of the Cellular Metabolism of Glycosylation for Recombinant Proteins Produced by Mammalian Cell Systems

Many biopharmaceuticals are now produced as secreted glycoproteins from mammalian cell culture. The glycosylation profile of these proteins is essential to ensure structural stability and biological and clinical activity. However, the ability to control the glycosylation is limited by our understanding of the parameters that affect the heterogeneity of added glycan structures. It is clear that the glycosylation process is affected by a number of factors including the 3-dimensional structure of the protein, the enzyme repertoire of the host cell, the transit time in the Golgi and the availability of intracellular sugar-nucleotide donors. From a process development perspective there are many culture parameters that can be controlled to enable a consistent glycosylation profile to emerge from each batch culture. A further, but more difficult goal is to control the culture conditions to enable the enrichment of specific glycoforms identified with desirable biological activities. The purpose of this paper is to discuss the cellular metabolism associated with protein glycosylation and review the attempts to manipulate, control or engineer this metabolism to allow the expression of human glycosylation profiles in producer lines such as genetically engineered Chinese hamster ovary (CHO) cells.     

S-layer nanoglycobiology of bacteria

Cell surface layers (S-layers) are common structures of the bacterial cell envelope with a lattice-like appearance that are formed by a self-assembly process. Frequently, the constituting S-layer proteins are modified with covalently linked glycan chains facing the extracellular environment. S-layer glycoproteins from organisms of the Bacillaceae family possess long, O-glycosidically linked glycans that are composed of a great variety of sugar constituents. The observed variations already exceed the display found in eukaryotic glycoproteins. Recent investigations of the S-layer protein glycosylation process at the molecular level, which has lagged behind the structural studies due to the lack of suitable molecular tools, indicated that the S-layer glycoprotein glycan biosynthesis pathway utilizes different modules of the well-known biosynthesis routes of lipopolysaccharide O-antigens. The genetic information for S-layer glycan biosynthesis is usually present in S-layer glycosylation (slg) gene clusters acting in concert with housekeeping genes. To account for the nanometer-scale cell surface display feature of bacterial S-layer glycosylation, we have coined the neologism 'nanoglycobiology'. It includes structural and biochemical aspects of S-layer glycans as well as molecular data on the machinery underlying the glycosylation event. A key aspect for the full potency of S-layer nanoglycobiology is the unique self-assembly feature of the S-layer protein matrix. Being aware that in many cases the glycan structures associated with a protein are the key to protein function, S-layer protein glycosylation will add a new and valuable component to an 'S-layer based molecular construction kit'. In our long-term research strategy, S-layer nanoglycobiology shall converge with other functional glycosylation systems to produce 'functional' S-layer neoglycoproteins for diverse applications in the fields of nanobiotechnology and vaccine technology. Recent advances in the field of S-layer nanoglycobiology have made our overall strategy a tangible aim of the near future.     

Identification of the N-linked glycosylation sites of vitamin K-dependent carboxylase and effect of glycosylation on carboxylase function

The vitamin K-dependent carboxylase is an integral membrane protein which is required for the post-translational modification of a variety of vitamin K-dependent proteins. Previous studies have suggested carboxylase is a glycoprotein with N-linked glycosylation sites. In this study, we identify the N-glycosylation sites of carboxylase by mass spectrometric peptide mapping analyses combined with site-directed mutagenesis. Our mass spectrometric results show that the N-linked glycosylation in carboxylase occurs at positions N459, N550, N605, and N627. Eliminating these glycosylation sites by changing asparagine to glutamine caused the mutant carboxylase to migrate faster on SDS-PAGE gels, adding further evidence that these sites are glycosylated. In addition, the mutation studies identified N525, a site that cannot be recovered by mass spectroscopy analysis, as a glycosylation site. Furthermore, the potential glycosylation site at N570 is glycosylated only if all five natural glycosylation sites are simultaneously mutated. Removal of the oligosaccharides by glycosidase from wild-type carboxylase or by elimination of the functional glycosylation sites by site-directed mutagenesis did not affect either the carboxylation or epoxidation activity when the small FLEEL pentapeptide was used as a substrate, suggesting that N-linked glycosylation is not required for the enzymatic function of carboxylase. In contrast, when site N570 and the five natural glycosylation sites were mutated simultaneously, the resulting carboxylase protein was degraded. Our results suggest that N-linked glycosylation is not essential for carboxylase enzymatic activity but is important for protein folding and stability.     

Protein glycosylation in infectious disease pathobiology and treatment

A host of bacteria and viruses are dependent on O-linked and N-linked glycosylation to perform vital biological functions. Pathogens often have integral proteins that participate in host-cell interactions such as receptor binding and fusion with host membrane. Fusion proteins from a broad range of disparate viruses, such as paramyxovirus, HIV, ebola, and the influenza viruses share a variety of common features that are augmented by glycosylation. Each of these viruses contain multiple glycosylation sites that must be processed and modified by the host post-translational machinery to be fusogenically active. In most viruses, glycosylation plays a role in biogenesis, stability, antigenicity and infectivity. In bacteria, glycosylation events play an important role in the formation of flagellin and pili and are vitally important to adherence, attachment, infectivity and immune evasion. With the importance of glycosylation to pathogen survival, it is clear that a better understanding of the processes is needed to understand the pathogen requirement for glycosylation and to capitalize on this requirement for the development of novel therapeutics.