Recent progress in chemical synthesis of bacterial surface glycans

With the continuing advancement of carbohydrate chemical synthesis, bacterial glycomes have become increasingly attractive and accessible synthetic targets. Although bacteria also produce carbohydrate-containing secondary metabolites, our review here will cover recent chemical synthetic efforts on bacterial surface glycans. The obtained compounds are excellent candidates for the development of improved structurally defined glycoconjugate vaccines to combat bacterial infections. They are also important probes for investigating glycan–protein interactions. Glycosylation strategies applied for the formation of some challenging glycosidic bonds of various uncommon sugars in a number of recently synthesized bacterial surface glycans are highlighted.     

Genomics and epigenomics of the human glycome

The majority of all proteins are glycosylated and glycans have numerous important structural, functional and regulatory roles in various physiological processes. While structure of the polypeptide part of a glycoprotein is defined by the sequence of nucleotides in the corresponding gene, structure of a glycan part results from dynamic interactions between hundreds of genes, their protein products and environmental factors. The composition of the glycome attached to an individual protein, or to a complex mixture of proteins, like human plasma, is stable within an individual, but very variable between individuals. This variability stems from numerous common genetic polymorphisms reflecting in changes in the complex biosynthetic pathway of glycans, but also from the interaction with the environment. Environment can affect glycan biosynthesis at the level of substrate availability, regulation of enzyme activity and/or hormonal signals, but also through gene-environment interactions. Epigenetics provides a molecular basis how the environment can modify phenotype of an individual. The epigenetic information (DNA methylation pattern and histone code) is especially vulnerable to environmental effects in the early intrauterine and neo-natal development and many common late-onset diseases take root already at that time. The evidences showing the link between epigenetics and glycosylation are accumulating. Recent progress in high-throughput glycomics, genomics and epigenomics enabled first epidemiological and genome-wide association studies of the glycome, which are presented in this mini-review.     

Insight into the Regulation of Glycan Synthesis in Drosophila Chaoptin Based on Mass Spectrometry

Background

A variety of N-glycans attached to protein are known to involve in many important biological functions. Endoplasmic reticulum (ER) and Golgi localized enzymes are responsible to this template-independent glycan synthesis resulting glycoforms at each asparagine residues. The regulation mechanism such glycan synthesis remains largely unknown.

Methodology/Principal Findings

In order to investigate the relationship between glycan structure and protein conformation, we analyzed a glycoprotein of Drosophila melanogaster, chaoptin (Chp), which is localized in photoreceptor cells and is bound to the cell membrane via a glycosylphosphatidylinositol anchor. Detailed analysis based on mass spectrometry revealed the presence of 13 N-glycosylation sites and the composition of the glycoform at each site. The synthetic pathway of glycans was speculated from the observed glycan structures and the composition at each N-glycosylation site, where the presence of novel routes were suggested. The distribution of glycoforms on a Chp polypeptide suggested that various processing enzymes act on the exterior of Chp in the Golgi apparatus, although virtually no enzyme can gain access to the interior of the horseshoe-shaped scaffold, hence explaining the presence of longer glycans within the interior. Furthermore, analysis of Chp from a mutant (RNAi against dolichyl-phosphate α-d-mannosyltransferase), which affects N-glycan synthesis in the ER, revealed that truncated glycan structures were processed. As a result, the distribution of glycoforms was affected for the high-mannose-type glycans only, whereas other types of glycans remained similar to those observed in the control and wild-type.

Conclusions/Significance

These results indicate that glycan processing depends largely on the backbone structure of the parent polypeptide. The information we obtained can be applied to other members of the LRR family of proteins.     

Glycans‐by‐design: Engineering bacteria for the biosynthesis of complex glycans and glycoconjugates

There is an urgent need for new tools that enable better understanding of the structure, recognition, metabolism, and biosynthesis of glycans as well as the production of biologically important glycans and glycoconjugates. With the discovery of glycoprotein synthesis in bacteria and functional transfer of glycosylation pathways between species, Escherichia coli cells have become a tractable host for both understanding glycosylation and the underlying glycan code of living cells as well as for expressing glycoprotein therapeutics and vaccines. Here, we review recent efforts to harness natural biological pathways and engineer synthetic designer pathways in bacteria for making complex glycans and conjugating these to lipids and proteins. The result of these efforts has been a veritable transformation of bacteria into living factories for scalable, bottom‐up production of complex glycoconjugates by design. Biotechnol. Bioeng. 2013; 110: 1550–1564. © 2013 Wiley Periodicals, Inc.     

The glycan code of the endoplasmic reticulum: asparagine-linked carbohydrates as protein maturation and quality-control tags

The majority of proteins that traverse the secretory pathway receive asparagine (Asn)-linked glycosylations. Glycans are bulky hydrophilic modifications that serve a variety of structural and functional roles within the cell. Here, we review the recent growing knowledge on the role of Asn-linked glycans as maturation and quality-control protein tags in the early secretory pathway. The carbohydrate composition encodes crucial information about the structure, localization and age of glycoproteins. The "glycan code" is encoded by a series of glycosidases and carbohydrate transferases that line the secretory pathway. This code is deciphered by carbohydrate-binding proteins that possess distinct carbohydrate binding properties and act as molecular chaperones or sorting receptors. These glycosidases and transferases work in concert with resident secretory pathway carbohydrate-binding proteins to form a network that assists in the maturation and trafficking of both native and aberrant glycoproteins within the cell.     

The polypeptide part of human chorionic gonadotrophin affects the kinetics of alpha 6-sialylation of its N-linked glycans but does not alter the branch specificity of CMP-NeuAc:Gal beta 1----4GlcNAc-R alpha 2----6-sialyltransferase.

Human chorionic gonadotrophin (hCG) is a heterodimeric glycoprotein hormone consisting of an alpha- and a beta-subunit, both containing two N-linked, complex-type glycans. Using this hormone as a model glycoprotein, the influence of its polypeptide part on the activity and specificity of bovine colostrum CMP-NeuAc:Gal beta 1----4GlcNAc-R alpha 2----6-sialyltransferase (alpha 6-sialyltransferase) was investigated. Initial rates of sialic acid incorporation into the desialylated glycans of hCG alpha and hCG beta in the heterodimer were higher with the alpha-subunit. This appeared to be due to a higher V which, together with a slightly lowered affinity (higher Km), resulted in a higher kinetic efficiency of the sialyltransferase for the glycans of this subunit. By contrast, the kinetic parameters did not differ significantly when the subunits were in the free form, indicating that the differences in the kinetics of sialylation found for the subunits in the heterodimeric state were not caused by the differences in N-linked carbohydrate structures of the subunits. It is proposed that these effects are due to conformational constraints which the polypeptide moieties put on the glycan chains upon dimerization. Furthermore, it was investigated whether the polypeptide of hCG would interfere with the sialyltransferase so as to alter the branch specificity of the enzyme. 1H-NMR spectroscopy (400 MHz) of the glycan chains, alpha 6-sialylated in vitro, showed that the enzyme highly prefers the galactosyl residue at the Gal beta 1----4GlcNAc beta 1----2-Man alpha 1----3Man branch for attachment of the first mol of sialic acid into the diantennary glycans of desialylated hCG.(ABSTRACT TRUNCATED AT 250 WORDS)     

High-mannose-type glycan modifications of dihydrofolate reductase using glycan-methotrexate conjugates

Various high-mannose-type glycan modifications of dihydrofolate reductase (DHFR) were achieved by ligand-based approach using glycan-methotrexate (MTX) conjugates as tight binding glycan bearing ligands for DHFR. The resulting glycan-MTX conjugates and the corresponding artificial glycoproteins could be useful as oligosaccharide- and glycoprotein-probes to perform quantitative analysis of glycan recognizing protein such as lectins, glycosyltransferases or glycosidases. Moreover, artificial glycoproteins having two different high-mannose-type glycans were developed for the first time by a combination of two different types of glycan modification strategies.     

Terminal sialic acids on CD44 N‐glycans can block hyaluronan binding by forming competing intramolecular contacts with arginine sidechains

Specific sugar residues and their linkages form the basis of molecular recognition for interactions of glycoproteins with other biomolecules. Seemingly small changes, like the addition of a single monosaccharide in the covalently attached glycan component of glycoproteins, can greatly affect these interactions. For instance, the sialic acid capping of glycans affects protein‐ligand binding involved in cell–cell and cell–matrix interactions. CD44 is a single‐pass transmembrane glycoprotein whose binding with its carbohydrate ligand hyaluronan (HA), an extracellular matrix component, mediates processes such as leukocyte homing, cell adhesion, and tumor metastasis. This binding is highly regulated by glycosylation of the N‐terminal extracellular hyaluronan‐binding domain (HABD); specifically, sialic acid capped N‐glycans of HABD inhibit ligand binding. However, the molecular mechanism behind this sialic acid mediated regulation has remained unknown. Two of the five N‐glycosyation sites of HABD have been previously identified as having the greatest inhibitory effect on HA binding, but only if the glycans contain terminal sialic acid residues. These two sites, Asn25 and Asn120, were chosen for in silico glycosylation in this study. Here, from extensive standard molecular dynamics simulations and biased simulations, we propose a molecular mechanism for this behavior based on spontaneously‐formed charge‐paired hydrogen bonding interactions between the negatively‐charged sialic acid residues and positively‐charged Arg sidechains known to be critically important for binding to HA, which itself is negatively charged. Such intramolecular hydrogen bonds would preclude associations critical to hyaluronan binding. This observation suggests how CD44 and related glycoprotein binding is regulated by sialylation as cellular environments fluctuate. Proteins 2014; 82:3079–3089. © 2014 Wiley Periodicals, Inc.     

A sensitive mapping strategy for monitoring the reproducibility of glycan processing in an HIV vaccine, RGP-160, expressed in a mammalian cell line

The external envelope glycoprotein (gp 160) of HIV-1 is a candidate for vaccines against AIDS. Most of the surface of the molecule is shielded by carbohydrate and the structures and locations of these glycans may be important in defining the immunogenicity of the viral coat. Here we report a sensitive mapping strategy for profiling and analysing the N-glycosylation of gp160, based on chemical release of glycans, fluorescent labelling and HPLC analysis. This approach has been validated in terms of establishing the reproducibility of all steps in the analytical procedure and on overall reproducibility on a run-to-run and day-to-day basis. The validated analysis technique was used to monitor the consistency of N-glycosylation of one rgp 160 vaccine candidate produced in bovine hamster kidney (BHK) cell culture. It was demonstrated that the variation in the glycan profiles of 6 different lots was not statistically significant.     

Polypeptide molecular weights of the (Na+,K+)-ATPase from porcine kidney medulla

The molecular weights of the polypeptide chains from (Na+,K+)-ATPase of porcine kidney medulla have been determined by analytical sedimentation equilibrium. The alpha-subunit molecular weight is 93 900, and the beta subunit is a glycoprotein with a polypeptide molecular weight of 32 300 (41 400 including protein and carbohydrate). Amino acid and carbohydrate compositions are presented together with related properties (i.e., partial specific volumes, extinction coefficients, and hydrophobic/hydrophilic amino acid content).     

Inhibition of mammalian glycan biosynthesis produces non-self antigens for a broadly neutralising, HIV-1 specific antibody

The HIV envelope has evolved a dense array of immunologically "self" carbohydrates that efficiently protect the virus from antibody recognition. Nonetheless, one broadly neutralising antibody, IgG1 2G12, has been shown to recognise a cluster of oligomannose glycans on the HIV-1 surface antigen gp120. Thus the self carbohydrates of HIV are now regarded as potential targets for viral neutralisation and vaccine design. Here, we show that chemical inhibition of mammalian glycoprotein synthesis, with the plant alkaloid kifunensine, creates multiple HIV (2G12) epitopes on the surface of previously non-antigenic self proteins and cells, including HIV gp120. This formally demonstrates the structural basis for self/non-self discrimination between viral and host glycans, by a neutralising antibody. Moreover, this study provides an alternative protein engineering approach to the design of a carbohydrate vaccine for HIV-1 by chemical synthesis.     

LC/MS analysis of complex multiglycosylated human α1-acid glycoprotein as a model for developing identification and quantitation methods for intact glycopeptide analysis

The site-specific characterization of the complex glycans in multiglycosylated proteins requires developing methods where the carbohydrates remain covalently bound to the protein. The complexity in the carbohydrate composition of α₁-acid glycoprotein (AAG) makes it an ideal model protein for such development. AAG has five N-asparaginyl-linked glycosylation sites, each varying in its bi-, tri-, and tetraantennary glycan content. We present an on-line liquid chromatography/mass spectrometry (LC/MS) method that uses high-low cone voltage switching for in-source fragmentation to determine the structures of the complex glycans present on each site for the two gene products of AAG. High cone voltage caused carbohydrate fragmentation, leading to the generation of signature carbohydrate ions that we used as markers to identify the glycopeptides. Low cone voltage produced minimal carbohydrate fragmentation and enabled the identification and quantification of the intact oligosaccharide structures on each glycopeptide based on its monoisotopic mass and intensity. Quantitation was accomplished by using the intensities of peaks from deconvoluted and deisotoped mass spectra or from the areas of the extracted ion chromatograms from the tryptic peptide maps. The combined results from the two methods can be used to better characterize and quantitate site heterogeneity in multiglycosylated proteins.     

A novel mechanism for LSECtin binding to Ebola virus surface glycoprotein through truncated glycans

LSECtin is a member of the C-type lectin family of glycan-binding receptors that is expressed on sinusoidal endothelial cells of the liver and lymph nodes. To compare the sugar and pathogen binding properties of LSECtin with those of related but more extensively characterized receptors, such as DC-SIGN, a soluble fragment of LSECtin consisting of the C-terminal carbohydrate-recognition domain has been expressed in bacteria. A biotin-tagged version of the protein was also generated and complexed with streptavidin to create tetramers. These forms of the carbohydrate-recognition domain were used to probe a glycan array and to characterize binding to oligosaccharide and glycoprotein ligands. LSECtin binds with high selectivity to glycoproteins terminating in GlcNAcbeta1-2Man. The inhibition constant for this disaccharide is 3.5 microm, making it one of the best low molecular weight ligands known for any C-type lectin. As a result of the selective binding of this disaccharide unit, the receptor recognizes glycoproteins with a truncated complex and hybrid N-linked glycans on glycoproteins. Glycan analysis of the surface glycoprotein of Ebola virus reveals the presence of such truncated glycans, explaining the ability of LSECtin to facilitate infection by Ebola virus. High mannose glycans are also present on the viral glycoprotein, which explains why DC-SIGN also binds to this virus. Thus, multiple receptors interact with surface glycoproteins of enveloped viruses that bear different types of relatively poorly processed glycans.     

Just a spoonful of sugar: Short glycans affect protein properties and functions

Glycosylation has a strong impact on the chemical and physical properties of proteins and on their activity. The heterogeneous nature of this modification complicates the elucidation of the role of each glycan, thus slowing down the progress in glycobiology. Nevertheless, the great advances recently made in protein engineering and in the chemical synthesis, and semisynthesis of glycoproteins are giving impulse to the field, fostering important discoveries. In this review, we report on the findings of the last two decades on the importance that the attachment site, linkage, and composition of short glycans have in affecting protein properties and functions.     

In vitro mutated phytohemagglutinin genes expressed in tobacco seeds: role of glycans in protein targeting and stability.

Phytohemagglutinin is a glycoprotein that accumulates in the protein storage vacuoles of bean seeds. The mature glycoprotein has a high-mannose and a complex glycan. We describe here the use of site-directed mutagenesis and expression of the mutated genes in transgenic tobacco to study the role of glycans in intracellular targeting. The reading frame for phytohemagglutinin-L was mutated so that either one or both of the glycosylation signals were disrupted to specifically prevent the attachment of asparagine-linked glycans. Expression of these genes with the beta-phaseolin promoter in the seeds of transgenic tobacco plants showed that phytohemagglutinin-L with only one glycan or without glycans was correctly targeted to the protein storage vacuoles of the seeds. Furthermore, the absence of either the complex glycan or the high-mannose glycan did not alter the processing of the other glycan. On the basis of these results, we propose that the targeting signal of this vacuolar protein is contained in its polypeptide domain and not in its glycans.     

Determination of glycan structures and molecular masses of the glycovariants of serum transferrin from a patient with carbohydrate deficient syndrome type II

Serum transferrin from a child with carbohydrate deficient syndrome type II was isolated by immunoaffinity chromatography and separated into minor and major fractions by fast protein liquid chromatography. The structure of the glycans released from the major fraction by hydrazinolysis was established by application of methanolysis and 1H-NMR spectroscopy. The results led to the identification of an N-acetyllactosamininic type monosialylated, monoantennary Man(1-3) linked glycan. By electrospray-mass spectrometry analysis, the whole serum transferrin was separated into at least seven species (I to VII) with molecular masses ranging from 77 958 to 79 130 Da. On the basis of a polypeptide chain molecular mass of 75 143 Da, it was calculated that the major transferrin species III (78 247 Da) contains two monosialylated monoantennary glycans. The molecular mass of transferrin species V and VI (78 678 and 78 971 Da) suggests that one of their two glycans contains an additional N-acetyllactosamine and a sialylated N-acetyllactosamine units, respectively. Transferrin species I and V were found to correspond to the desialylated forms of species III and VI. The abnormal glycan structures can be explained by a defect in the N-acetylglucosaminyltransferase II activity [Charuk et al. (1995) Eur J Biochem 230: 797-805].     

Candidate glycophospholipid precursor for the glycosylphosphatidylinositol membrane anchor of Trypanosoma brucei variant surface glycoproteins

Trypanosoma brucei variant surface glycoproteins are apparently synthesized with a hydrophobic carboxyl-terminal peptide that is cleaved and replaced by a complex glycosylphosphatidylinositol membrane anchor within 1 min of the completion of polypeptide synthesis. The rapidity of this carboxyl-terminal modification suggests the existence of a prefabricated core glycolipid that would be transferred en bloc to the variant surface glycoprotein polypeptide. We report the purification and chemical characterization of a glycolipid from T. brucei that has properties consistent with a role as a variant surface glycoprotein glycolipid donor. This candidate glycolipid precursor has been defined by thin-layer chromatography of extracts of trypanosomes metabolically labeled with radioactive myristic acid, ethanolamine, glucosamine, mannose, and phosphate and by enzymatic, chemical, and gas chromatographic-mass spectrometric analysis. Mild alkali released 100% of the myristic acid, and reaction with phospholipase A2 released 50%. Nitrous acid deamination generated dimyristylphosphatidylinositol, and periodate oxidation released phosphatidic acid. Treatment of purified glycolipid with phosphatidylinositol-specific phospholipase C released dimyristylglycerol and a water-soluble glycan that was sized on Bio-Gel P-4 columns. The candidate precursor contained mannose, myristic acid, phosphate, and ethanolamine with an unsubstituted amino group, but not galactose.     

The distribution of glycan structures in individual N-glycosylation sites in animal and plant glycoproteins

Glycopeptides representing each individual N-glycosylation site in six animal and plant glycoproteins (ovoinhibitor and ovotransferrin, orosomucoid, antitrypsin, phaseolin, and phytohemagglutinin) have been isolated and compared by mass spectrometric analysis. Since the isolation step separates each individual peptide regardless of the nature of the glycan attached to it, it is possible to observe the entire spectrum of glycans associated with each site from the mass spectrum of the corresponding glycopeptide. The three glycosylation sites in ovoinhibitor have very similar but not identical glycans; they are significantly different from those observed in the single site of ovotransferrin. The three sites in serum antitrypsin also have quite similar glycans, whereas the five sites in orosomucoid show considerable variation in both the nature and the relative amount of glycans. The two plant glycoproteins each have two sites with very different glycan structures. Except for the first and third glycosylation sites of antitrypsin which were found to have remarkably homogeneous glycans (97 and 90% of a biantennary complex structure), all the individual glycosylation sites contained heterogeneous mixtures of glycan structures. The results support the proposition that each N-linked glycan in a glycoprotein is affected by its unique protein environment to such an extent that each one may be displayed to the processing enzymes as a unique structural entity. On the basis of a limited number of observations of the glycan interfering with chymotryptic but not tryptic cleavage in the proximity of the glycan attachment site, it is proposed that hydrophobic interactions between the protein and the glycan may be involved in the conformational modulation of the glycans.     

Structural glycobiology of the major allergen of Artemisia vulgaris pollen, Art v 1: O-glycosylation influence on the protein dynamics and allergenicity

Art v 1 is the major allergen of mugwort (Artemisia vulgaris) pollen. It is formed by an N-terminal globular defensin-like part and a C-terminal proline-rich domain. As the structure and the dynamics of Art v 1 have been mostly described for its recombinant, non-glycosylated form, which does not occur in normal plant physiology, the present work intends to obtain a three-dimensional model for Art v 1 native O-glycosylation structure and to evaluate the influence of such glycans over the protein dynamics and allergenicity through molecular dynamics simulations in triplicates. Structural insights into the mutual recognition of Art v 1 protein and carbohydrate moieties recognition by antibodies were obtained, in which glycan chains remained close to the previously identified epitopes in the defensin-like domain, thus pointing to potential interferences with antibodies recognition. To our knowledge, this is the first structural report of an entire furanose-containing glycoprotein. As well, together with the previously determined NMR structures, the obtained results contribute in the comprehension of the effect of glycosylation over both proline-rich and defensin-like domains, providing an atomic representation of such alterations.