Nicolai Bovin

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Papers by Nicolai Bovin

Adhesion to oviduct glycans regulates porcine sperm Ca2+ influx and viability

PLOS ONE

Porcine sperm binding to oviduct cells and glycans as supplements to traditional laboratory semen analysis

Journal of animal science, Jan 25, 2018

Accurate semen evaluation is necessary to maintain high reproductive efficiency but difficult to ... more Accurate semen evaluation is necessary to maintain high reproductive efficiency but difficult to accomplish. The objective was to determine if the ability to bind oviduct cells or oviduct glycans are useful supplements to traditional semen analyses. Measuring binding to specific soluble glycans is less laborious than assessing binding to oviduct cell aggregates and more suitable for routine use. Previous work has shown that sperm binding to oviduct cells improves fertility prediction, possibly by estimating the ability of sperm to form an oviduct reservoir. The two oviduct glycan motifs, biantennary 6-sialylated N-acetyllactosamine (bi-SiaLN) and LewisX trisaccharide (LeX), that bind boar spermatozoa with high affinity and specificity were tested. Semen from 30 boars was shipped overnight for laboratory analysis and for inseminations to determine fertility (n=3 replicates). Oviduct cell binding and traditional sperm analyses including motility and morphology were completed. Addition...

YesU from Bacillus subtilis preferentially binds fucosylated glycans

Scientific reports, Jan 3, 2018

The interaction of carbohydrate-binding proteins (CBPs) with their corresponding glycan ligands i... more The interaction of carbohydrate-binding proteins (CBPs) with their corresponding glycan ligands is challenging to study both experimentally and computationally. This is in part due to their low binding affinity, high flexibility, and the lack of a linear sequence in carbohydrates, as exists in nucleic acids and proteins. We recently described a function-prediction technique called SPOT-Struc that identifies CBPs by global structural alignment and binding-affinity prediction. Here we experimentally determined the carbohydrate specificity and binding affinity of YesU (RCSB PDB ID: 1oq1), an uncharacterized protein from Bacillus subtilis that SPOT-Struc predicted would bind high mannose-type glycans. Glycan array analyses however revealed glycan binding patterns similar to those exhibited by fucose (Fuc)-binding lectins, with SPR analysis revealing high affinity binding to Lewis and lacto-N-fucopentaose III. Structure based alignment of YesU revealed high similarity to the legume lecti...

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Histo-blood group antigen-binding specificities of human rotaviruses are associated with gastroenteritis but not with in vitro infection

Scientific reports, Jan 28, 2018

Human strains of rotavirus A (RVAs) recognize fucosylated glycans belonging to histo-blood group ... more Human strains of rotavirus A (RVAs) recognize fucosylated glycans belonging to histo-blood group antigens (HBGAs) through their spike protein VP8*. Lack of these ligands due to genetic polymorphisms is associated with resistance to gastroenteritis caused by P[8] genotype RVAs. With the aim to delineate the contribution of HBGAs in the process, we analyzed the glycan specificity of VP8* proteins from various P genotypes. Binding to saliva of VP8* from P[8] and P[4] genotypes required expression of both FUT2 and FUT3 enzymes, whilst binding of VP8* from the P[14] genotype required FUT2 and A enzymes. We further defined a glycan motif, GlcNAcβ3Galβ4GlcNAc, recognized by P[6] clinical strains. Conversion into Lewis antigens by the FUT3 enzyme impaired recognition, explaining their lower binding to saliva of Lewis positive phenotype. In addition, the presence of neutralizing antibodies was associated with the presence of the FUT2 wild type allele in sera from young healthy adults. Noneth...

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Rapid one-step biotinylation of biological and non-biological surfaces

Scientific reports, Jan 12, 2018

We describe a rapid one-step method to biotinylate virtually any biological or non-biological sur... more We describe a rapid one-step method to biotinylate virtually any biological or non-biological surface. Contacting a solution of biotin-spacer-lipid constructs with a surface will form a coating within seconds on non-biological surfaces or within minutes on most biological membranes including membrane viruses. The resultant biotinylated surface can then be used to interact with avidinylated conjugates, beads, vesicles, surfaces or cells.

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The use of plant lectins to regulate H1N1 influenza A virus receptor binding activity

PloS one, 2018

We applied an in vitro selection approach using two different plant lectins that bind to α2,3- or... more We applied an in vitro selection approach using two different plant lectins that bind to α2,3- or α2,6-linked sialic acids to determine which genetic changes of the A/California/04/09 (H1N1) virus alter hemagglutinin (HA) receptor binding toward α2,3- or α2,6-linked glycans. Consecutive passages of the A/California/04/09 virus with or without lectins in human lung epithelial Calu-3 cells led to development of three HA1 amino acid substitutions, N129D, G155E, and S183P, and one mutation in the neuraminidase (NA), G201E. The S183P mutation significantly increased binding to several α2,6 SA-linked glycans, including YDS, 6'SL(N), and 6-Su-6'SLN, compared to the wild-type virus (↑3.6-fold, P < 0.05). Two other HA1 mutations, N129D and G155E, were sufficient to significantly increase binding to α2,6-linked glycans, 6'SLN and 6-Su-6'SLN, compared to S183P (↑4.1-fold, P < 0.05). These HA1 mutations also increased binding affinity for 3'SLN glycan compared to the w...

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Publisher Correction: Network inference from glycoproteomics data reveals new reactions in the IgG glycosylation pathway

Nature communications, Feb 13, 2018

Correction to: Nature Communications (2017) 8:1231. doi:10.1038/s41467-017-01525-0.

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Effect of sorting boar spermatozoa by sex chromosomes on oviduct cell binding

Theriogenology, Jan 21, 2017

This study examined the hypothesis that flow sorting sperm by sex chromosomes affects oviduct cel... more This study examined the hypothesis that flow sorting sperm by sex chromosomes affects oviduct cell binding which would influence formation of the sperm reservoir in the oviduct. The sperm-rich fraction from boars (n = 5) was collected, sperm were stained with Hoechst 33342 and sorted. Sperm were sorted based on the presence of either an X or Y chromosome and placed into the following treatments: 1) sperm selected for the Y chromosome, 2) selected for the X, 3) an equal mixture of sorted X and Y, and 4) a control of non-sorted sperm from the same collection. Samples were tested for oviduct cell binding within 12 h of sorting. Additionally, sperm were analyzed for motility characteristics, acrosome status, and binding to the two oviduct glycan motifs that bind porcine sperm, biantennary 6-sialylated N-acetyllactosamine on a mannose core (bi-SiaLN) and sulfated LeX trisaccharide (suLeX). The disaccharide found within both glycan motifs, N-acetyllactosamine (LacNAc), was used as a contr...

Lectin activity of the pneumococcal pilin proteins

Scientific reports, Jan 19, 2017

Streptococcus pneumoniae is a leading cause of morbidity and mortality globally. The Pilus-1 prot... more Streptococcus pneumoniae is a leading cause of morbidity and mortality globally. The Pilus-1 proteins, RrgA, RrgB and RrgC of S. pneumoniae have been previously assessed for their role in infection, invasive disease and as possible vaccine candidates. In this study we have investigated the glycan binding repertoire of all three Pilus-1 proteins, identifying that the tip adhesin RrgA has the broadest glycan recognition of the three proteins, binding to maltose/cellobiose, α/β linked galactose and blood group A and H antigens. RrgB only bound mannose, while RrgC bound a subset of glycans also recognized by RrgA. Adherence of S. pneumoniae TIGR4 to epithelial cells was tested using four of the oligosaccharides identified through the glycan array analysis as competitive inhibitors. The blood group H trisaccharide provided the best blocking of S. pneumoniae TIGR4 adherence. Adherence is the first step in disease, and host glycoconjugates are a common target for many adhesins. This study ...

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Host specific glycans are correlated with susceptibility to infection by lagoviruses, but not with their virulence

Journal of virology, Jan 29, 2017

The rabbit hemorrhagic disease virus (RHDV) and the European brown hare syndrome virus (EBHSV) ar... more The rabbit hemorrhagic disease virus (RHDV) and the European brown hare syndrome virus (EBHSV) are two lagoviruses from the family Caliciviridae that cause fatal diseases in two leporid genera, Oryctolagus and Lepus, respectively. In the last few years, several examples of host jumps of lagoviruses among leporids were recorded. In addition, a new pathogenic genotype of RHDV emerged and many non-pathogenic strains of lagoviruses have been described. The molecular mechanisms behind host shifts and the emergence of virulence are unknown. Since RHDV uses glycans of the histo-blood group antigen type as attachment factors to initiate infection, we studied if glycan specificities of the new pathogenic RHDV genotype, non-pathogenic lagoviruses and EBHSV potentially play a role in determining host range and virulence of lagoviruses. We observed binding to A, B or H antigens of the histo-blood group family for all strains known to primarily infect European rabbits (Oryctolagus cuniculus), th...

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Network inference from glycoproteomics data reveals new reactions in the IgG glycosylation pathway

Nature communications, Nov 14, 2017

Immunoglobulin G (IgG) is a major effector molecule of the human immune response, and aberrations... more Immunoglobulin G (IgG) is a major effector molecule of the human immune response, and aberrations in IgG glycosylation are linked to various diseases. However, the molecular mechanisms underlying protein glycosylation are still poorly understood. We present a data-driven approach to infer reactions in the IgG glycosylation pathway using large-scale mass-spectrometry measurements. Gaussian graphical models are used to construct association networks from four cohorts. We find that glycan pairs with high partial correlations represent enzymatic reactions in the known glycosylation pathway, and then predict new biochemical reactions using a rule-based approach. Validation is performed using data from a GWAS and results from three in vitro experiments. We show that one predicted reaction is enzymatically feasible and that one rejected reaction does not occur in vitro. Moreover, in contrast to previous knowledge, enzymes involved in our predictions colocalize in the Golgi of two cell line...

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Approaches to the selective benzylation of the primary hydroxyl group

Bulletin of the Academy of Sciences of the USSR Division of Chemical Science

The selective acylation, alkylation, and glycosylation of carbohydrates has received considerable... more The selective acylation, alkylation, and glycosylation of carbohydrates has received considerable attention [i]. In the case of acylation and glycosylation, some characteristic features of the reactivity of hydroxyl groups have been found, and general methods of selective glycosylation have been proposed for many types of aglycones. The same is true of acylation, but this applies for the most part to acylation of primary hydroxyl groups in the presence of secondary groups. On the other hand, alkylation, and in particular benzylation, of polyols islessselective [i], and seldom affords preparative yields of mono-O-benzyl derivatives. Furthermore, cases are known in which benzylation of the secondary OH group occurs more readily than that of the primary group [2, 3]. Lower selectivity is typical of the classical methods of alkylation (in the presence of NaH, Ba(OH) 2, NaOH, or Ag20), and of phasetransfer catalyzed alkylations [4]. Two indirect methods have been put forward for the indirect synthesis of 6-O-alkylated hexopyranoses. The first of these [5, 6] involves nucleophilic replacement of the 6-O-tosylates by sodium alkoxide, the tosylates in turn being obtained by selective tosylation.

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Methylation of carbohydrates with methyl iodide in presence of dimsilpotassium

Bulletin of the Academy of Sciences of the USSR Division of Chemical Science

... For the TLC we used a silica gel with a particle size of 5-40 ~, which contained 13% of gypsu... more

The Relevance of Neoglycoconjugates for Histology and Pathology

Trends in Glycoscience and Glycotechnology

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1,6-Anhydro-N-acetyl-β-D-glucosamine in the oligosaccharide syntheses: I. Synthesis of 3-acetate and 3-benzoate of 1,6-anhydro-N-acetyl-β-D-glucosamine via the 4-O-trityl derivative

Russian Journal of Bioorganic Chemistry

3-Aminopropyl glycoside of 3,2'-di-O-α-L-fucosyl-N-acetyllactosamine (Le y tetrasaccharide) was s... more 3-Aminopropyl glycoside of 3,2'-di-O-α-L-fucosyl-N-acetyllactosamine (Le y tetrasaccharide) was synthesized. The glycosyl donor, 2-O-acetyl-2,4,6-triO -benzoyl-α-D-galactopyranosyl bromide, was coupled with glycosyl acceptor, 1,6-anhydro-2-acetamido-2-deoxy-β-D-glucopyranose or its 3-O-acetyl derivative, to give the corresponding N-acetyllactosamine derivatives in 20 and 71% yields, respectively. The glycosyl donor was synthesized from 1,2-di-O-acetyl-3,4,6-tri O-benzoyl-D-galactopyranose, which was obtained by the treatment of benzobromogalactose with sodium borohydride to yield 1,2-O-benzylidene derivative and subsequent removal of benzylidene group and acetylation. Acidic methanolysis of the disaccharide derivatives resulted in the selective removal of one or both acetyl groups to give the disaccharide acceptor bearing hydroxy groups at C3 of the glucosamine residue and C2 of the galactose residue. The introduction of fucose residues in these positions by the treatment with tetrabenzylfucopyranosyl bromide resulted in a tetrasaccharide derivative, which was converted into 3,2'-di-O-α-L-fucopuranosyl-1,6-anhydro-N-acetyllactosamine peracetate after substitution of acetyl groups for benzoyl and benzyl groups. Opening of the anhydro ring by acetolysis resulted in peracetate, which was then converted into the corresponding oxazoline derivative by two steps. Glycosydation of the oxazoline derivative with 3-trifluoroacetamidopropan-1-ol and removal of O-acetyl and N-trifluoroacetyl protective groups resulted in a free spacered Le y tetrasaccharide.

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The study of the substrate specificity of rat-brain fucosyltransferase using synthetic acceptors

Russian Journal of Bioorganic Chemistry

The substrate specificity of fucosyltransferase (FT) from rat forebrain and cerebellum was studie... more The substrate specificity of fucosyltransferase (FT) from rat forebrain and cerebellum was studied using synthetic aceeptors. Of 16 acceptors tested, only those containing the GalI31-4GIcNAcfll-R fragment were subjected to enzymic fucosylation. The isomer with a 1-3 bond as well as lactose and oligosaccharides with an additional Neu5Ac residue attached to Gal or a Fuc residue attached to GIcNAc were not fucosylated, whereas Fuc~l-2Gal[31-4GlcNAc displayed the same substrate properties as Gall31-4GIcNAc. FT from the cerebellum and forebrain was shown to have a specificity similar to that of mammalian Fr IV. The activity of the cerebellum FT with all types of substrates was higher than that of FT isolated from the forebrain, the specificity profiles being similar.

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Synthesis of disaccharide neu5Gcα(2–6)galNAcα as a spacer glycoside

Russian Journal of Bioorganic Chemistry

The first synthesis of the Neu5Gc analogue of SiaT n disaccharide, which can be detected in breas... more The first synthesis of the Neu5Gc analogue of SiaT n disaccharide, which can be detected in breast tumors by immunochemical methods, is reported. The regioselective sialylation of (3-trifluoroacetamidopropyl)-2-azido-2-deoxy-α-D-galactopyranoside with peracetate of the methyl ester ofN-acetoxyacetyl-neuraminic acid β-ethylthioglycoside in the presence ofN-iodosuccinimide and trifluoromethanesulfonic acid (or its trimethylsilyl ester) resulted in the derivatives of α- and β-sialyl(2→6)galactosaminide in 39 and 32% yields, respectively. The catalytic hydrogenolysis of the azido group and subsequentN- andO-acetylation of the α-anomer gave the peracetate of trifluoroacetamidopropyl glycoside. Removal of the protective groups led to glycoside Neu5Gcα2→6GalNAcα-O(CH2)3NH2. Using the Neu5Gc derivative with acetoxyacetyl groups at positions O9 and O4 as a donor increases the α-selectivity of sialylation to afford the α- and β-anomers in 69 and 8% yields, respectively.

Altered (neo-) lacto series glycolipid biosynthesis impairs α2-6 sialylation on N-glycoproteins in ovarian cancer cells

Scientific reports, Mar 30, 2017

The (neo-) lacto series glycosphingolipids (nsGSLs) comprise of glycan epitopes that are present ... more The (neo-) lacto series glycosphingolipids (nsGSLs) comprise of glycan epitopes that are present as blood group antigens, act as primary receptors for human pathogens and are also increasingly associated with malignant diseases. Beta-1, 3-N-acetyl-glucosaminyl-transferase 5 (B3GNT5) is suggested as the key glycosyltransferase for the biosynthesis of nsGSLs. In this study, we investigated the impact of CRISPR-Cas9 -mediated gene disruption of B3GNT5 (∆B3GNT5) on the expression of glycosphingolipids and N-glycoproteins by utilizing immunostaining and glycomics-based PGC-UHPLC-ESI-QTOF-MS/MS profiling. ∆B3GNT5 cells lost nsGSL expression coinciding with reduction of α2-6 sialylation on N-glycoproteins. In contrast, disruption of B4GALNT1, a glycosyltransferase for ganglio series GSLs did not affect α2-6 sialylation on N-glycoproteins. We further profiled all known α2-6 sialyltransferase-encoding genes and showed that the loss of α2-6 sialylation is due to silencing of ST6GAL1 expressio...

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Immuno-PCR technology for detection of natural human antibodies against Le(c) disaccharide

Glycoconjugate journal, 2017

The development of an immuno-PCR assay for quantitation of low amounts of anti-glycan human antib... more The development of an immuno-PCR assay for quantitation of low amounts of anti-glycan human antibodies is described. The sensitivity of the assay for determination of low-affinity anti-Le(C) IgM has been found to be 4 ng/ml (~100 pg per sample), thus being two orders of magnitude higher compared to the conventional ELISA with the same antigen.

Naturally occurring anti-glycan antibodies binding to Globo H-expressing cells identify ovarian cancer patients

Journal of ovarian research, Jan 10, 2017

Glycosphingolipids are important compounds of the plasma membrane of mammalian cells and a number... more Glycosphingolipids are important compounds of the plasma membrane of mammalian cells and a number of them have been associated with malignant transformation and progression, reinforcing tumour aggressiveness and metastasis. Here we investigated the levels of naturally occurring anti-glycan antibodies to Globo H in blood plasma obtained from high-grade serous ovarian cancer patients (SOC) and women without gynaecological malignancies (control) using suspension glycan array technology employing chemically synthesized glycans as antibody targets. We found that anti-human Globo H IgG antibodies were able to significantly discriminate SOC from controls (P < 0.05). A combination with the clinically used tumour marker CA125 increased the diagnostic performance (AUC 0.8711). We next compared suspension array with standard flow cytometry in plasma samples and found that the level of anti-Globo H antibodies highly correlated (r = 0.992). The incubation of plasma-derived anti-glycan antibod...

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