Koen M. Halkes

Gold Glyconanoparticles as Probes to Explore the Carbohydrate- Mediated Self-Recognition of Marine Sponge Cells

Cell aggregation in the red-beard marine sponge Microciona prolifera is mediated by a 2 10 kDa proteoglycan-like macromolecular aggregation factor (MAF), and is based on two highly polyvalent functional properties; a Ca-dependent proteoglycan self-interaction and a Ca-independent cell-binding activity. MAF, the first circular proteoglycan described, is composed of two N-glycosylated proteins, MAFp3 and MAFp4, with twenty units of each glycoprotein forming the central ring and the radiating arms, respectively. Each MAFp3 carries one or two copies of a 200 kDa acidic glycan, g-200, whereas each MAFp4 carries about 50 copies of a 6 kDa glycan, g-6. The MAFp4 arms of the sunburst-like proteoglycan are linked to cell-surface binding receptors, while the MAFp3 ring exposes the g-200 glycans so that they can engage in the Ca-dependent self-association (for a detailed review, see ref. [4]). By making use of MAF-specific monoclonal antibodies, it could be demonstrated that the self-association of MAF occurs through highly repetitive epitopes on the g-200 glycan. One of these epitopes was shown to be the sulfated disaccharide GlcpNAc3S(b1–3)Fucp. To gain insight into the role of carbohydrate interactions in MAF self-aggregation, we designed a challenging system for mimicking the g-200 self-association. By using the synthetic sulfated disaccharide, multivalently presented as a bovine serum albumin conjugate, and surface plasmon resonance spectroscopy, it was shown that Ca-dependent carbohydrate self-recognition is a major force in the g-200 association phenomenon. Gold glyconanoparticles have been successfully used as inert multivalent systems to explore either carbohydrate self-interactions or carbohydrate binding to proteins. In the present study, water-soluble gold glyconanoparticles coated with synthetic carbohydrates related to the sulfated disaccharide fragment (Scheme 1) were used as multivalent systems to investigate the g-200 glycan–glycan interaction by transmission electron microscopy (TEM). Very recently, an NMR study of intact MAF glycans suggested the presence of a-Fuc residues. However, earlier structural analysis of oligosaccharide fragments obtained from a partial acid hydrolysate of the g-200 glycan could not identify the anomeric configuration of the fucose residue in these fragments. 16] Therefore, gold glyconanoparticles coated with the aor the b-anomer (Au-1a and Au-1b) of the native sulfated disaccharide epitope were used in the aggregation experiments. The importance of each of the two monosaccharide units for the self-recognition process of the disaccharide epitope was determined by studying the gold glyconanoparticles Au-2 and Au-3. The three gold glyconanoparticle systems Au-4 (a-l-Fucp replaced by a-l-Galp), Au-5 (bd-GlcpNAc3S replaced by b-d-GlcpNAc), and Au-6 (b-dGlcpNAc3S replaced by b-d-Glcp3S) were used to evaluate the relevance of the modified sites in the self-recognition process. Scheme 1. Gold glyconanoparticles Au-1a/b to Au-6, related to the MAF sulfated disaccharide epitope.

SPR Studies of Carbohydrate–Protein Interactions: Signal Enhancement of Low-Molecular-Mass Analytes by Organoplatinum(II)-Labeling

The relatively insensitive surface plasmon resonance (SPR) signal detection of low‐molecular‐mass analytes that bind with weak affinity to a protein—for example, carbohydrate–lectin binding—is hampering the use of biosensors in interaction studies. In this investigation, low‐molecular‐mass carbohydrates have been labeled with an organoplatinum(II) complex of the type [PtCl(NCNR)]. The attachment of this complex increased the SPR response tremendously and allowed the detection of binding events between monosaccharides and lectins at very low analyte concentrations. The platinum atom inside the organoplatinum(II) complex was shown to be essential for the SPR‐signal enhancement. The organoplatinum(II) complex did not influence the specificity of the biological interaction, but both the signal enhancement and the different binding character of labeled compounds when compared with unlabeled ones makes the method unsuitable for the direct calculation of biologically relevant kinetic parameters. However, the labeling procedure is expected to be of high relevance for qualitative binding studies and relative affinity ranking of small molecules (not restricted only to carbohydrates) to receptors, a process of immense interest in pharmaceutical research.

Critical Elements of Oligosaccharide Acceptor Substrates for the Pasteurella multocida Hyaluronan Synthase

Three-dimensional structures are not available for polysaccharide synthases and only minimal information on the molecular basis for catalysis is known. The Pasteurella multocida hyaluronan synthase (PmHAS) catalyzes the polymerization of the alternating β1,3-N-acetylglucosamine-β1,4-glucuronic acid sugar chain by the sequential addition of single monosaccharides to the non-reducing terminus. Therefore, PmHAS possesses both GlcNAc-transferase and glucuronic acid (GlcUA)-transferase activities. The recombinant Escherichia coli-derived PmHAS enzyme will elongate exogenously supplied hyaluronan chains in vitro with either a single monosaccharide or a long chain depending on the UDP-sugar availability. Competition studies using pairs of acceptors with distinct termini (where one oligosaccharide is a substrate that may be elongated, whereas the other cannot) were performed here; the lack of competition suggests that PmHAS contains at least two distinct acceptor sites. We hypothesize that the size of the acceptor binding pockets of the enzyme corresponds to the size of the smallest high efficiency substrates; thus we tested the relative activity of a series of authentic hyaluronan oligosaccharides and related structural analogs. The GlcUA-transferase site readily elongates (GlcNAc-GlcUA)2, whereas the GlcNAc-transferase elongates GlcUA-Glc-NAc-GlcUA. The minimally sized oligosaccharides, elongated with high efficiency, both contain a trisaccharide with two glucuronic acid residues that enabled the identification of a synthetic, artificial acceptor for the synthase. PmHAS behaves as a fusion of two complete glycosyltransferases, each containing a donor site and an acceptor site, in one polypeptide. Overall, this information advances the knowledge of glycosaminoglycan biosynthesis as well as assists the creation of various therapeutic sugars for medical applications in the future.

Synthesis of a spacer-containing tetrasaccharide representing a repeating unit of the capsular polysaccharide of Streptococcus pneumoniae type 6B

Abstract The synthesis is reported of the spacer-containing tetrasaccharide α-D-Galp-(1→3)-α- d -Glc p -(1→3)-α- l -Rhap-(1→4)- d -RibOH-(5→phosphate→CH 2 CH 2 CH 2 NH 2 ( 1 ), using a 2+2 block synthesis approach.

Immunodiagnostically applicable monoclonal antibodies to the circulating anodic antigen of Schistosoma mansoni bind to small, defined oligosaccharide epitopes

Gut-associated glycoproteins constitute a major group of the circulating excretory antigens produced by human Schistosoma species. The O-glycans of the relatively abundant circulating anodic antigen (CAA) from S. mansoni carry long stretches of unique →6(GlcAβ1→3)GalNAcβ1→ repeats. Specific anti-carbohydrate monoclonal antibodies (mAbs) are essential tools for the immunodiagnostic detection of CAA in the serum or urine of Schistosoma-infected subjects. In order to define the epitopes recognised by these anti-CAA mAbs, we screened a series of protein-coupled synthetic di- to pentasaccharide building blocks of the CAA polysaccharide for immunoreactivity, using ELISA and surface plasmon resonance spectroscopy. It was shown that anti-CAA IgM mAbs preferentially recognise →6(GlcAβ1→3)GalNAcβ1→ disaccharide units. Interestingly, no mouse anti-CAA mAbs of the IgG class were found that bind to the synthetic epitopes, although many of the IgG mAbs tested do recognise native CAA in a carbohydrate-dependent manner. In addition, both IgM and IgG class antibodies could be detected in human infection sera using the synthetic CAA fragments. These synthetic schistosome glycan epitopes and their matching set of specific mAbs are useful tools that further the development of diagnostic methods and are helpful in defining the immunological responses of the mammalian hosts to schistosome glycoconjugates.

Synthesis of hyaluronic-acid-related oligosaccharides and analogues, as their 4-methoxyphenyl glycosides, having N-acetyl-β-d-glucosamine at the reducing end

To contribute to the possibilities to study the ability of oligosaccharide fragments of hyaluronic acid to induce angiogenesis, several hyaluronic-acid-related oligosaccharides and their 6-O-sulfated analogues were synthesised as their 4-methoxyphenyl glycosides having 2-acetamido-2-deoxy-D-glucopyranose at the reducing end. In all syntheses described, the D-glucopyranosyluronic acid residue was obtained by oxidation at C-6 of a corresponding D-glucopyranosyl residue after construction of the oligosaccharide backbone, using pyridinium dichromate and acetic anhydride.

Assessing the inhibitory potency of galectin ligands identified from combinatorial (glyco)peptide libraries using surface plasmon resonance spectroscopy

Combinatorial (glyco)peptide libraries offer the possibility to define effective inhibitors of protein (lectin)-glycan interactions. If a (glyco)peptide surpasses the inhibitory potency of the free sugar, then the new peptide-lectin contacts underlying the affinity enhancement may guide further rational drug design. Focusing on the adhesion/growth regulatory human galectins 1 and 3, a screening of three combinatorial solid-phase (glyco)peptide libraries, containing Gal(beta1-O)Thr, Gal(beta1-S)Cys/Gal(beta1-N)Asn, and Lac(beta1-O)Thr, with the fluorescently labeled lectins had led to a series of lead compounds. To define the inhibitory potency of a selection of resynthesized (glyco)peptides systematically, a surface plasmon resonance-based inhibition assay with immobilized asialofetuin was set up. (Glyco)Peptides with up to 66-fold potency relative to free lactose as inhibitor were characterized. The presence of lactose in the most effective glycopeptides indicated the presence of affinity-enhancing peptide-lectin contacts. In addition to drug design, they may be helpful for fine-structural analysis of the binding sites.

The application of neoglycopeptides in the development of sensitive surface plasmon resonance-based biosensors

The development of a biosensor based on surface plasmon resonance is described for the detection of carbohydrate-binding proteins in solution on a Biacore 2000 instrument, using immobilized glycopeptides as ligands. Their selection was based on previous screenings of solid-phase glycopeptide libraries with Ricinus communis agglutinin (RCA(120)) and human adhesion/growth-regulatory galectin-1 (h-Gal-1). Glycopeptides were immobilized on Au sensor chips functionalized with mixed self-assembled monolayers of different ratios of 11-mercapto-1-undecanol and 11-mercaptoundecanoic acid, and of 3-mercapto-1-propanol and 11-mercaptoundecanoic acid. The biosensors were optimized for the detection of RCA(120), and a detection limit of 0.13 nM was obtained. Subsequent experiments with h-Gal-1 indicated a detection limit of at least 0.9 nM for this lectin. Additionally, the effect of interfering proteins on the sensitivity of the optimized biosensor was investigated.

Synthesis and conjugation of oligosaccharide fragments related to the immunologically reactive part of the circulating anodic antigen of the parasite Schistosoma mansoni

The immunoreactive part of the circulating anodic antigen (CAA) from the parasite Schistosoma mansoni is a threonine-linked polysaccharide consisting of →6)-[β-D-GlcpA-(1→3)]-β-D-GalpNAc-(1→ repeating disaccharides. In the framework of an immunochemical project, as a follow-up of earlier synthesized di- to tetrasaccharide CAA fragments, the synthesis of a spacer-containing pentasaccharide fragment, 3-(2-aminoethylthio)propyl (2-acetamido-2-deoxy-β-D-galactopyranosyl)-(1→6)-[(β-D-glucopyranosyluronic acid)-(1→3)]-(2-acetamido-2-deoxy-β-D-galactopyranosyl)-(1→6)-[(β-D-glucopyranosyluronic acid)-(1→3)]-2-acetamido-2-deoxy-β-D-galactopyranoside, is described. Moreover, 1-O-[3-(2-aminoethylthio)propyl]-N-acetyl-β-D-galactosamine was synthesized. Oxidation steps in the synthesis of tri- to pentasaccharide CAA fragments were performed using pyridinium dichromate and acetic anhydride. TEMPO-catalyzed oxidations were explored in the synthesis of 1-O-[6-aminohexyl]-β-D-glucuronic acid and 3-aminopropyl (2-acetamido-2-deoxy-β-D-galactopyranosyl)-(1→6)-[(β-D-glucopyranosyluronic acid)-(1→3)]-2-acetamido-2-deoxy-β-D-galactopyranoside, affording short reaction times and high yields. All synthesized compounds, including the earlier described 3-(2-aminoethylthio)propyl-spacered di-, tri-, and tetrasaccharide CAA fragments, were conjugated to BSA using squaric diester chemistry with coupling efficiencies in the range of 30–90%. The efficiency decreased when larger oligosaccharides were coupled to BSA. Finally, conformational analyses of the tri- and tetrasaccharide fragments were performed using Molecular Mechanics (MM) and Molecular Dynamics (MD) calculations.

Identification of a GDP-Fuc:Galβ1-3GalNAc-R (Fuc to Gal) α1-2 fucosyltransferase and a GDP-Fuc:Galβ1-4GlcNAc (Fuc to GlcNAc) α1-3 fucosyltransferase in connective tissue of the snail Lymnaea stagnalis

Connective tissue of the freshwater pulmonateLymnaea stagnalis was shown to contain fucosyltransferase activity capable of transferring fucose from GDP-Fuc in α1–2 linkage to terminal Gal of type 3 (Galβ1–3GalNAc) acceptors, and in α1–3 linkage to GlcNAc of type 2 (Galβ1–4GlcNAc) acceptors. The α1–2 fucosyltransferase was active with Galβ1–3GalNAcβ1-OCH2CH=CH2 (Km=12 mM,Vmax=1.3 mU ml−1) and Galβ1–3GalNAc (Km=20 mM,Vmax=2.1 mU ml−1), whereas the α1–3 fucosyltransferase was active with Galβ1–4GlcNAc (Km=23 mM,Vmax=1.1 mU ml−1). The products formed from Galβ1–3GalNAcβ1-OCH2CH=CH2 and Galβ1–4GlcNAc were purified by high performance liquid chromatography, and identified by 500 MHz1H-NMR spectroscopy and methylation analysis to be Fucα1–2Galβ1–3GalNAcβ1-OCH2CH=CH2 and Galβ1–4(Fucα1–3)GlcNAc, respectively. Competition experiments suggest that the two fucosyltransferase activities are due to two distinct enzymes.