Investigating the candidacy of lipopolysaccharide-based glycoconjugates as vaccines to combat <Emphasis Type="Italic">Mannheimia haemolytica</Emphasis>

Inner core lipopolysaccharide (LPS) has been shown to be conserved in the majority of veterinary strains from the species Mannheimia haemolytica, Actinobacillus pleuropneumoniae and Pasteurella multocida and as such is being considered as a possible vaccine antigen. The proof-in-principle that a LPS-based antigen could be considered as a vaccine candidate has been demonstrated from studies with monoclonal antibodies raised to the inner core LPS of Mannheimia haemolytica, which were shown to be both bactericidal and protective in a mouse model of disease. In this study we confirm and extend the candidacy of the inner core LPS by demonstrating that it is possible to elicit functional antibodies against Mannheimia haemolytica wild-type strains following immunisation of rabbits with glycoconjugates elaborating the conserved inner core LPS antigen. The present study describes a conjugation strategy that uses amidases produced by Dictyostelium discoideum, targeting the amino functionality created by the amidase activity as the attachment point on the LPS molecule. To protect the amino functionality on the phosphoethanolamine (PEtn) residue of the inner core, we developed a novel blocking and unblocking strategy with t-butyl oxycarbonyl. A maleimide-thiol linker strategy with the thiol linker on the carboxyl residues of the carrier protein and the maleimide linker on the carbohydrate resulted in a high loading of carbohydrates per carrier protein. Immunisation derived antisera from rabbits recognised fully extended Mannheimia haemolytica LPS and whole cells from serotypes 1 and 2, despite a somewhat immunodominant response to the linkers also being observed. Moreover, bactericidal activity was demonstrated to a strain elaborating the immunising carbohydrate antigen and crucially to wild-type cells of serotypes 1 and 2, thus further supporting the consideration of inner core LPS as a potential vaccine antigen to combat disease caused by Mannheimia haemolytica.     

Molecular modeling provides insights into the loading of sialic acid‐containing antigens onto CRM197: the role of chain flexibility in conjugation efficiency and glycoconjugate architecture

Vaccination is the most cost-effective way to control disease caused by encapsulated bacteria; the capsular polysaccharide (CPS) is the primary virulence factor and vaccine target. Neisseria meningitidis (Nm) serogroups B, C, Y and W all contain sialic acid, a common surface feature of human pathogens. Two protein-based vaccines against serogroup B infection are available for human use while four tetravalent conjugate vaccines including serogroups C, W and Y have been licensed. The tetravalent Menveo® conjugate vaccine is well-defined: a simple monomeric structure of oligosaccharides terminally conjugated to amino groups of the carrier protein CRM₁₉₇. However, not only is there a surprisingly low limit for antigen chain attachment to CRM₁₉₇, but different serogroup saccharides have consistently different CRM₁₉₇ loading, the reasons for which are unclear. Understanding this phenomenon is important for the long-term goal of controlling conjugation to prepare conjugate vaccines of optimal immunogenicity. Here we use molecular modeling to explore whether antigen flexibility can explain the varying antigen loading of the conjugates. Because flexibility is difficult to separate from other structural factors, we focus on sialic-acid containing CPS present in current glycoconjugate vaccines: serogroups NmC, NmW and NmY. Our simulations reveal a correlation between Nm antigen flexibility (NmW?>?NmC?>?NmY) and the number of chains attached to CRM₁₉₇, suggesting that increased flexibility enables accommodation of additional chains on the protein surface. Further, in silico models of the glycoconjugates confirm the relatively large hydrodynamic size of the saccharide chains and indicate steric constraints to further conjugation.

     

Biochemical and biological characteristics of cross-reacting material 197 (CRM197), a non-toxic mutant of diphtheria toxin: Use as a conjugation protein in vaccines and other potential clinical applications

The biochemical and biological characteristics of CRM₁₉₇ are reviewed. Polysaccharide protein conjugate vaccines represent an important technological advancement that allowed for protection against dangerous diseases in vulnerable populations such as infants. The first carrier proteins, diphtheria and tetanus toxoids, were chosen in the context of an extensive body of information describing their immunogenicity and safety profiles in clinical use. These carriers perform well, and they require detoxification. A non-toxic mutant of diphtheria toxin, cross-reacting material 197 (CRM₁₉₇), is a useful carrier protein with several manufacturing and other potential advantages over toxoids. For over a decade, several important and widely used routine childhood glycoconjugate vaccines against serious illnesses, including Haemophilus influenzae type b and pneumococcal disease, have employed CRM₁₉₇ as carrier protein. Additional clinical applications of CRM₁₉₇, as in chemotherapy, also exist.     

Investigating the candidacy of LPS-based glycoconjugates to prevent invasive meningococcal disease: conjugates based on core oligosaccharides

In this study we have prepared glycoconjugates with core oligosaccharides (OS) from the lipopolysaccharide (LPS) of Neisseria meningitidis, thus avoiding the neo-epitopes of the deacylated lipid A region of the derived LPS molecule identified in our previous studies. A comprehensive investigation was performed with glycoconjugates prepared from the most extended to the most truncated core OS still maintaining the conserved inner core epitope. As previously, we have established reproducible bactericidal killing of the homologous antigen elaborating strain, but a failure to kill wild-type strains. In these studies it was evident that the linker molecules used in the conjugation methodologies were dominating the immune response. However, when galE core OS based conjugates were prepared without utilizing linkers, via direct reductive amination, we failed to generate an immune response to even the homologous antigen. We also identified that immunisation with the galE antigen via linker methodologies provoked an immune response that was dependent upon key residues of the conserved inner core OS structure, whereas the immune responses to lgtB and lgtA antigens did not involve the inner core OS. This comprehensive study has, despite our best efforts, cast significant doubt as to the utility of the conserved inner core region of the meningococcal LPS as a potential vaccine antigen.     

The potential of dextran-based glycoconjugates for development of Helicobacter pylori vaccine

We have recently demonstrated that synthetic glycoconjugates based on delipidated lipopolysaccharide (LPS) of Helicobacter pylori and containing an α(1-6)-glucan chain induced broadly cross-reactive functional antibodies in immunized animals. To investigate the candidacy of α(1-6)-glucan as an alternative vaccine strategy we prepared glycoconjugates based on dextrans produced by lactic acid bacteria Leuconostoc mesenteroides B512F and consisting of linear α(1-6)-glucan chains with limited branching. Three dextrans with averaged molecular masses of 5,000 Da, 3,500 Da and 1,500 Da, respectively, were modified with a diamino group-containing linker and conjugated to a carrier protein, tetanus toxoid (TT) or diphtheria toxoid (DT), and their immunological properties investigated. The conjugates were immunogenic in both rabbits and mice and induced specific IgG responses against α(1-6)-glucan-expressing H. pylori LPS. Studies performed with post-immune sera of mice and rabbits immunized with dextran-based conjugates demonstrated cross-reactivity with LPS from typeable and non-typeable strains of H. pylori and selected mutants. The post-immune sera from rabbits that received the conjugates exhibited functional activity against α(1-6)-glucan-positive strains of H. pylori. These data provide evidence that dextran-based conjugates may offer a simplified approach to the development of carbohydrate-based vaccines against H. pylori.     

Synthesis of chitooligomer-based glycoconjugates and their binding to the rat natural killer cell activation receptor NKR-P1

NKR-P1 protein is an important activating receptor at the surface of the rat natural killer cells. GlcNAc and chitooligomers were identified as strong activation ligands in vitro and in vivo. Their clustering brings about increase of their affinity to the NKR-P1 by 3–6 orders. Here we describe novel methodology for preparation of neoglycoproteins based on BSA carying the chitooligomers (n = 2–5). Further on we developed novel methodology of the coupling of glycosylamines via aromatic-SCN activated linker both to protein or synthetic cores. Inhibition studies of chitooligomer glycoconjugates with the NKR-P1 receptor show that our neoglycoproteins are very strong ligands with high binding affinity (–log IC₅₀ = 13–15). In analogy with our previous observations with GlcNAc clustered on protein or PAMAM backbones the synthetic chitooligomer clusters should provide considerably better ligands in the in vivo antitumor treatment.     

Investigating the potential of conserved inner core oligosaccharide regions of Moraxella catarrhalis lipopolysaccharide as vaccine antigens: accessibility and functional activity of monoclonal antibodies and glycoconjugate derived sera

We investigated the conservation and antibody accessibility of inner core epitopes of Moraxella catarrhalis lipopolysaccharide (LPS) in order to assess their potential as vaccine candidates. Two LPS mutants, a single mutant designated lgt2 and a double mutant termed lgt2/lgt4, elaborating truncated inner core structures were generated in order to preclude expression of host-like outer core structures and to create an inner core structure that was shared by all three serotypes A, B and C of M. catarrhalis. Murine monoclonal antibodies (mAbs), designated MC2-1 and MC2-10 were obtained by immunising mice with the lgt2 mutant of M. catarrhalis serotype A strain. We showed that mAb MC2-1 can bind to the core LPS of wild-type (wt) serotype A, B and C organisms and concluded that mAb MC2-1 defines an immunogenic inner core epitope of M. catarrhalis LPS. We were unsuccessful in obtaining mAbs to the lgt2/lgt4 mutant. MAb MC2-10 only recognised the lgt2 mutant and the wt serotype A strain, and exhibited a strong requirement for the terminal N-acetyl-glucosamine residue of the lgt2 mutant core oligosaccharide, suggesting that this residue was immunodominant. Subsequently, we showed that both mAbs MC2-1 and MC2-10 could facilitate bactericidal killing of the lgt2 mutant, however neither mAb could facilitate bactericidal killing of the wt serotype A strain. We then confirmed and extended the candidacy of the inner core LPS by demonstrating that it is possible to elicit functional antibodies against M. catarrhalis wt strains following immunisation of rabbits with glycoconjugates elaborating the conserved inner core LPS antigen. The present study describes three conjugation strategies that either uses amidases produced by Dictyostelium discoideum, targeting the amino functionality created by the amidase activity as the attachment point on the LPS molecule, or a strong base treatment to remove all fatty acids from the LPS, thus creating amino functionalities in the lipid A region to conjugate via maleimide-thiol linker strategies targeting the carboxyl residues of the carrier protein and the free amino functionalities of the derived lipid A region of the carbohydrate resulted in a high loading of carbohydrates per carrier protein from these carbohydrate preparations. Immunisation derived antisera from rabbits recognised fully extended M. catarrhalis LPS and whole cells. Moreover, bactericidal activity was demonstrated to both the immunising carbohydrate antigen and importantly to wt cells, thus further supporting the consideration of inner core LPS as a potential vaccine antigen to combat disease caused by M. catarrhalis.     

Investigating the candidacy of a lipoteichoic acid-based glycoconjugate as a vaccine to combat Clostridium difficile infection

A lipoteichoic acid has recently been shown to be conserved in the majority of strains from Clostridium difficile and as such is being considered as a possible vaccine antigen. In this study we examine the candidacy of the conserved lipoteichoic acid by demonstrating that it is possible to elicit antibodies against C. difficile strains following immunisation of rabbits and mice with glycoconjugates elaborating the conserved lipoteichoic acid antigen. The present study describes a conjugation strategy that utilises an amino functionality, present at approximately 33 % substitution of the N-acetyl-glucosamine residues within the LTA polymer repeating unit, as the attachment point for conjugation. A maleimide-thiol linker strategy with the maleimide linker on the carboxyl residues of the carrier protein and the thiol linker on the carbohydrate was employed. Immunisation derived antisera from rabbits and mice, recognised all strains of C. difficile vegetative cells examined, despite an immune response to the linkers also being observed. These sera recognised live cells in an immunofluorescence assay and were also able to recognise the spore form of the bacterium. This study has illustrated that the LTA polymer is a highly conserved surface polymer of C. difficile that is easily accessible to the immune system and as such merits consideration as a vaccine antigen to combat C. difficile infection.     

Chemical evidence for covalent linkages of a semisynthetic glycoconjugate vaccine forHaemophilus influenzae type b disease

We have defined the nature of the covalent linkages in aHaemophilus influenzae type b oligosaccharide-CRM₁₉₇ conjugate vaccine, designated HbOC. The conjugate was acid hydrolyzed to release a novel amino-acid derivative,N-(2-hydroxyethyl)lysine (OHEt-Lys), identifiable with an amino-acid analyzer. This amino-acid derivative was formed by reduction of Schiff bases formed betweenH. influenzae type b oligosaccharides (HbO) and the lysyl -amino groups of CRM₁₉₇ (a non-toxic, cross-reactive variant of diphtheria toxin), followed by acid hydrolysis of HbOC. Quantification of OHEt-Lys per CRM₁₉₇ molecule allowed the determination of a covalency ratio, a useful parameter for evaluating the stoichiometry and consistency of HbOC preparations. Covalent association between HbO and CRM₁₉₇ was also demonstrated by the coincidence of immunoreactivity of gelelectrophoresed HbOC on a Western blot probed with anti-CRM₁₉₇ and anti-saccharide antisera.     

Chemoenzymatic synthesis of immunogenic meningococcal group C polysialic acid-tetanus Hc fragment glycoconjugates

Vaccination with meningococcal glycoconjugate vaccines has decreased the incidence of invasive meningitis worldwide. These vaccines contain purified capsular polysaccharides attached to a carrier protein. Because of derivatization chemistries used in the process, conjugation of polysaccharide to protein often results in heterogeneous mixtures. Well-defined vaccines are needed to determine the relationship between vaccine structure and generated immune response. Here, we describe efforts to produce well-defined vaccine candidates by chemoenzymatic synthesis. Chemically synthesized lactosides were substrates for recombinant sialyltransferase enzymes from Camplyobacter jejuni and Neisseria meningitidis serogroup C. These resulting oligosialic acids have the same α(2-9) sialic acid repeat structure as Neisseria polysaccharide capsule with the addition of a conjugatable azide aglycon. The degree of polymerization (DP) of carbohydrate products was controlled by inclusion of the inhibitor CMP-9-deoxy-NeuNAc. Polymers with estimated DP?<?47 (median DP 25) and DP?<?100 (median DP 51) were produced. The receptor binding domain of the tetanus toxin protein (TetHc) was coupled as a carrier to the enzymatically synthesized oligosialic acids. Recombinant TetHc was derivatized with an alkyne squarate. Protein modification sites were determined by trypsin proteolysis followed by LC/MS-MS^E analysis of peptides. Oligosialic acid azides were conjugated to modified TetHc via click chemistry. These chemoenzymatically prepared glycoconjugates were reactive in immunoassays with specific antibodies against either group C polysaccharide or TetHc. Sera of mice immunized with oligosialic acid-TetHc glycoconjugates contained much greater levels of polysaccharide-reactive IgG than the sera of control mice receiving unconjugated oligosialic acids. There was no apparent difference between glycoconjugates containing oligosaccharides of DP?<?47 and DP?<?100. These results suggest that chemoenzymatic synthesis may provide a viable method for making defined meningococcal vaccine candidates.     

Preparation,characterization and immunogenicity of HIV-1 related high-mannose oligosaccharides-CRM<Subscript>197</Subscript> glycoconjugates

The dense glycan shield on the surface of human immunodeficiency virus type 1 (HIV-1) gp120 masks conserved protein epitopes and facilitates virus entry via interaction to glycan binding proteins on susceptible host cells. The broadly neutralizing monoclonal antibody 2G12 binds a cluster of high-mannose oligosaccharides on the gp120 subunit of HIV-1 Env protein. This oligomannose epitope is currently being considered for the design of a synthetic vaccine. The cluster nature of the 2G12 epitope suggests that a multivalent antigen presentation is important to develop a carbohydrate-based vaccine candidate. In this work we describe the development of neoglycoconjugates displaying clustered HIV-1 related oligomannose carbohydrates. We exploited flexible polyamidoamine (PAMAM) scaffold to generate four- and eight-valent sugar clusters of HIV-1-related oligomannose antigens Man₄, Man₆ and Man₉. The multivalent presentation of oligomannoses increased the avidity of Man₄ and Man₉ to 2G12. The synthetic glycodendrons were then covalently coupled to the protein carrier CRM₁₉₇, formulated with the adjuvant MF59, and used to immunize two animal species. Oligomannose-specific IgG antibodies were generated; however, the antisera failed to recognize recombinant HIV-1 gp120 proteins. We conclude that further structural vaccinology work is needed to identify an antigen presentation that closely matches in vivo the structure of the epitope mapped by 2G12.     

A single-step method for the production of sugar hydrazides: intermediates for the chemoselective preparation of glycoconjugates

The ability to selectively conjugate carbohydrate molecules to a protein is a key step in the preparation of conjugate vaccines, while facile methods for linking carbohydrates to polymers or solid surfaces to produce diagnostic probes and functional microarrays are also sought. Here, we describe a simple, single-step method of producing glycosylhydrazides from unprotected sugars, which were then linked in a controlled manner to a desired carrier, through an appropriate linker. The method was chemoselective and did not require coupling reagents, and the native pyranose form of the reducing end residue was retained. Initially, mono- and disaccharide hydrazides were produced from the corresponding reducing sugars and linked to BSA through a bifunctional linker. Final exemplification of the procedure was demonstrated by the preparation of a LewisY tetrasaccharide protein conjugate, which was recognized by a LewisY monoclonal antibody indicating the preservation of the natural conformation of the tetrasaccharide in the final construct. It is envisaged that this method will have general applicability to a variety of functionally diverse reducing sugars and provide a route to highly defined glycoconjugates, without the need for elaborate synthetic strategies.     

O:2-CRM197 Conjugates against Salmonella Paratyphi A

Enteric fevers remain a common and serious disease, affecting mainly children and adolescents in developing countries. Salmonella enterica serovar Typhi was believed to cause most enteric fever episodes, but several recent reports have shown an increasing incidence of S. Paratyphi A, encouraging the development of a bivalent vaccine to protect against both serovars, especially considering that at present there is no vaccine against S. Paratyphi A. The O-specific polysaccharide (O:2) of S. Paratyphi A is a protective antigen and clinical data have previously demonstrated the potential of using O:2 conjugate vaccines. Here we describe a new conjugation chemistry to link O:2 and the carrier protein CRM₁₉₇, using the terminus 3-deoxy-D-manno-octulosonic acid (KDO), thus leaving the O:2 chain unmodified. The new conjugates were tested in mice and compared with other O:2-antigen conjugates, synthesized adopting previously described methods that use CRM₁₉₇ as carrier protein. The newly developed conjugation chemistry yielded immunogenic conjugates with strong serum bactericidal activity against S. Paratyphi A.     

Synthesis of 8-hydroxyquinoline glycoconjugates and preliminary assay of their β1,4-GalT inhibitory and anti-cancer properties

8-Hydroxyquinoline scaffold is a privileged structure used in designing a new active agents with therapeutic potential. Its connections with the sugar unit is formed to improve the pharmacokinetic properties. The broad spectrum of activity of quinoline derivatives, especially glycoconjugates, is often associated with the ability to chelate metal ions or with the ability to intercalate into DNA. Simple and effective methods of synthesis glycoconjugates of 8-hydroxyquinoline and 8-hydroxyquinaldine derivatives, containing an O-glycosidic bond or a 1,2,3-triazole linker in their structure, have been developed. The obtained glycoconjugates were tested for their ability to inhibit β-1,4-Galactosyltransferase, as well as inhibit cancer cell proliferation. It was found that used glycoconjugation strategy influenced both improvement of activity and improvement of the bioavailability of 8-HQ derivatives. Their activity depends on type of attached sugar, presence of protecting groups in sugar moiety and presence of a linker between sugar and quinolone aglycone.     

Partial characterisation of high-molecular weight glycoconjugates in the trail mucus of the freshwater pond snail Lymnaea stagnalis

We have studied the glycoconjugates in trail mucus of the pond snail Lymnaea stagnalis. The mucus was dissolved with 6 M guanidinium hydrochloride (GuHCl) and the major component was comprised of very high-M_r glycoconjugates that were eluted in the void volume of a Sepharose CL-4B gel-filtration column. This high-M_r material was pooled and thereafter subjected to density gradient centrifugation first in 4 M GuHCl/CsCl and subsequently 0.2 M GuHCl/CsCl to further remove non-glycosylated proteins and DNA. The harvested glycoconjugate pool chromatographed in the void volume of Sepharose CL-2B. However, reduction of disulfide bonds lowered the molecular size of approximately 80% of the void material yielding a major fragment and some minor smaller fragments in gel chromatography. The reduced glycoconjugates were digested with papain and yielded high molecular weight, proteinase-resistant glycopeptides. This fragmentation pattern is similar to that found for oligomeric gel-forming mucins in mammals and the amino acid composition (60% Ser/Thr) and sugar analysis of the glycopeptides is consistent with mucin-like molecules, there being no significant amounts of xylose or uronic acids. The residual 20% of the preparation, which apparently resisted reduction and protease digestion, had a similar amino acid composition to the bulk, but was somewhat different in sugar composition, containing some xylose and a significant amount of glucuronic acid. The two groups of molecules had very different morphologies in the electron microscope. Taken together, these data suggest that trail mucus is a complex mixture of at least two families of protein-glycoconjugate molecules based upon the gel-forming mucin and proteoglycan families, though we cannot rule out that polysaccharides may also be present.     

Intraparticle concentration gradients for substrate and acidic product in immobilized cephalosporin C amidase and their dependencies on carrier characteristics and reaction parameters

Cephalosporin C amidase was covalently attached using a protein loading of 7.0–200 mg protein/g dry carrier on four epoxy‐activated Sepabeads differing in particle size and pore diameter. Initial‐rate kinetic analysis showed that for Sepabeads with small pore diameters (30–40 nm), the apparent K_M of the amidase for hydrolysis of cephalosporin C at 37°C and pH 8.0 increased ～3‐fold in response to increased particle size (～120–400 µm) and increased amount of immobilized enzyme (7.0–70 mg protein/g dry carrier) while maximum specific activity (3.2 U/mg protein; 25% of free amidase) was affected only by particle size. In contrast, for Sepabeads with wide pores (150–250 nm), the K_M was independent of the enzyme loading. Internal effectiveness factors calculated from observable Thiele modulus reflected the dependence of K_M on geometrical parameters of the particles. A new method for determination of the overall intraparticle pH was developed based on luminescence lifetime measurements in the frequency domain. Sepabeads were doubly labeled using a lipophilic variant of the pH‐sensitive dye fluorescein, and Ru(II) tris(4,7‐diphenyl‐1,10‐phenantroline) whose phosphorescence properties are independent of pH. Luminescent lifetime measurements of doubly labeled particle suspensions showed superior signal‐to‐noise ratio compared to fluorescence intensity‐based measurements using singly labeled particles. The difference at apparent steady state (ΔpH) between bulk (external pH) and intraparticle pH (internal pH) was as large as ～0.6 units. The ΔpH was dependent on substrate concentration, particle size, and pore diameter. Therefore, these results characterize the role of carrier characteristics and reaction parameters in the formation of concentration gradients for substrate and acidic product during hydrolysis of cephalosporin C by immobilized amidase. The strong pH dependence of the immobilized amidase underscores the importance of considering intraparticle pH gradients in the design of an efficient carrier‐bound biocatalyst. Biotechnol. Bioeng. 2010;106: 528–540. © 2010 Wiley Periodicals, Inc.     

A Vaccine Approach for the Prevention of Infections by Multidrug-resistant Enterococcus faecium

The incidence of multidrug-resistant Enterococcus faecium hospital infections has been steadily increasing. With the goal of discovering new vaccine antigens, we systematically fractionated and purified four distinct surface carbohydrates from E. faecium endocarditis isolate Tx16, shown previously to be resistant to phagocytosis in the presence of human serum. The two most abundant polysaccharides consist of novel branched heteroglycan repeating units that include signature sugars altruronic acid and legionaminic acid, respectively. A minor high molecular weight polysaccharide component was recognized as the fructose homopolymer levan, and a glucosylated lipoteichoic acid (LTA) was identified in a micellar fraction. The polysaccharides were conjugated to the CRM₁₉₇ carrier protein, and the resulting glycoconjugates were used to immunize rabbits. Rabbit immune sera were evaluated for their ability to kill Tx16 in opsonophagocytic assays and in a mouse passive protection infection model. Although antibodies raised against levan failed to mediate opsonophagocytic killing, the other glycoconjugates induced effective opsonic antibodies, with the altruronic acid-containing polysaccharide antisera showing the greatest opsonophagocytic assay activity. Antibodies directed against either novel heteroglycan or the LTA reduced bacterial load in mouse liver or kidney tissue. To assess antigen prevalence, we screened a diverse collection of blood isolates (n = 101) with antibodies to the polysaccharides. LTA was detected on the surface of 80% of the strains, and antigens recognized by antibodies to the two major heteroglycans were co-expressed on 63% of these clinical isolates. Collectively, these results represent the first steps toward identifying components of a glycoconjugate vaccine to prevent E. faecium infection.     

Interaction of aromatic imino glycoconjugates with jacalin: experimental and computational docking studies

Altering the lectin properties by chemically modified glycoconjugates can have profound effect on their biological applications. In the present case, jacalin has been chosen to study the binding aspects toward glycoconjugates modified by connecting aromatic moieties through imine conjugation at their C-1- or C-2-positions. Out of 10 glycoconjugates, the galactosyl-naphthyl imine (1c) was found to be most effective toward agglutination inhibition (260 times better than galactose), quenching fluorescence intensity, and exhibiting greater binding (K_a, 1.3 × 10⁴ M⁻¹) with jacalin. The specific binding of galactose conjugates and the nonspecific binding of other conjugates have been demonstrated based on ITC. Changes in the secondary structures have been addressed by far- and near-UV CD spectroscopy. The present studies demonstrated that galactose-based conjugates bind at carbohydrate recognition domain (CRD) mainly through polar interactions in addition to exhibiting some nonpolar/hydrophobic interactions, whereas the conjugates other than galactose primarily interact through hydrophobic interactions. Binding of galactosyl conjugates at CRD has been further demonstrated by rigid docking.     

Purification and characterization of phosphorylase from tubers ofDioscorea dumentorum

Phosphorylase from tubers ofDioscorea dumentorum was fractionated by (NH₄)₂SO₄ precipitation and further purified to apparent homogeneity. Two forms of phosphorylase, Dd₁ and Dd₂ were obtained with different K_m, optimum temperatures and pH values. They obeyed Michaelis-Menten kinetics, they were activated by AMP, Mg²⁺ and Ca²⁺ but inhibited by ATP, ADP, ADP-glucose and Na₂SO₄. The molecular masses found for Dd₁ and Dd₂ were 102 000 and 195 000 respectively. SDS gel electrophoresis indicated that Dd₂ is a dimer and Dd₁ is a monomer. Both phosphorylase forms contained pyridoxal-5′-phosphate as their prosthetic group, which was essential for their activity. Dd₂ is more active in starch synthesis while Dd₁ is more active in starch degradation. The affinity of the enzyme forms for P_i is not a measure of the rate of glucan degradation. The decline in activities of the enzymes between pH 5.5 and 7.5 was due to unsuitable ionic forms of the enzymes and not due to irreversible denaturation. Both forms of phosphorylase showed double displacement reaction mechanism in direction of phosphorolysis, while in direction of synthesis, sequential mechanism was indicated. Indoleacetic acid activated the enzyme forms in direction of starch degradation but acted as an inhibitor in direction of glucan synthesis. The results of this study suggest that Dd₂ may have some synthetic function while Dd₁ has a degradative role. The effect of pH on the phosphorylase activity suggests histidine as the amino acid residue around the active site that might be involved in enzyme catalysis.     

Use of glycoconjugates for trypanosomatid taxonomy

Glycoconjugates from five trypanosomatid genera—Crithidia, Herpetomonas, Endotrypanum, Leishmania, and Trypanosoma—were extracted with Triton X-114 and analyzed by sodium dodecyl sulfate polyacrylamide gel electrophoresis followed by periodic acid-Schiff staining. Most of the glycoconjugates were detected in the hydrophobic phase, indicating the presence of anchored glycoconjugates. All the trypanosomatids expressed a glycoconjugate with a low molecular weigh (below 20 kDa) in this phase. In each species, however, a characteristic and specific pattern of glycoconjugates was also observed in both phases. In the hydrophobic phase: 14–29 kDa lycoconjugates in C. guilhermei; 24–70 kDa in C. fasciculata, C. luciliae, E. schaudinni, and T. cruzi Y and G strains; 45–66 kDa in C. oncopelti and H. samuelpessoai; above 36 kDa in T. dionisii; 20–24 kDa, 36–45 kDa, and 70 kDa in L. tarentolae and T. mega. In the hydrophilic phase, typical glycoproteins were observed in some trypanosomatids: 60 kDa in T. mega and T. cruzi Y strain; 70 kDa in H. samuelpessoai; 66 kDa in C. oncopelti; 20–70 kDa in C. luciliae. These findings suggest that Triton X-114-extracted glycoconjugates could be useful markers for trypanosomatid taxonomy.