Protein-carbohydrate interaction : On the mode of binding of aromatic moieties to concanavalin A, the phytohemagglutinin of the jack bean

A number of meta-alkylphenyl [beta]--glucopyranosides were synthesized and their ability to inhibit the concanavalin A-polysaccharide system was examined. The binding constants of these compounds as well as other substituted phenyl [amalgamation or coproduct]--glucopyranosides were related to the hydrophobic ([pi]) and electronic ([sigma]) nature of the substituents utilizing the equations devised by Hansch S and Hammett[paragraph sign] respectively.Regression analysis of these relationships revealed that: (1) no linear correlation between the binding constants and the electronic properties of the aromatic substituents was evident; (2) the molecular volume of mono-ortho-substituents does not significantly effect the binding of aromatic [beta]--glucopyranosides to concanavalin A; and (3) the hydrophobic nature ([pi]) of ortho- and meta- but not para-substituents is closely associated with the binding of aryl [beta]--glucosides to concanavalin A.It is proposed that apolar binding involving hydrophobic interactions associated with ortho and meta but not with the para positions of the aromatic nucleus are the predominant forces involved in the binding of the phenyl moiety of phenyl [beta]--glucosides to concanavalin A.Peer Reviewedhttp://deepblue.lib.umich.edu/bitstream/2027.42/33567/1/0000068.pd

Quantitative studies on the interaction of concanavalin A, the carbohydrate-binding protein of the jack bean, with model carbohydrate-protein conjugates

The interaction of concanavalin A with a comprehensive series of carbohydrate-bovine serum albumin comjugates was investigated by agar gel diffusion and quantitative precipitation techniques. These studies showed that when conjugated to bovine serum albumin, those sugars which inhibited concanavalin A-polysaccharide interaction formed a precipitate with concanavalin A. The unexpected reactivity of the p-phenylazo [ss]--glucopyranoside-bovine serum albumin conjugate is rationalized on the basis of inhibition data employing several aryl and alkylglycosides.Peer Reviewedhttp://deepblue.lib.umich.edu/bitstream/2027.42/33889/1/0000154.pd

Application of Nanotrap technology for high sensitivity measurement of urinary outer surface protein A carboxyl-terminus domain in early stage Lyme borreliosis

Objectives: Prompt antibiotic treatment of early stage Lyme borreliosis (LB) prevents progression to severe multisystem disease. There is a clinical need to improve the diagnostic specificity of early stage Lyme assays in the period prior to the mounting of a robust serology response. Using a novel analyte harvesting nanotechnology, Nanotrap particles, we evaluated urinary Borrelia Outer surface protein A (OspA) C-terminus peptide in early stage LB before and after treatment, and in patients suspected of late stage disseminated LB. Method: We employed Nanotrap particles to concentrate urinary OspA and used a highly specific anti-OspA monoclonal antibody (mAb) as a detector of the C-terminus peptides. We mapped the mAb epitope to a narrow specific OspA C-terminal domain OspA236-239 conserved across infectious Borrelia species but with no homology to human proteins and no cross-reactivity with relevant viral and non-Borrelia bacterial proteins. 268 urine samples from patients being evaluated for all categories of LB were collected in a LB endemic area. The urinary OspA assay, blinded to outcome, utilized Nanotrap particle pre-processing, western blotting to evaluate the OspA molecular size, and OspA peptide competition for confirmation. Results: OspA test characteristics: sensitivity 1.7 pg/mL (lowest limit of detection), % coefficient of variation (CV) = 8 %, dynamic range 1.7-30 pg/mL. Pre-treatment, 24/24 newly diagnosed patients with an erythema migrans (EM) rash were positive for urinary OspA while false positives for asymptomatic patients were 0/117 (Chi squared p < 10-6). For 10 patients who exhibited persistence of the EM rash during the course of antibiotic therapy, 10/10 were positive for urinary OspA. Urinary OspA of 8/8 patients switched from detectable to undetectable following symptom resolution post-treatment. Specificity of the urinary OspA test for the clinical symptoms was 40/40. Specificity of the urinary OspA antigen test for later serology outcome was 87.5 % (21 urinary OspA positive/24 serology positive, Chi squared p = 4.072e-15). 41 of 100 patients under surveillance for persistent LB in an endemic area were positive for urinary OspA protein. Conclusions: OspA urinary shedding was strongly linked to concurrent active symptoms (e.g. EM rash and arthritis), while resolution of these symptoms after therapy correlated with urinary conversion to OspA negative

Synthesis of high-affinity, hydrophobic monosaccharide derivatives and study of their interaction with concanavalin A, the pea, the lentil, and fava bean lectins

Concanavalin A (Con A) and agglutinins from the pea (PSA), lentil (LCH), and fava bean (VFA) constitute a group of -mannose/-glucose binding legume lectins. In addition to their sugar binding specificity, these lectins also contain sites that bind hydrophobic ligands. The present study explores a class of nonpolar binding sites reportedly present adjacent to the carbohydrate binding site in PSA, LCH, and VFA. A series of 2-O- and 3-O-substituted nitrobenzoyl and nitrobenzyl derivatives of methyl [alpha]--glucopyranoside and methyl [alpha]--mannopyranoside were synthesized. Evaluation of their binding to Con A, PSA, LCH, and VFA was carried out by the technique of hapten inhibition of precipitation reaction. The hapten inhibition assay results reveal that the presence of a methyl or methylene group at the O-2 or O-3 position of the sugar is essential for hydrophobic interaction with PSA, LCH, and VFA. The substitution of methyl by nitrobenzyl leads to enhanced binding (1.7-16.7 times for the 2-O-substituted compounds and 7.9-40.5 times for the 3-O-substituted compounds) with the m-nitrobenzyl group contributing to maximum binding. A hydrophobic interaction is also involved between Con A and 2-O-nitrobenzyl derivatives, resulting in enhanced binding, but the corresponding 3-O-isomers bind poorly due probably to steric reasons. These results may be rationalized on the basis of the recently published X-ray data of Con A and VFA. The nitrobenzyl derivatives, after transformation to their azido analogs, have potential applications in the photoaffinity labeling of these lectins.Peer Reviewedhttp://deepblue.lib.umich.edu/bitstream/2027.42/29695/1/0000027.pd

D-Glucuronic acid: A non-inhibitor of the concanavalin A system

D-Glucuronic acid is demonstrated not to inhibit the concanavalin A system; previous claims to the contrary are shown to be due to a pH effect. Concanavalin A does not precipitate biopolymers containing nonreducing, terminal [alpha]-D-glucopyranosiduronic acid residues.Peer Reviewedhttp://deepblue.lib.umich.edu/bitstream/2027.42/34081/1/0000360.pd

Cross-linked guaran: A versatile immunosorbent for D-galactopyranosyl binding lectins

Peer Reviewedhttp://deepblue.lib.umich.edu/bitstream/2027.42/21677/1/0000065.pd

An "extension" of the carbohydrate binding specificity of concanavalin A

Evidence based on the quantitative precipitin method and hapten inhibition technique demonstrates that concanavalin A may interact with internal 2-O-linked [alpha]--mannopyranosyl residues as may occur in glycoproteins and polysaccharides.Peer Reviewedhttp://deepblue.lib.umich.edu/bitstream/2027.42/33828/1/0000085.pd

Carbohydrate-binding specificity of the daffodil (Narcissus pseudonarcissus) and amaryllis (Hippeastrum hybr.) bulb lectins

The carbohydrate binding specificity of the daffodil (Narcissus pseudonarcissus; NPA) and amaryllis (Hippeastrum hybr.; HHA) lectins, isolated from extracts of their bulbs by affinity chromatography on immobilized mannose, was studied by quantitative precipitation, sugar hapten inhibition, and affinity chromatography on the immobilized lectins. These lectins gave strong precipitation reactions with several yeast mannans, but did not precipitate with [alpha]--glucans (e.g., dextrans and glycogen). Interestingly, both lectins reacted strongly with yeast galactomannans having multiple nonreducing terminal [alpha]--galactosyl groups, a synthetic linear [alpha]-1,6-mannan, and an [alpha]-1,3-mannan (DP = 30). Treatment of the linear [alpha]-1,3-mannan with periodate, resulting in oxidation of the terminal, nonreducing mannosyl group, did not reduce its reactivity with NPA or HHA. Taken together, these observations suggest that NPA and HHA react not only with terminal but also with internal [alpha]--mannosyl residues. Sugar hapten inhibition studies showed these lectins to possess the greatest specific activity for [alpha]--mannosyl units whereas -Glc and -GlcNAc did not inhibit either lectin precipitation system. Of the oligosaccharides tested, the best inhibitor of NPA interaction was [alpha]-1,6-linked mannotriose, which was twice as good an inhibitor as Man[alpha] 1,6Man[alpha]-O-Me and 10 times better than methyl [alpha]--mannoside. On the other hand, oligosaccharides containing either 1,3- or 1,6-linked mannosyl units were good inhibitors of the HHA-mannan precipitation system (6- to 20-fold more active than -Man). These results indicate that both lectins appear to possess an extended binding site(s) complementary to at least three 1,6-linked [alpha]-mannosyl units. Various glycosylasparagine glycopeptides which contain [alpha]-1,6-Man units were retarded on the immobilized NPA column. On the other hand, those containing either [alpha]-1,3- or [alpha]-1,6-mannosyl residues were retarded on the immobilized HHA column; Man5-GlcNAc2-Asn [containing two Man[alpha] 1,3 (Man[alpha] 1,6) units] bound to the HHA column. On the contrary, glycopeptides with hybrid type glycan chains were not retarded on either column. These two new lectins which differ in their fine sugar binding specificity from each other, and also from the snowdrop lectin, should prove to be useful probes for the detection and preliminary characterization of glycoconjugates on cell surfaces and in solution.Peer Reviewedhttp://deepblue.lib.umich.edu/bitstream/2027.42/28546/1/0000345.pd