Genetically encoded fragment-based discovery of glycopeptide ligands for carbohydrate-binding proteins.

We describe an approach to accelerate the search for competitive inhibitors for carbohydrate-recognition domains (CRDs). Genetically encoded fragment-based discovery (GE-FBD) uses selection of phage-displayed glycopeptides to dock a glycan fragment at the CRD and guide selection of synergistic peptide motifs adjacent to the CRD. Starting from concanavalin A (ConA), a mannose (Man)-binding protein, as a bait, we narrowed a library of 10(8) glycopeptides to 86 leads that share a consensus motif, Man-WYD. Validation of synthetic leads yielded Man-WYDLF that exhibited 40-50-fold enhancement in affinity over methyl α-d-mannopyranoside (MeMan). Lectin array suggested specificity: Man-WYD derivative bound only to 3 out of 17 proteins­ConA, LcH, and PSA­that bind to Man. An X-ray structure of ConA:Man-WYD proved that the trimannoside core and Man-WYD exhibit identical CRD docking, but their extra-CRD binding modes are significantly different. Still, they have comparable affinity and selectivity for various Man-binding proteins. The intriguing observation provides new insight into functional mimicry of carbohydrates by peptide ligands. GE-FBD may provide an alternative to rapidly search for competitive inhibitors for lectins.

Oligosaccharides and peptide displayed on an amphiphilic polymer enable solid phase assay of hapten specific antibodies.

Copovidone, a copolymer of vinyl acetate and N-vinyl-2-pyrrolidone, was synthesized via reversible addition-fragmentation chain transfer (RAFT) polymerization, and after deacetylation the polymer was functionalized by introduction of amino, azide, and alkyne pendant groups to allow attachment of glycans and peptide. Candida albicans ß-mannan trisaccharides 1 and 2 and M. tuberculosis arabinan hexasaccharide 3 with appropriate tethers were conjugated to the polymers by squarate or click chemistry. C. albicans T-cell peptide 4 bearing a C-terminal ε-azidolysine was also conjugated to copovidone by click chemistry. The resulting conjugates provide convenient non-protein-based antigens that are readily adsorbed on ELISA plates, and display excellent characteristics for assay of antibody binding to the haptenic group of interest. Copovidone and BSA glycoconjugates exhibited similar adsorption characteristics when used to coat ELISA plates, and both conjugates were optimal when used as coating solutions at low nanogram/mL concentrations. Provided that the copovidone conjugated glycan is stable to acid, assay plates can be easily processed for reuse at least three times without detectable variation or degradation in ELISA readout.

Discovery of light-responsive ligands through screening of a light-responsive genetically encoded library.

Light-responsive ligands are useful tools in biochemistry and cell biology because the function of these ligands can be spatially and temporally controlled. Conventional design of such ligands relies on previously available data about the structure of both the ligand and the receptor. In this paper, we describe de novo discovery of light-responsive ligands through screening of a genetically encoded light-responsive library. We ligated a photoresponsive azobenzene core to a random CX7C peptide library displayed on the coat protein of M13 phage. A one-pot alkylation/reduction of the cysteines yielded a photoresponsive library of random heptapeptide macrocycles with over 2 × 10(8) members. We characterized the reaction on-phage and optimized the yield of the modifications in phage libraries. Screening of the library against streptavidin yielded three macrocycles that bind to streptavidin in the dark and cease binding upon irradiation with 370 nm light. All ligands restored their binding properties upon thermal relaxation and could be turned ON and OFF for several cycles. We measured dissociation constants, Kd, by electrospray ionization mass spectrometry (ESI-MS) binding assay. For ligand ACGFERERTCG, the Kd of cis and trans isomers differed by 22-fold; an incomplete isomerization (85%), however, resulted in the apparent difference of 4.5-fold between the dark and the irradiated state. We anticipate that the selection strategy described in this report can be used to find light-responsive ligands for many targets that do not have known natural ligands.

Poly(N-vinyl-2-pyrrolidone-co-vinyl alcohol), a versatile amphiphilic polymeric scaffold for multivalent probes.

A convenient scaffold based on poly(N-vinyl-2-pyrrolidone-co-vinyl alcohol) is proposed for presenting ligands in multivalent format. This amphiphilic polymer supports synthesis of conjugates in both organic and aqueous media, permits enzymatic processing of the ligand precursor, and, finally, offers a choice of formats for evaluation of biological activity either as a soluble inhibitor or as a capture reagent after deposition on a hydrophobic surface or standard microtiter plates.

Supramolecular complexing of membane Siglec CD22 mediated by a polyvalent heterobifunctional ligand that templates on IgM.

We report the synthesis and in vitro evaluation of a multivalent homing device, a polymer which contains preordered pendant groups with dual specificity, a trisaccharide moiety, which is specific for the siglec CD22, and an antibody specific hapten, nitrophenol. The device efficiently attracts antihapten IgM to the surface of human lymphoma B cells as well as to CD22-conjugated magnetic beads by mediating the formation of a ternary complex on the surface of the target.

Bifunctional CD22 ligands use multimeric immunoglobulins as protein scaffolds in assembly of immune complexes on B cells.

CD22 is a B cell-specific sialic acid-binding immunoglobulin-like lectin (Siglec) whose function as a regulator of B cell signaling is modulated by its interaction with glycan ligands bearing the sequence NeuAc alpha2-6Gal. To date, only highly multivalent polymeric ligands (n = 450) have achieved sufficient avidity to bind to CD22 on native B cells. Here we demonstrate that a synthetic bifunctional molecule comprising a ligand of CD22 linked to an antigen (nitrophenol; NP) can use a monoclonal anti-NP IgM as a decavalent protein scaffold to efficiently drive assembly of IgM-CD22 complexes on the surface of native B cells. Surprisingly, anti-NP antibodies of lower valency, IgA (n = 4) and IgG (n = 2), were also found to drive complex formation, though with lower avidity. Ligands bearing alternate linkers of variable length and structure were constructed to establish the importance of a minimal length requirement, and versatility in the structural requirement. We show that the ligand drives assembly of IgM complexes exclusively on the surface of B cells and not other classes of white blood cells that do not express CD22, which lends itself to the possibility of targeting B cells in certain hematopoietic malignancies.

N-acetyllactosamine conjugated to gold nanoparticles inhibits enteropathogenic Escherichia coli colonization of the epithelium in human intestinal biopsy specimens.

We previously reported that the bundle-forming pilus-mediated localized adherence of enteropathogenic Escherichia coli to HEp-2, T84, and Caco-2 cells is inhibited by N-acetyllactosamine neoglycoconjugates. The results presented here extend this observation to the epithelium of biopsy specimens obtained from the human adult duodenum, terminal ileum, and colon.

A comparison of mineral affinity of bisphosphonate-protein conjugates constructed with disulfide and thioether linkages.

Chemical conjugation of bisphosphonates (BPs) to therapeutic proteins is an effective means to impart mineral affinity to proteins. Such conjugates can be implanted with mineral-based matrices to control the local delivery kinetics of the proteins. BPs linked to proteins with reversible (i.e., cleavable) linkages are desirable over conjugates with stable linkages to release the protein in free form. This study conducted a direct comparison of mineral affinity of BP-protein conjugates linked together with cleavable disulfide and non-cleavable thioether linkages. Bovine serum albumin (BSA) was used as a model protein and the desired conjugates were created with N-succinimidyl-3-(2-pyridyldithio)propionate (disulfide) and succinimidyl-4-(N-maleimido-methyl)cyclohexane-1-carboxylate (thioether) linkers. The disulfide-linked conjugates were cleaved in the presence of a major thiol constituent of serum, cysteine. The imparted mineral affinity, as assessed by hydroxyapatite binding in vitro, was lost upon the cleavage of the disulfide-linked aminoBP. The presence of the serum did not accelerate the cleavage of disulfide-linked conjugates. The aminoBP-BSA conjugates formed with disulfide and thioether linkages were subcutaneously implanted in rats with two different mineral-based matrices to assess protein loss from the matrices. All conjugates exhibited a higher retention in mineral matrices as compared to unmodified BSA. However, no significant differences in in situ pharmacokinetics of the disulfide- and thioether-linked conjugates were observed. We conclude that disulfide-linked BP conjugates were readily cleavable by the amino acid cysteine in vitro, but in vivo cleavage of the disulfide-linked conjugates was not evident when the proteins were implanted adsorbed to mineral-based matrices. BP-protein conjugates with faster-cleaving tethers might be required to significantly influence the release of the BP conjugates from the mineral matrices.

Ligand-assisted aggregation of proteins. Dimerization of serum amyloid P component by bivalent ligands.

A comprehensive series of solution and crystallographic studies reveal how simple, achiral, bivalent ligands of the cyclic pyruvate of glycerol promote face-to-face complex formation of the pentraxin, serum amyloid P component (SAP) into decamers. SAP, a protein of the human innate immune system, is universally present in amyloids, including cerebral amyloid deposits found in the brain of Alzheimer disease patients. Removal of SAP through a specific aggregation mechanism mediated by multivalent ligands appears to provide therapeutic benefit in the progression of this disease. Crystallographic studies reveal that in our novel series of ligands only the methyl and carboxylate moieties of the pyruvate ketal directly interact with the protein, but the geometric constraints imposed by the tether dictate which of two chair conformations are adopted by the pyruvate dioxane ring. Solution studies, as interpreted through a simple thermodynamic model, account for the distribution of pentameric and decameric bound states at different ligand concentrations and indicate that differences in the flexibility of the tether determine the geometry and stability of the specific aggregates formed between SAP and two different bivalent ligands. The factors affecting the design of ligands promoting face-to-face protein dimerization as well as potential biological implications are discussed.