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Site-directed conjugation of "clicked" glycopolymers to form glycoprotein mimics: binding to mammalian lectin and induction of immunological function.

ABSTRACT: Synthesis of well-defined neoglycopolymer-protein biohybrid materials and a preliminary study focused on their ability of binding mammalian lectins and inducing immunological function is reported. Crucial intermediates for their preparation are well-defined maleimide-terminated neoglycopolymers (M(n) = 8-30 kDa; M(w)/M(n) = 1.20-1.28) presenting multiple copies of mannose epitope units, obtained by combination of transition-metal-mediated living radical polymerization (TMM LRP) and Huisgen [2+3] cycloaddition. Bovine serum albumin (BSA) was employed as single thiol-containing model protein, and the resulting bioconjugates were purified following two independent protocols and characterized by circular dichroism (CD) spectroscopy, SDS PAGE, and SEC HPLC. The versatility of the synthetic strategy presented in this work was demonstrated by preparing a small library of conjugating glycopolymers that only differ from each other for their relative epitope density were prepared by coclicking of appropriate mixtures of mannopyranoside and galactopyranoside azides to the same polyalkyne scaffold intermediate. Surface plasmon resonance binding studies carried out using recombinant rat mannose-binding lectin (MBL) showed clear and dose-dependent MBL binding to glycopolymer-conjugated BSA. In addition, enzyme-linked immunosorbent assay (ELISA) revealed that the neoglycopolymer-protein materials described in this work possess significantly enhanced capacity to activate complement via the lectin pathway when compared with native unmodified BSA.

SUBMITTER: Geng J

PROVIDER: S-EPMC2702692 | biostudies-literature | 2007 Dec

REPOSITORIES: biostudies-literature

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Site-directed conjugation of "clicked" glycopolymers to form glycoprotein mimics: binding to mammalian lectin and induction of immunological function.

Geng Jin J Mantovani Giuseppe G Tao Lei L Nicolas Julien J Chen Gaojian G Wallis Russell R Mitchell Daniel A DA Johnson Benjamin R G BR Evans Stephen D SD Haddleton David M DM

Journal of the American Chemical Society 20071117 49

Synthesis of well-defined neoglycopolymer-protein biohybrid materials and a preliminary study focused on their ability of binding mammalian lectins and inducing immunological function is reported. Crucial intermediates for their preparation are well-defined maleimide-terminated neoglycopolymers (M(n) = 8-30 kDa; M(w)/M(n) = 1.20-1.28) presenting multiple copies of mannose epitope units, obtained by combination of transition-metal-mediated living radical polymerization (TMM LRP) and Huisgen [2+3] ...[more]

PMID: 18020332

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Project description:Colorectal cancer (CRC) remains a major worldwide cause of cancer-related morbidity and mortality largely due to the insidious onset of the disease. The current clinical procedures utilized for disease diagnosis are invasive, unpleasant, and inconvenient; hence, the need for simple blood tests that could be used for the early detection of CRC. In this work, we have developed methods for glycoproteomics analysis to identify plasma markers with utility to assist in the detection of colorectal cancer (CRC). Following immunodepletion of the most abundant plasma proteins, the plasma N -linked glycoproteins were enriched using lectin affinity chromatography and subsequently further separated by nonporous silica reversed-phase (NPS-RP)-HPLC. Individual RP-HPLC fractions were printed on nitrocellulose coated slides which were then probed with lectins to determine glycan patterns in plasma samples from 9 normal, 5 adenoma, and 6 colorectal cancer patients. Statistical tools, including principal component analysis, hierarchical clustering, and Z-statistics analysis, were employed to identify distinctive glycosylation patterns. Patients diagnosed with colorectal cancer or adenomas were shown to have dramatically higher levels of sialylation and fucosylation as compared to normal controls. Plasma glycoproteins with aberrant glycosylation were identified by nano-LC-MS/MS, while a lectin blotting methodology was used to validate proteins with significantly altered glycosylation as a function of cancer progression. The potential markers identified in this study for diagnosis to distinguish colorectal cancer from adenoma and normal include elevated sialylation and fucosylation in complement C3, histidine-rich glycoprotein, and kininogen-1. These potential markers of colorectal cancer were subsequently validated by lectin blotting in an independent set of plasma samples obtained from 10 CRC patients, 10 patients with adenomas, and 10 normal subjects. These results demonstrate the utility of this strategy for the identification of N -linked glycan patterns as potential markers of CRC in human plasma, and may have the utility to distinguish different disease states.

Dataset Information

Site-directed conjugation of "clicked" glycopolymers to form glycoprotein mimics: binding to mammalian lectin and induction of immunological function.

Publications

Site-directed conjugation of "clicked" glycopolymers to form glycoprotein mimics: binding to mammalian lectin and induction of immunological function.

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