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Development and application of a novel recombinant Aleuria aurantia lectin with enhanced core fucose binding for identification of glycoprotein biomarkers of hepatocellular carcinoma.

ABSTRACT: The Aleuria aurantia lectin (AAL) derived from orange peel fungus contains five fucose-binding sites that recognizes fucose bound in ?-1,2, ?-1,3, ?-1,4, and ?-1,6 linkages to N-acetylglucosamine and galactose. Recently, we have created several recombinant AAL (rAAL) proteins that had altered binding affinity to fucose linkages. In this report, we further characterize the binding specificity of one of the mutated lectins, N224Q lectin. This lectin was characterized by lectin Western blotting, surface plasmon resonance, and glycan microarray and shown to have increased binding to fucosylated glycan. Subsequently, we used this lectin to identify secreted fucosylated glycoproteins from a fetal hepatic cell line. Proteomic analysis revealed several glycoproteins secreted by the fetal cell line that were bound by N224Q lectin. These findings were confirmed by subsequent proteomic analysis of human serum from control patients or patients with hepatocellular carcinoma. These represent candidate oncofetal markers for liver cancer.

SUBMITTER: Norton P

PROVIDER: S-EPMC5869706 | biostudies-literature | 2016 Dec

REPOSITORIES: biostudies-literature

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Development and application of a novel recombinant Aleuria aurantia lectin with enhanced core fucose binding for identification of glycoprotein biomarkers of hepatocellular carcinoma.

Norton Pamela P Comunale Mary Ann MA Herrera Harmin H Wang Mengjun M Houser Josef J Wimmerova Michaela M Romano Patrick R PR Mehta Anand A

Proteomics 20161128 24

The Aleuria aurantia lectin (AAL) derived from orange peel fungus contains five fucose-binding sites that recognizes fucose bound in α-1,2, α-1,3, α-1,4, and α-1,6 linkages to N-acetylglucosamine and galactose. Recently, we have created several recombinant AAL (rAAL) proteins that had altered binding affinity to fucose linkages. In this report, we further characterize the binding specificity of one of the mutated lectins, N224Q lectin. This lectin was characterized by lectin Western blotting, su ...[more]

PMID: 27650323

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Project description:BackgroundThe production of N-linked glycoproteins in genetically amenable bacterial hosts offers great potential for reduced cost, faster/simpler bioprocesses, greater customisation, and utility for distributed manufacturing of glycoconjugate vaccines and glycoprotein therapeutics. Efforts to optimize production hosts have included heterologous expression of glycosylation enzymes, metabolic engineering, use of alternative secretion pathways, and attenuation of gene expression. However, a major bottleneck to enhance glycosylation efficiency, which limits the utility of the other improvements, is the impact of target protein sequon accessibility during glycosylation.ResultsHere, we explore a series of genetic and process engineering strategies to increase recombinant N-linked glycosylation, mediated by the Campylobacter-derived PglB oligosaccharyltransferase in Escherichia coli. Strategies include increasing membrane residency time of the target protein by modifying the cleavage site of its secretion signal, and modulating protein folding in the periplasm by use of oxygen limitation or strains with compromised oxidoreductase or disulphide-bond isomerase activity. These approaches achieve up to twofold improvement in glycosylation efficiency. Furthermore, we also demonstrate that supplementation with the chemical oxidant cystine enhances the titre of glycoprotein in an oxidoreductase knockout strain by improving total protein production and cell fitness, while at the same time maintaining higher levels of glycosylation efficiency.ConclusionsIn this study, we demonstrate that improved protein glycosylation in the heterologous host could be achieved by mimicking the coordination between protein translocation, folding and glycosylation observed in native host such as Campylobacter jejuni and mammalian cells. Furthermore, it provides insight into strain engineering and bioprocess strategies, to improve glycoprotein yield and titre, and to avoid physiological burden of unfolded protein stress upon cell growth. The process and genetic strategies identified herein will inform further optimisation and scale-up of heterologous recombinant N-glycoprotein production.

Dataset Information

Development and application of a novel recombinant Aleuria aurantia lectin with enhanced core fucose binding for identification of glycoprotein biomarkers of hepatocellular carcinoma.

Publications

Development and application of a novel recombinant Aleuria aurantia lectin with enhanced core fucose binding for identification of glycoprotein biomarkers of hepatocellular carcinoma.

Similar Datasets

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