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Mar 15, 2014 (Vol. 34, No. 6)

Cultivating the Glycome to Improve Therapeutics

  • Information-packed and bearing extraordinary diversity, glycans decorate more than 50% of mammalian secreted and cell surface proteins.

    This fine architecture also presents challenges for their appropriate addition or reconstruction in engineered glycoproteins.

    The recent meeting of the American Institute of Chemical Engineers in San Francisco featured presentations describing new advances in deciphering glycosylation reaction networks, optimizing biosimilar expression in Chinese hamster ovary (CHO) cells, and bioengineering plants and bacteria to serve as viable alternatives to traditional insect and mammalian expression systems.

    Glycosylated proteins play critical structural and functional roles in a broad range of biological processes. These processes include protein folding, cell development, immunity, pathogen invasion, and cancer metastasis.

    Glycosylation also regulates the pharmacokinetics and pharmacodynamics of bio-therapeutics. New developments in analytical tools, especially mass spectrometry, are generating vast amounts of data and deriving whole glycomes from cells and tissue.

  • Glycomics and Computer Modeling

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    At the State University of New York at Buffalo, glycosylation network analysis starts with the collection of glycomics, enzyme, and pathway data. This information is subsequently processed via network inference algorithms and quantitative modeling methods to reconstruct cellular glycosylation reaction networks.

    At the State University of New York at Buffalo, scientists in the department of chemical and biological engineering have been developing computer algorithms for the study of these glycosylation processes. These scientists include Sriram Neelamegham, Ph.D., professor, and Gang Liu, Ph.D., research assistant professor.

    Dr. Liu explained that while “the glycomes of many cells and tissues are now available in public databases, and such data are also being collected by independent investigators,” data analysis remains a challenge. “The quantitative determination of the underlying biosynthetic pathways that drive cell/tissue-specific glycomes is not possible without computer models. In particular, since glycomics experiments do not account for glycosyltransferase enzyme specificity and concentration data, it is difficult to derive a direct linkage between cell/tissue-specific enzyme activity and corresponding phenotype.”

    To address this problem, the scientists developed an application package called GNAT (Glycosylation Network Analysis Toolbox). This program, which is freely available online, provides a streamlined approach for the construction, visualization, and simulation of glycosylation reaction networks.

    Development of the application, explained Dr. Liu, involved three tasks: “First, we gathered glycan structure data from mass spectrometry experiments and enzyme specificity data using the IUBMB (International Union of Biochemistry and Molecular Biology), BRENDA (Comprehensive Enzyme Information System), and KEGG (Kyoto Encyclopedia of Genes and Genomes) databases. Second, we implemented a network construction algorithm by combining glycan structure and species-specific enzyme data. This allowed the synthesis of complete glycosylation reaction networks. Third, we developed ways to refine the model using the open-source toolbox.”

    The team tested their models on two cell lines. “We assessed N-glycosylation in Chinese hamster ovary cells and its mutants,” recalled Dr. Liu. “We also assessed O-glycosylation networks in a human promyelocytic leukemia cell line, HL-60.”

    “In both of these experiments, the toolbox was able to extract core pathways involved during glycosylation. Thus, it is possible to obtain greater insight from high-throughput mass spectrometry data using quantitative modeling. Such knowledge can aid the engineering of specific glycoforms on therapeutic proteins, and it can also enhance biomedical investigation of human diseases.”

  • Plant-Based Systems

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    In vitro glycosylation strategy for plant-made glycoproteins [University of California/Davis]

    Expression of appropriately glycosylated proteins in mammalian cells can be a challenging and expensive task. A less expensive alternative is the use of genetically manipulated plant-based systems. But can plant cells, with their different glycosylation machinery, decorate mammalian proteins properly?

    A team of researchers at the University of California, Davis, are tackling this complex problem. Led by Raymond Rodriguez, Ph.D., a professor of molecular and cellular biology, and Karen A. McDonald, Ph.D., a professor of chemical engineering and materials science, the Davis team is carrying out an interdisciplinary program—combining plant science, chemistry, chemical engineering, and molecular biology—to achieve its goal of in vitro, post-production sialylation of an important human enzyme.

    “We performed collaborative studies in which plant cells are used to express recombinant human butyrylcholinesterase (rhBChE), a serum-based bioscavenger for neurotoxic organophosphates (OP) like the deadly compound sarin,” said Dr. McDonald. “Current therapies are designed to elevate serum levels of BChE, but a single dose can cost as much as $10,000. We performed studies to express rhBChE in plants utilizing a novel means to create appropriate modifications in post-production.”

    The Davis team utilized viral amplicon-based gene expression systems that compared rhBChE production via Tobacco mosaic virus versus Cucumber mosaic virus in Nicotiana benthamiana, a relative of tobacco and a commonly used model organism in plant research.

    According to Dr. Rodriguez, “Development of rhBChE is a pressing national security issue. The U.S. government is seeking economical ways to mass produce this enzyme. However, BChE is a real challenge as it is a tetrameric protein with each monomer housing nine potential N-glycosylation sites. We targeted the protein to different subcellular compartments and found important differences in how the protein was glycosylated.”

    Another novel aspect of the studies dealt with in vitro sialylation, the final “polishing” step for N-glycans. “Sialylation of glycoproteins is an end-stage modification that is critical for many processes such as maintaining stability, protein half-life, and immunogenic properties,” commented Xi Chen, Ph.D., professor of chemistry. “Since plants are incapable of sialylating glycoproteins, we employed post-production multistep enzymatic reactions to systematically add sialic acid to the termini of the N-glycans.”

    Dr. McDonald noted that although the Davis team is in the early stages of bioengineering plant-based systems, the results are already encouraging. “We will continue to optimize these systems and also incorporate computational modeling of glycoproteins. Given that more than 30% of all commercial biopharmaceuticals are glycoproteins, our results suggest plant-based systems are viable alternatives to standard mammalian and insect expression systems.”

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