Carbohydrates are ubiquitous in nature and play an essential role in a variety of important biological processes. In contrast to the in-depth understanding of the molecular mechanisms of nucleic acid and protein biosynthesis, polysaccharide biosynthesis is poorly understood. In spite of the remarkable structural diversity of polysaccharides, only three major elongation/polymerization mechanisms, namely wzy- dependent, ABC-transporter dependent and synthase-dependent, are utilized based on previous extensive genetic studies. Among them, the wzy-dependent pathway is the most widely used in nature. It has been implicated in the biosynthesis of a variety of hetero-polysaccharides such as O-polysaccharides, capsular polysaccharides, exo- polysaccharides, colonic acids and enterobacterial common antigens. The current working model for the wzy-dependent pathway is that the individual repeating oligosaccharide unit is synthesized in the cytoplasm by the sequential action of specific glycosyltransferases. The repeating unit is then transported to the periplasmic side of the membrane where it is polymerized into a polysaccharide by the polymerase Wzy. The chain length of the polymer is regulated by the Wzz protein. So far, with the exception of gene knockout experiments, this rough biosynthetic picture has never been experimentally tested in vitro. Most of the critical enzymes in the hypothesized pathway have never been biochemically characterized and their mechanisms of action (especially for the O-polysaccharide polymerization, regulation and transport) are rather speculative. In the past three years, using E. coli O86 O-polysaccharide as a model system, we have made significant progress towards understanding polysaccharide biosynthesis. In this proposed research period, we plan to focus on the following three specific aims:
Aim 1. Chemo-enzymatic synthesis of O-unit-PP-lipid substrates/analogs. These chemically defined substrates will be used for probing Wzy polymerization reaction mechanism.
Aim 2. Investigation of polymerase Wzy. This section includes development of in vitro assays to obtain reliable kinetics and investigation of lipid specificity and chain elongation direction.
Aim 3. Investigation of Wzz and probing chain length regulation. This section includes the investigation of Wzy-Wzz interaction, Wzz-polysaccharide interaction and Wzz oligomeric states using a series of biochemical and biophysical approaches. In summary, this long-term research program will uncover the detailed biosynthetic mechanism of complex carbohydrate polymers. An in-depth understanding of biosynthesis of polysaccharides will have a considerable impact on research and development of novel antibiotics, vaccines, immuno-suppressors, and anti-inflammatory agents.

Public Health Relevance

Many pathogenic bacteria species are surrounded by a capsular structure that is assembled from structurally diverse, high molecular weight polysaccharides. These cell surface polysaccharides play an essential role in mediating interactions between bacterial cells and their environment, and are recognized as an important pathogenic factor. Thus understanding how the polysaccharides are biosynthesized in the cell in molecular level is a fundamentally important biological question with significant medical implications. For example, it can provide new targets for the development of new antibiotics against emerging drug resistance problems;new methods to detect new pathogenic bacteria;new strategies to generate polysaccharide-based vaccines, immuno-suppressors and anti-inflammatory agents.

Agency
National Institute of Health (NIH)
Institute
National Institute of General Medical Sciences (NIGMS)
Type
Research Project (R01)
Project #
7R01GM085267-02
Application #
7906823
Study Section
Synthetic and Biological Chemistry A Study Section (SBCA)
Program Officer
Jones, Warren
Project Start
2009-08-03
Project End
2011-05-31
Budget Start
2010-08-01
Budget End
2011-05-31
Support Year
2
Fiscal Year
2010
Total Cost
$309,971
Indirect Cost
Name
Ohio State University
Department
Biochemistry
Type
Schools of Arts and Sciences
DUNS #
832127323
City
Columbus
State
OH
Country
United States
Zip Code
43210
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Li, Shanshan; Zhu, He; Wang, Jiajia et al. (2016) Comparative analysis of Cu (I)-catalyzed alkyne-azide cycloaddition (CuAAC) and strain-promoted alkyne-azide cycloaddition (SPAAC) in O-GlcNAc proteomics. Electrophoresis 37:1431-6

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