Proteoglycans (PGs) are ubiquitous on mammalian cell surfaces and in the extracellular matrix. PGs are made up of heparan sulfate or chondroitin sulfate glycosaminoglycan chains covalently attached to the core protein through tetrasaccharide linkers. PGs play important roles in many biological processes. However, due to the heterogeneity of naturally existing PGs, it is extremely challenging to purify well-defined structures to study the structure and activity relationship. Traditionally, the biological functions of PGs are believed to be dictated by the glycosaminoglycan chains attached. Evidence is emerging that the core protein may significantly impact the glycan activities. During the last grant period, homogeneous heparan sulfate and chondroitin sulfate glycopeptides have been synthesized, providing access to these complex molecules for the first time. However, the synthesis required total over 100 chemical steps to complete a glycopeptide with a heparan sulfate chain, which limited the synthetic output. Furthermore, with the current synthetic strategy, some of the common structural features in PGs are not accessible. To overcome these challenges, in this renewal application, the power of the biosynthetic enzymes will be harnessed to greatly expand the capability for PG synthesis.
In aim 1, the key enzymes will be produced to generate the tetrasaccharide linkage region with the core peptide, and to extend the glycan chain. Automated synthesis strategy will be developed to further expedite the synthesis.
In aim 2, ligation strategies will be established to synthesize glyco poly- peptides with multiple glycan chains. This will provide a powerful tool to extend the poly-peptide backbone, and to probe the hypothesis that the core protein can modulate glycan activities.
In aim 3, synthesis of a chondroitin sulfate proteoglycan bikunin like glycoprotein will be completed. Bikunin is an approved drug to treat the potentially life-threatening sepsis conditions, and its mechanism is currently unclear. Aided by the structurally well-defined synthetic bikunin, the molecular mechanisms of the anti-inflammatory activities of bikunin will be established, demonstrating the power of synthesis in expanding the understanding of the interesting biological properties of these complex molecules.
Proteoglycans important roles in many biological processes such as inflammation, cell proliferation and cancer development. In this project, new methods will be developed to greatly expedite the synthesis of proteoglycan like molecules bearing homogeneous carbohydrate chains. The availability of these complex molecules can significantly aid in the better understanding of their fascinating biological functions, and provide potential targets for the development of novel therapeutics.
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