Glycosaminoglycans (GAGs) are complex acidic polysaccharides that are located at the interface between virtually every eukaryotic cell and its extracellular matrix (ECM). Numerous studies in the recent years have identified roles for GAGs fundamental biological processes. GAGs are far more chemically diverse than DNA and proteins and this diversity together with the physiological context of GAGs facilitates their interactions with numerous proteins and signaling molecules. An emerging paradigm from these developments is that specific binding between GAGs and these proteins modulate the activity of the proteins and thus their biological functions. The important role of GAGs in numerous biological processes necessitates a detailed understanding of GAG structure-function relationships. Such an understanding would provide new insights into fundamental biological processes. However, decoding GAG structure function relationships has been traditionally complicated by the chemical complexity, heterogeneity and polydispersity of GAGs. Over the years, significant advances have been made by our group and others in developing tools and technologies for accurate characterization of fine chemical structure of GAGs which have motivated several studies investigating GAG structure-function relationships in important biological processes. The current grant cycle marked a transformation in our ability to sequence isolated GAG oligosaccharides to characterizing complex mixtures of GAG oligosaccharides with varying chain length and composition, which is how GAGs are naturally present either as therapeutic modalities (i.e. heparin) or at the cell surface. A serious global health crisis associated with the administration of heparin in the clinic emerged in early March 2008. Adverse allergy-type reactions were observed in patients who were administered specific batches (or lots) of heparin- a GAG mixture that is widely used as an anticoagulant for a variety of clinical indications. Through our understanding of heparin as a complex GAG mixture, we employed a series of orthogonal tools developed as a part of this grant cycle to identify an unnatural GAG contaminant in the lots of heparin that led to the adverse side effects. Our participation in helping solve this crisis reaffirmed our strategy to deal with GAGs as complex mixtures. Additionally, it laid the foundation for defining the future of our GAG research strategy. It is becoming clear to us that we need to stay in pursuit of developing additional enzymatic and analytical tools for a comprehensive characterization of GAG mixtures. More importantly, this experience demonstrated the need to deploy these tools with the goal of building stringent structural characterization filters that facilitate rapid identification of additional impurities or contaminants in GAG-based therapeutics such as heparins, should they be present. An integral part of our strategy for comprehensive structural characterization of GAG mixtures involves expansion of the informatics platform for GAGs that we have built as a part of this grant cycle. Based on the above motivation and rationale, we seek to develop enzymatic, analytical and informatics tools with the primary goal of developing robust benchmarks for characterization of GAG-based therapeutics such as heparins and low-molecular weight heparins.
In the past three grant cycles, we have made substantial progress in understanding the structurefunction relationships of glycosaminoglycans (GAGs) in fundamental biological processes, specifically, in anticoagulation and cancer. The recent global health crisis associated with administration of heparin (which is a type of GAG) has reiterated the need for additional tools for a comprehensive structural characterization of such complex GAG-based therapeutic agents. In this proposal, we seek to build on our efforts from the previous cycles to develop enzymatic, analytical and informatics tools with emphasis on ?benchmarking? the characterization of GAG-based therapeutic agents such as heparins.
|Tharakaraman, Kannan; Watanabe, Satoru; Chan, Kuan Rong et al. (2018) Rational Engineering and Characterization of an mAb that Neutralizes Zika Virus by Targeting a Mutationally Constrained Quaternary Epitope. Cell Host Microbe 23:618-627.e6|
|Quinlan, Devin S; Raman, Rahul; Tharakaraman, Kannan et al. (2017) An inter-residue network model to identify mutational-constrained regions on the Ebola coat glycoprotein. Sci Rep 7:45886|
|Raguram, Aditya; Sasisekharan, V; Sasisekharan, Ram (2017) AChiralPentagonalPolyhedralFramework forCharacterizingVirusCapsidStructures. Trends Microbiol 25:438-446|
|Ramakrishnan, Boopathy; Viswanathan, Karthik; Tharakaraman, Kannan et al. (2016) A Structural and Mathematical Modeling Analysis of the Likelihood of Antibody-Dependent Enhancement in Influenza. Trends Microbiol 24:933-943|
|Macchi, Eleonora; Rudd, Timothy R; Raman, Rahul et al. (2016) Nuclear Magnetic Resonance and Molecular Dynamics Simulation of the Interaction between Recognition Protein H7 of the Novel Influenza Virus H7N9 and Glycan Cell Surface Receptors. Biochemistry 55:6605-6616|
|Raman, Rahul; Tharakaraman, Kannan; Sasisekharan, V et al. (2016) Glycan-protein interactions in viral pathogenesis. Curr Opin Struct Biol 40:153-162|
|Shriver, Zachary; Sasisekharan, Ram (2015) Capillary electrophoretic analysis of isolated sulfated polysaccharides to characterize pharmaceutical products. Methods Mol Biol 1229:161-71|
|Zhu, Xueyong; Viswanathan, Karthik; Raman, Rahul et al. (2015) Structural Basis for a Switch in Receptor Binding Specificity of Two H5N1 Hemagglutinin Mutants. Cell Rep 13:1683-91|
|Robinson, Luke N; Tharakaraman, Kannan; Rowley, Kirk J et al. (2015) Structure-Guided Design of an Anti-dengue Antibody Directed to a Non-immunodominant Epitope. Cell 162:493-504|
|Tharakaraman, Kannan; Subramanian, Vidya; Viswanathan, Karthik et al. (2015) A broadly neutralizing human monoclonal antibody is effective against H7N9. Proc Natl Acad Sci U S A 112:10890-5|
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