Chemoenzymatic synthesis of heparan sulfates, and investigations of their role in cell signaling, human health and disease
Authors
Suflita, Matthew
ORCID
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Other Contributors
Linhardt, Robert J.
Ligon, Lee
Koffas, Mattheos A. G.
Dordick, Jonathan
Ligon, Lee
Koffas, Mattheos A. G.
Dordick, Jonathan
Issue Date
2018-05
Keywords
Biology
Degree
PhD
Terms of Use
Attribution-NonCommercial-NoDerivs 3.0 United States
This electronic version is a licensed copy owned by Rensselaer Polytechnic Institute, Troy, NY. Copyright of original work retained by author.
This electronic version is a licensed copy owned by Rensselaer Polytechnic Institute, Troy, NY. Copyright of original work retained by author.
Full Citation
Abstract
Heparan sulfate and heparin are linear polysaccharides, called glycosaminoglycans, which possess many important biological and pharmacological activities. While heparin has historically received most of the scientific attention for its anticoagulant activity, interest has steadily grown in the multi-faceted role heparan sulfate plays in normal and pathophysiology. Cell surface heparan sulfate binds signaling proteins such as fibroblast growth factors and promotes the formation of signaling complexes. In endothelial cells, heparan sulfate in the glycocalyx forms a physical barrier critical for endothelial function, and its degradation in response to direct injury or septic shock leads to tissue damaging inflammation.
We next sought to determine if urinary indices of GAG fragmentation are associated with outcomes in patients with critical illnesses such as septic shock or acute respiratory distress syndrome. Indices of GAG fragmentation correlated with both the development of renal dysfunction over the 72 hours after urine collection and with hospital mortality. This association remained after controlling for severity of illness and was similarly observed using the inexpensive dimethylmethylene blue assay. These predictive findings were corroborated using urine samples previously collected at three consecutive time points from patients with acute respiratory distress syndrome.
The last chapter details two in vivo studies of the role of heparan sulfate in disease. We first examined the reconstitution of heparan sulfate in the endothelial glycocalyx following sepsis-induced degradation. Homeostatic pulmonary endothelial glycocalyx reconstitution occurred rapidly after non-septic degradation and was associated with induction of the heparan sulfate biosynthetic enzyme exostosin-1. In contrast, sepsis was characterized by loss of pulmonary exostosin-1 expression and delayed glycocalyx reconstitution. Rapid glycocalyx recovery after non-septic degradation was dependent upon induction of fibroblast growth factor receptor 1 expression and was augmented by fibroblast growth factor-promoting effects of circulating heparan sulfate fragments released during glycocalyx degradation. While sepsis-released heparan sulfate fragments maintained this ability to activate fibroblast growth factor receptor 1, sepsis was associated with the downstream absence of reparative pulmonary endothelial fibroblast growth factor receptor 1 induction.
Heparin is a critically important drug, with an accordingly high demand. The heparin contamination crisis of 2008 caused over 200 deaths worldwide, and illustrated the problems associated with animal sourced heparin. Chemical synthesis of glycosaminoglycans is largely precluded by their structural complexity, which led our lab to develop methods for the chemoenzymatic synthesis of heparan sulfates toward a bioengineered heparin replacement for current animal source heparin.
This thesis will explore methods for chemoenzymatic synthesis of heparan sulfates, and their role in fibroblast growth factor signaling and sepsis pathology. We first present the scalable production of recombinant heparin biosynthetic enzymes used in chemoenzymatic synthesis of heparan sulfates. Next, we demonstrate the successful application of fluorous-tagged sugars in an iterative chemoenzymatic synthesis of heparan sulfate oligosaccharides, and a one-pot chemoenzymatic synthesis of anticoagulant heparin. We then applied chemoenzymatic methods to synthesize a library of structurally defined heparan sulfate oligosaccharides with domain structure, which were used in combination with a cell-based signaling assay to investigate the structure-activity relationship of heparan sulfate domains in fibroblast growth factor signaling in vitro. We found clear differences in signaling requirements for fibroblast growth factors 1 and 2, which suggests the possibility of defined heparan sulfate therapeutics targeted to specific fibroblast growth factor signaling pathways.
We next sought to determine if urinary indices of GAG fragmentation are associated with outcomes in patients with critical illnesses such as septic shock or acute respiratory distress syndrome. Indices of GAG fragmentation correlated with both the development of renal dysfunction over the 72 hours after urine collection and with hospital mortality. This association remained after controlling for severity of illness and was similarly observed using the inexpensive dimethylmethylene blue assay. These predictive findings were corroborated using urine samples previously collected at three consecutive time points from patients with acute respiratory distress syndrome.
The last chapter details two in vivo studies of the role of heparan sulfate in disease. We first examined the reconstitution of heparan sulfate in the endothelial glycocalyx following sepsis-induced degradation. Homeostatic pulmonary endothelial glycocalyx reconstitution occurred rapidly after non-septic degradation and was associated with induction of the heparan sulfate biosynthetic enzyme exostosin-1. In contrast, sepsis was characterized by loss of pulmonary exostosin-1 expression and delayed glycocalyx reconstitution. Rapid glycocalyx recovery after non-septic degradation was dependent upon induction of fibroblast growth factor receptor 1 expression and was augmented by fibroblast growth factor-promoting effects of circulating heparan sulfate fragments released during glycocalyx degradation. While sepsis-released heparan sulfate fragments maintained this ability to activate fibroblast growth factor receptor 1, sepsis was associated with the downstream absence of reparative pulmonary endothelial fibroblast growth factor receptor 1 induction.
Heparin is a critically important drug, with an accordingly high demand. The heparin contamination crisis of 2008 caused over 200 deaths worldwide, and illustrated the problems associated with animal sourced heparin. Chemical synthesis of glycosaminoglycans is largely precluded by their structural complexity, which led our lab to develop methods for the chemoenzymatic synthesis of heparan sulfates toward a bioengineered heparin replacement for current animal source heparin.
This thesis will explore methods for chemoenzymatic synthesis of heparan sulfates, and their role in fibroblast growth factor signaling and sepsis pathology. We first present the scalable production of recombinant heparin biosynthetic enzymes used in chemoenzymatic synthesis of heparan sulfates. Next, we demonstrate the successful application of fluorous-tagged sugars in an iterative chemoenzymatic synthesis of heparan sulfate oligosaccharides, and a one-pot chemoenzymatic synthesis of anticoagulant heparin. We then applied chemoenzymatic methods to synthesize a library of structurally defined heparan sulfate oligosaccharides with domain structure, which were used in combination with a cell-based signaling assay to investigate the structure-activity relationship of heparan sulfate domains in fibroblast growth factor signaling in vitro. We found clear differences in signaling requirements for fibroblast growth factors 1 and 2, which suggests the possibility of defined heparan sulfate therapeutics targeted to specific fibroblast growth factor signaling pathways.
Description
May 2018
School of Science
School of Science
Department
Dept. of Biological Sciences
Publisher
Rensselaer Polytechnic Institute, Troy, NY
Relationships
Rensselaer Theses and Dissertations Online Collection
Access
CC BY-NC-ND. Users may download and share copies with attribution in accordance with a Creative Commons Attribution-Noncommercial-No Derivative Works 3.0 License. No commercial use or derivatives are permitted without the explicit approval of the author.