This dissertation represents an effort to develop tools to study the glycosylation of a mucinous protein called P-selectin glycoprotein ligand-1 (PSGL-1). This protein is a key adhesion molecule in inflammation and immunity. During the inflammatory response, leukocyte recruitment is a critical step that maintains homeostasis but can also lead to subsequent tissue injury, sometimes associated with cardiovascular diseases. Among the many factors contributing to leukocyte recruitment, carbohydrate ligand binding to the selectin family of cell adhesion molecules (L-, E- and P-selectin) is an early step of leukocyte adhesion. Since all three selectins interact with a leukocyte cell-surface protein PSGL-1 with high affinity under fluid flow conditions and since antagonizing selectin-PSGL-1 interactions significantly reduces the rate of leukocyte adhesion in vitro and in vivo, the current work focuses on developing recombinant PSGL-1 proteins to study the O-glycan located near the N-terminus. While previous studies have investigated the glycosylation of this protein, they looked at the glycosylation on the entire PSGL-1 protein rather than a single functional site located near the N-terminus at Thr-57. Because PSGL-1 has copious amounts of glycans, deciphering the glycosylation at a single site would be challenging. The work presented here shows the development of recombinant PSGL-1 proteins to overcome this challenge. These proteins were expressed in various platforms including E. coli, CHO cells, HEK293T cells and HL-60 cells. The promoter and signal peptide were optimized for high protein production. Methodologies in mass spectrometry were developed to analyze glycopeptides. This method was used to further identify ST6GalNAc2 as a glycosyltransferase which can alter selectin-binding function and chemokine binding function of PSGL-1. Further, the same recombinant PSGL-1 protein could also be used to engineer mesenchymal stem cells to home more effectively to sites of inflammation.
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|Commitee:||Andreadis, Stelios T., Park, Sheldon J.|
|School:||State University of New York at Buffalo|
|Department:||Chemical and Biological Engineering|
|School Location:||United States -- New York|
|Source:||DAI-B 74/10(E), Dissertation Abstracts International|
|Subjects:||Cellular biology, Biomedical engineering, Chemical engineering|
|Keywords:||Adhesion molecules, Cell homing, Glycosylation, Glycosyltransferase, Psgl-1, Selectins, Stem cells|
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