Item – Theses Canada

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OCLC number
1032921029
Link(s) to full text
LAC copy
LAC copy
Author
Liu, Hao.
Title
Identification and Characterization of the Receptor for the Soluble Fibrinogen Like Protein 2 (FGL2).
Degree
Ph. D. -- University of Toronto, 2010
Publisher
Toronto : University of Toronto, 2010.
Description
1 online resource
Notes
Includes bibliographical references.
Abstract
The multi-functional FGL2 can be expressed as either a type II membrane-associated glycoprotein or a secreted tetrameric molecule. As an important effector of regulatory T cells, secreted FGL2 inhibits dendritic cell maturation and T cell proliferation. The mechanism of its immunomodulatory function remains unclear. The goals of this thesis are to identify receptor(s) of secreted FGL2, key biological functions and signaling pathways, and mechanism of FGL2 oligomerization. Soluble FGL2 was critical for all studies, and the production of recombinant FGL2 was compared in E. coli, insect cells and mammalian cells. Soluble and stable FGL2 was secreted only by mammalian cells, indicating the importance of post-translational modification. In flow cytometry and surface plasmon resonance assays, recombinant FcFGL2 and albumin tagged FGL2 fusion proteins bound to Fc gamma RIIB and Fc gamma RIII receptors expressed by antigen presenting cells (APCs), including lipopolysaccharide (LPS)-stimulated B lymphocytes, endothelial cells, thioglycollate-stimulated peritoneal macrophages, and bone marrow-derived dendritic cells (BM-DCs). The binding of recombinant FGL2 to Fc gamma RIIB and Fc gamma RIII was specific, dependent on receptor expression and blocked by anti-Fc gamma RIIB/III antibody. FcFGL2 inhibited the maturation of BM-DC derived from fc gamma riib wild type mice but not from fc gamma riib knock out mice. It also induced apoptosis of the A20 mouse B cell line (Fc gamma#xF067;RIIB+), but not the A20IIA1.6 cell line (Fc gamma RIIB-). The activation of caspases induced by FcFGL2 binding to A20 cells was confirmed by flow cytometry, Western blotting and analysis of DNA fragmentation. The role of Fc gamma#xF067;RIIB in FGL2-mediated immunosuppression was confirmed in vivo. Infusion of FcFGL2 into fc gamma riib+/+, but not fc gamma riib-/- C57BL/6J mice (H-2b) inhibited the rejection of fully mismatched BALB/cJ (H-2d) skin and heart allografts. Studies on the mechanism of FGL2 oligomerization employed site-directed mutagenesis and revealed that cysteines at positions 94, 97, 184, and 187 were critical. Mutation of these cysteines resulted in secretion of monomeric FGL2. Computer modeling of FGL2 tetramers predicted an asymmetric arrangement that was similar to the structure of multimeric ficolin. The data presented in this thesis provide mechanistic insights into the immunosuppressive activity of soluble FGL2, and a foundation for the development of a novel and potentially highly effective immunosuppressive therapy.
Other link(s)
hdl.handle.net
tspace.library.utoronto.ca
Subject
fibrinogen like protein 2.
immunoregulation.
0307.