Characterization ofhsp110 andgrp170 interactions with scavenger receptors
Facciponte, John G.
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Hsp110 is a cytosolic chaperone protein and grp170 is a related endoplasmic reticulum chaperone protein. Both can induce immune responses against tumors when complexed to tumor antigens and injected into mice. Mechanisms by which hsp110 and grp170 elicit immune responses is still evasive. However, antigen presenting cells (APCs) and their surface receptors are implicated in playing a role for hsp70 and gp96. This dissertation studies various aspects of hsp110 and grp170 interactions with APCs. The first part of the thesis investigates the interactions of hsp110 and grp170 with receptors on APCs. We fluorochrome-labeled recombinant hsp110 and grp170 along with hsp70, and then performed binding experiments using APCs. Binding experiments with hsp110 and RAW264.7 macrophages by flow cytometry revealed saturation, self-competition and specificity, suggesting receptor-ligand interactions. We hypothesize that hsp110 and grp170, part of the "hsp70 superfamily", bind to various scavenger receptors. We initially tested Scavenger Receptor-A (SR-A) transfected CHO cells. Hsp110 and grp170 bind specifically to SR-A expressing CHO cells, suggesting that SR-A is a receptor for these chaperones. Thioglycolate-elicited macrophages from SR-A knockout mice show reduced hsp110 binding compared to wild-type macrophages. Importantly, hsp110 binding to these SR-A deficient macrophages can be inhibited by agents such as fucoidin and poly I, which are known to bind to several scavenger receptors. We then examined Scavenger Receptor expressed on Endothelial Cells (SREC). CHO cells were stably transfected with SREC, and hsp110 and grp170 were found to bind saturably with scavenger receptor specificity. Hsp110 binding to thioglycolate-elicited macrophages can be partially inhibited with SREC antibodies. ELISPOT in vitro assays show that hsp110 coupled to intracellular domain (ICD) of HER2/ neu can generate immune responses to ICD antigen and the scavenger receptor family can play a role in crosspriming the ICD antigen. Hsp110 was also found to saturably bind to scavenger receptor LOX-1, but not to CLA-1, suggesting HSP-scavenger receptor selectivity. The second part of the thesis addresses how hsp110 and grp170 bind to several structurally diverse scavenger receptors. Domain deletion mutants of various HSPs were generated, purified and fluorochrome-labeled. Domain deletion mutants that can chaperone antigen can also bind to RAW264.7 macrophages. Here, we define the chaperoning function of HSPs as the ability to bind and stabilize partially denatured substrate proteins. We hypothesize that chaperoning function can contribute to scavenger receptor binding. Grp170 α-helical mutant can chaperone substrates, while hsp110 and hsp70 α-helical mutants do not chaperone. We show here that the grp170 α-helical mutant can bind to SR-A and SREC expressing CHO cells in a saturable scavenger receptor-specific manner, while the hsp70 and hsp110 mutants cannot saturably bind. The last section of this thesis examines the role that lipid rafts play in hsp110 binding to thioglycolate-elicited macrophages. We found that cholesterol depletion can partially inhibit hsp110 binding, suggesting that lipid rafts play a role in hsp110 binding. Sequestration of cholesterol with β-cyclodextrin inhibited hsp110 binding on SREC expressing CHO cells, suggesting that SREC is associated with lipid rafts. Data suggests that lipid rafts play a role in SREC stability on the plasma membrane of thioglycolate-elicited macrophages. In contrast, SR-A was characterized biochemically to be excluded from lipid rafts on various APCs or CHO expressing SR-A. Cumulatively, these results indicate that hsp110 and grp170 bind to multiple scavenger receptors on antigen presenting cells and to SR-A and SREC in particular. Chaperoning activity of hsp110 and grp170 may contribute to scavenger receptor binding. These results are immunologically relevant because this information can be used to target and regulate scavenger receptors for optimal antigen presentation and activation of the adaptive immune response. Uncovering how these chaperones function immunologically is important to basic cellular immunology and can be applied in the future to improve HSP-based clinical trials.