Mechanisms of Molecular Chaperone Surface Binding and Endocytosis: Insights into the Molecular Basis for GRP94 Immune Function
Extracellular GRP94 can elicit both innate and adaptive immune responses by interacting with endocytic and signaling receptors on professional antigen presenting cells (pAPCs). CD91 was the first receptor proposed to facilitate GRP94-mediated immune responses. Using a GRP94 affinity matrix, a CD91 fragment was isolated from the detergent-solubilized membranes of a pAPC cell line. It was then demonstrated that CD91 ligands could inhibit GRP94-mediated peptide cross-presentation, suggesting that CD91 played a critical role in this process. While these studies implied that CD91 could function as a GRP94 endocytic receptor, later works suggested that CD91 may not recognize GRP94 at the cell surface. These opposing observations have lead to a significant controversy surrounding the identity of CD91 as an endocytic receptor for GRP94. Because the ability of CD91 to directly mediate GRP94 surface binding and uptake has not been established, the studies included in this dissertation have focused on evaluating the ability of CD91 to facilitate three processes that are necessary for GRP94-mediated peptide cross-presentation: surface binding, internalization, and processing.
These studies utilized a recombinantly-expressed N-terminal domain of GRP94 (GRP94.NTD), which was previously shown to have nearly identical biological activity to full length GRP94. The ability of CD91 to directly bind and internalize GRP94.NTD was examined using murine embryonic fibroblast (MEF) cell lines whose expression of CD91 was either reduced via siRNA, or eliminated by genetic disruption of the CD91 locus. Binding competition experiments were also conducted. Together, these studies reveal that CD91 does not directly interact with GRP94 at the cell surface. The ability of CD91 to directly facilitate GRP94 internalization was examined using various internalization and internalization competition assays. These studies demonstrated that GRP94.NTD and the CD91 ligand RAP were internalized through spatially and kinetically distinct pathways, that CD91 was not necessary for GRP94.NTD internalization, and that RAP did not inhibit GRP94 endocytosis. Together, these studies strongly suggest that CD91 does not directly facilitate GRP94 internalization. When these studies were extended to DC2.4 mouse dendritic cells, the CD91 ligand RAP reduced GRP94.NTD internalization/process by ~15%. This suggests that CD91 may indirectly facilitate GRP94 internalization in pAPC cell lines. Lastly, cross-presentation studies were utilized to examine the ability of various CD91 ligands to influence GRP94.NTD-mediated peptide cross-presentation through a post-uptake mechanism using the DC2.4/OT-1 system. Although it was discovered that DC2.4 cells can internalize and process GRP94.NTD/peptide complexes through fluid-phase endocytosis, CD91 ligands did not significantly inhibit GRP94-mediated peptide cross-presentation by DC2.4 cells. These studies demonstrate that CD91 does not play a primary role in GRP94-mediated peptide cross-presentation.
In the course of these studies, cell surface heparan sulfate proteoglycans (HSPGs) were identified as novel cell surface binding sites for GRP94.NTD on MEF cells. This conclusion was established using three distinct experimental approaches. GRP94.NTD surface binding was significantly decreased following heparin pre-treatment, following incubation with the sulfation inhibitor sodium chlorate, and following digestion with extracellular heparinase II. Conversely, these treatments did not significantly influence GRP94.NTD binding to RAW264.7 mouse macrophage-like cells. This suggested that GRP94.NTD-HSPG cell surface interactions may require the expression of a specific type of cell surface HSPG that is not expressed by RAW264.7 cells. However, additional studies strongly suggested that GRP94.NTD-HSPG cell surface interactions were mediated by the heparan sulfate-containing side chains rather than the presence of a specific cell surface HSPG core protein.
This dissertation focuses on the critical re-examination of CD91 functions in GRP94 surface binding, uptake, and cross-presentation. Together, these results clarify conflicting data on CD91 function in GRP94 surface binding and endocytosis. This dissertation also describes the identification of cell surface HSPGs as GRP94 binding sites on MEF cells. These studies extend the diversity of surface receptors that recognize of GRP94, and suggest that cell surface HSPG-dependent interactions may contribute to the biology of GRP94-elicited immune responses.
Health Sciences, Immunology
cell surface heparan sulfate proteoglycan
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