Identification of the Peptide-binding Site in the Heat Shock Chaperone/Tumor Rejection Antigen gp96 (Grp94)

Abstract

Heat shock protein (HSP)-peptide complexes from tumor cells elicit specific protective immunity when injected into inbred mice bearing the same specific type of tumor. The HSP-mediated specific immunogenicity also occurs with virus-infected cells. The immune response is solely due to endogenous peptides noncovalently bound to HSP. A vesicular stomatitis virus capsid-derived peptide ligand bearing a photoreactive azido group was specifically bound by and cross-linked to murine HSP glycoprotein (gp) 96. The peptide-binding site was mapped by specific proteolysis of the cross-links followed by analysis of the cross-linked peptides using a judicious combination of SDS-gel electrophoresis, mass spectrometry, and amino acid sequencing. The minimal peptide-binding site was mapped to amino acid residues 624 -630 in a highly conserved region of gp96. A model of the peptide binding pocket of gp96 was constructed based on the known crystallographic structure of major histocompatibility complex class I molecule bound to a similar peptide. The gp96-peptide model predicts that the peptide ligand is held in a groove formed by ␣-helices and lies on a surface consisting of antiparallel ␤-sheets. Interestingly, in this model, the peptide binding pocket abuts the dimerization domain of gp96, which may have implications for the extraordinary stability of peptide-gp96 complexes, and for the faithful relay of peptides to major histocompatibility complex class I molecule for antigen presentation.Specific protective immunity results when heat shock protein (HSP) 1 -peptide complexes purified from tumor cells are injected into inbred mice bearing the same specific type of tumor (1-6). The HSP-mediated specific immunogenicity is also seen with virus-infected cells (7-9). This paradigm is the basis for a new therapeutic strategy against human cancers (9, 10). The immune response is directed against peptides noncovalently bound to the HSP and not to the HSP (for reviews, see Refs. 11 and 12)). HSPs that form the immunogenic peptide complexes include gp96 (GRP94) (13) and calreticulin (14), which reside in the endoplasmic reticulum (ER). Cytosolic HSP70-(15, 16) and HSP90-(17) peptide complexes are also immunogenic. The ER-resident chaperone gp96 (GRP94) has been the most extensively studied from an immunological standpoint (13, 18 -21). It is an abundant stress protein that displays dual functionality: it directs peptides into the immune response pathway and it assists in protein folding. The role of gp96 in the immune response is not well understood at the molecular level. gp96 binds a variety of peptides in vitro and in vivo with little or no apparent amino acid sequence specificity (13,(22)(23)(24). gp96 also binds ATP but the role of nucleotide in peptide loading/unloading is unclear (22,25). Highly purified HSP90, the cytosolic paralog of gp96, probably binds and uses ATP (26). This is in contrast to chaperones HSP70 and BiP (ER paralog of HSP70), where it is clear that ATP binding is important for the release of peptide substrates (r...

Generation of Murine CTL by a Hepatitis B Virus-Specific Peptide and Evaluation of the Adjuvant Effect of Heat Shock Protein Glycoprotein 96 and Its Terminal Fragments

“…It was reported that both the N-and C-terminal fragments of gp96 are able to bind peptides (22,23), with the N-terminal fragment behaving at a similar capacity as the full-length gp96 (19). However, whether the terminal fragments can serve as an adjuvant as effectively as the full-length gp96 remains to be determined.…”

“…It was reported that gp96 might contain a peptidebinding element within its N-and C-terminal domains (22,23), but whether the peptide binding domains can generate immune effects is not known. To address this issue, two terminal fragments of gp96, N333 (22-355 aa) and C190 (561-751 aa), were cloned into pGEX-6p1 expression vector, expressed in bacteria, and purified on a gel filtration column after removing GST as described in Materials and Methods (Fig.…”

Radicicol-sensitive Peptide Binding to the N-terminal Portion of GRP94

“…Both the putative N-terminal domain and the acidic domain are required for binding of nucleotides and inhibitors to GRP94 (15). A peptide-binding site was defined within the C-terminal domain of GRP94 by cross-linking pyrene-modified VSV8 (16,17), an octapeptide known to be presented via GRP94 to T cells (3). This site, centered around amino acids 624 -630, was modeled as a shallow surface groove formed by ␣ helices, resembling the peptide-binding site of a major histocompatibility locus protein (16).…”

“…A peptide-binding site was defined within the C-terminal domain of GRP94 by cross-linking pyrene-modified VSV8 (16,17), an octapeptide known to be presented via GRP94 to T cells (3). This site, centered around amino acids 624 -630, was modeled as a shallow surface groove formed by ␣ helices, resembling the peptide-binding site of a major histocompatibility locus protein (16). Site-directed mutations showed that VSV8 binding to this site is dependent on aromatic side chains for binding affinity (17).…”

Glycoprotein Folding in the Endoplasmic Reticulum

“…Those of us who know the protein as gp96 will be more familiar with its high-affinity peptide binding properties (Li and Srivastava, 1993) and its potential role as an accessory molecule in loading antigens onto MHC class I molecules in the ER. Gp96 has a peptide binding site located in a conserved region of the protein between amino acids 624 and 630 (Linderoth et al, 2000). Studies have now shown that cellular peptide-gp96 complexes, probably derived from dying, infected cells, are taken up by antigen-presenting cells.…”