The 170 kDa glucose-regulated protein (grp170) is an endoplasmic reticulum resident protein that shares some sequence homology with both the hsp70 and hsp110 heat shock protein (hsp) families, yet is representative of a third and unique family of stress proteins. Despite observations indicating important roles in normal cellular functions, the in vitro chaperone properties of grp170 have not been rigorously examined. We have cloned mouse grp170 and expressed the recombinant protein in a baculovirus expression system. The function of recombinant grp170 was then assessed by determining its ability to bind to and prevent aggregation of heat-denatured luciferase. Grp170 maintains heat-denatured luciferase in a soluble state in the absence of ATP. In the presence of rabbit reticulocyte lysate, grp170 can refold and partially restore function to denatured luciferase. The chaperoning function of grp170 was also studied using domain deletion mutants, designed using the crystal structure of DnaK and the theoretical secondary structure of hsp110 as guides. Unlike hsp70 and hsp110, grp170 appears to have two domains capable of binding denatured luciferase and inhibiting its heat-induced aggregation. The two domains were identified as being similar to the classical beta-sandwich peptide binding domain and the C-terminal alpha-helical domain in hsp70 and hsp110. The ability of the C-terminal region to bind peptides is a unique feature of grp170.

When used as vaccines, tumor-derived stress proteins can elicit antitumor immune responses. For members of the hsp70 superfamily, like grp170, this seems to be due to (a) the chaperoning of antigenic peptide by the stress protein and (b) the binding of the stress protein to receptor(s) on antigenpresenting cells (APC) and subsequent antigen presentation. This suggests that domains exist on the stress protein for each function. In this study, we determine the ability of grp170 and its structural domains to (a) bind to and present melanoma-associated antigen gp100 to the immune system and (b) to bind to receptors on APCs. A direct correlation between chaperone function, binding to APCs in a receptorlike manner, and antitumor immunity was observed. Two mutants that share no common sequence, yet are both effective in their antitumor activities, compete with one another for APC binding. Studies of other members of the hsp70 superfamily, hsp110 and hsp70, or their domain deletion mutants, further confirmed that APC binding segregates with chaperoning function and not sequence. Therefore, these studies suggest that molecular chaperoning is involved in stress protein interactions with APCs, antigen binding, and in eliciting antitumor immunity, thus bridging this ancient function of stress proteins in prokaryotes to their ability to elicit immunity in higher organisms. (Cancer Res 2006; 66(2): 1161-8)

Summary GD3, a ganglioside expressed on melanoma, is the only tumour‐associated glycolipid described to date that can induce a CD1d‐restricted natural killer T (NKT)‐cell response. We analysed the fine specificity of GD3‐reactive NKT cells and discovered that immunization with GD3 induced two populations of GD3‐reactive NKT cells. One population was CD4+ CD8− and was specific for GD3; the other population was CD4− CD8− and cross‐reacted with GM3 in a CD1d‐restricted manner, but did not cross‐react with GM2, GD2, or lactosylceramide. This indicated that the T‐cell receptors reacting with GD3 recognize glucose‐galactose linked to at least one N‐acetyl‐neuraminic acid but will not accommodate a terminal N‐acetylgalactosamine. Immunization with GM2, GM3, GD2, or lactosylceramide did not induce an NKT‐cell response. Coimmunization of GM3‐loaded antigen‐presenting cells (APCs) with GD3‐loaded APCs suppressed the NKT‐cell response to GD3 in a CD1d‐restricted manner. This suppressive effect was specific for GM3 and was a local effect lasting 2–4 days. In vitro, GM3‐loaded APCs also suppressed the interleukin‐4 response, but not the interferon‐γ response, of NKT cells to α‐galactosylceramide. However, there was no effect on the T helper type 2 responses of conventional T cells. We found that this suppression was not mediated by soluble factors. We hypothesize that GM3 induces changes to the APC that lead to suppression of T helper type 2‐like NKT‐cell responses.