The coordination chemistry of Hg() with tris[(1-methylimidazol-2-yl)methyl]amine (TMIMA) was investigated. The structures of [Hg(TMIMA) 2 ](ClO 4 ) 2 (1), [Hg(TMIMA)(NCCH 3 )](ClO 4 ) 2 (2) and [Hg(TMIMA)Cl] 2 (HgCl 4 ) (3) were characterized by X-ray crystallography. Complex 1 has six strong Hg-N imidazoyl bonds ranging from 2.257(5) to 2.631(6) Å. Ligand geometry suggests the Hg-N (NR 3 ) distances of 2.959(6) Å in 1 reflects weak bonding interactions. This complex has a 199 Hg chemical shift of Ϫ1496 ppm, significantly upfield from nitrogen coordination complexes with lower coordination numbers. The five-coordinate complex 2 has Hg-N (NR 3 ) , Hg-N imidazoyl and Hg-N acetonitrile bond lengths of 2.642(8), 2.198(5) and 2.264(11) Å, respectively. Complex 3 is also five coordinate, with Hg-N (NR 3 ) , Hg-Cl and average Hg-N imidazoyl distances in the cations of 2.758(7), 2.424(2) and 2.29(4) Å, respectively. Conditions for slow exchange on the J(HgH) coupling time-scale were found for both 1 : 1 metal-to-ligand complexes in acetonitrile-d 3 . Observed heteronuclear coupling constants were similar to those associated with Hg() substituted proteins with histidine-metal bonds. Solution and solid-state comparisons to the Hg() coordination chemistry of tetradenate pyridyl ligands are made. Relevance to development of 199 Hg NMR as a metallobioprobe is discussed.

alpha(2)M* targets antigens to APCs for rapid internalization, processing, and presentation. When used as an antigen-delivery vehicle, alpha(2)M* amplifies MHC class II presentation, as demonstrated by increased antibody titers. Recent evidence, however, suggests that alpha(2)M* encapsulation may also enhance antigen-specific CTL immunity. In this study, we demonstrate that alpha(2)M*-delivered antigen (OVA) enhances the production of specific in vitro and in vivo CTL responses. Murine splenocytes expressing a transgenic TCR specific for CTL peptide OVA(257-264) (SIINFEKL) demonstrated up to 25-fold greater IFN-gamma and IL-2 secretion when treated in vitro with alpha(2)M*-OVA compared with soluble OVA. The frequency of IFN-gamma-producing cells was increased approximately 15-fold, as measured by ELISPOT. Expansion of the OVA-specific CD8+ T cell population, as assayed by tetramer binding and [3H]thymidine incorporation, and OVA-specific cell-mediated cytotoxicity, as determined by a flow cytometric assay, were also enhanced significantly by alpha(2)M*-OVA. Furthermore, significant CTL responses were observed at antigen doses tenfold lower than those required with OVA alone. Finally, we also observed enhanced humoral and CTL responses by naïve mice following intradermal immunization with alpha(2)M*-OVA. These alpha(2)M*-OVA-immunized mice demonstrated increased protection against a s.c.-implanted, OVA-expressing tumor, as demonstrated by delayed tumor growth and prolonged animal survival. The observation that alpha(2)M*-mediated antigen delivery elicits specific CTL responses suggests the cross-presentation of antigen onto MHC class I. These results support alpha(2)M* as an effective antigen-delivery system that may be particularly useful for vaccines based on weakly immunogenic subunits or requiring dose sparing.

Extracellular GRP94 (gp96) can initiate both innate and adaptive immune responses through interactions with antigen presenting cell surface receptors. Following the identification of CD91 as a receptor functioning in the cross-presentation of GRP94-associated peptides, scavenger receptors SR-A and SREC-I were demonstrated to function in GRP94 cell surface binding and endocytosis, lending controversy to the assignment of CD91 as the unique GRP94 endocytic receptor. To assess CD91 function in GRP94 surface binding and endocytosis, these parameters were examined in murine embyronic fibroblast (MEF) cell lines whose expression of CD91 was either reduced via RNAi or eliminated by genetic disruption of the CD91 locus. Reduction or loss of CD91 expression abrogated the binding and uptake of the CD91 ligand receptor-associated protein (RAP); surface binding and uptake of an N-terminal domain of GRP94 (GRP94.NTD) was unaffected. GRP94.NTD surface binding was markedly suppressed following treatment of MEF cell lines with heparin, the sulfation inhibitor sodium chlorate, or heparinase II, demonstrating that heparin sulfate proteoglycans can function in GRP94.NTD surface binding. The role of CD91 in the cross-presentaton of GRP94-associated peptides was examined in the DC2.4 dendritic cell line. In DC2.4 cells, which express CD91, GRP94.NTD-peptide cross-presentation was insensitive to RAP or activated α2-macroglobulin and occurred primarily via a fluid phase uptake pathway. In summary, these data clarify conflicting data on CD91 function in GRP94 surface binding, endocytosis and peptide cross-presentation and identify HSPGs as novel GRP94 cell surface binding sites.