Identification and characterization of a misfolded monomeric serpin formed at physiological temperature.

The native serpin state is kinetically trapped. However, under mildly destabilizing conditions, the conformational landscape changes, and a number of nonnative conformations with increased stability can be readily formed. The ability to undergo structural change is due to intrinsic strain within the serpin's tertiary fold, which is utilized for proteinase inhibition but renders the protein susceptible to aberrant folding and self-association. The relationship between these various conformations is poorly understood. Antichymotrypsin (ACT) is an inhibitory serpin that readily forms a number of inactive conformations, induced via either environmental stress or interaction with proteinases. Here we have used a variety of biophysical and structural techniques to characterize the relationship between some of these conformations. Incubation of ACT at physiological temperature results in the formation of a range of conformations, including both polymer and misfolded monomer. The ability to populate these nonnative states and the native conformation reflects an energy landscape that is very sensitive to the solution conditions. X-ray crystallography reveals that the misfolded monomeric conformation is in the delta conformation. Further polymerization and seeding experiments show that the delta conformation is an end point in the misfolding pathway of ACT and not an on-pathway intermediate formed during polymerization. The observation that ACT readily forms this inactive conformation at physiological temperature and pH suggests that it may have a role in both health and disease.

Cytotoxic T lymphocyte recognition of HLA-A/B antigens introduced into EL4 cells by cell-liposome fusion.

HLA-A2 and -B7 antigens were introduced into EL4 (H-2b) cells by cell-liposome fusion and were used as targets or stimulators for cytotoxic T lymphocytes (CTL) generated in C57B1/6 (H-2b) mice. It was found that such EL4-HLA cells were not recognized by CTL that had been raised against either a human cell line bearing these HLA antigens or the purified HLA-A2 and -B7 antigens reconstituted into liposomes. In addition, EL4-HLA cells were not capable of inducing CTL that could recognize a human cell line bearing HLA-A2 and -B7 antigens. Instead, EL4-HLA cells induced CTL that specifically lysed EL4-HLA cells and not human cells expressing HLA-A2 and -B7. CTL recognition required the presence of HLA antigens on the EL4 cell surface and was inhibited by antibodies against either H-2b or HLA-A/B. Monoclonal antibody binding studies showed that the expected polymorphic determinants of the HLA-A2 and -B7 antigens were still present on EL4-HLA cells. However, the specificity of CTL or their precursors that are capable of recognizing HLA-A2 or -B7 was altered after these antigens became associated with the EL4 surface. Possible explanations for these results are discussed.

Introduction of HLA-A/B antigens into lymphoid cell membranes by cell-liposome fusion.

Conditions were optimized for surface binding by mouse lymphblastoid cell lines of liposomes containing purified human histocompatibility antigens. Antigens were reconstituted into lipid vesicles by detergent dialysis, and were incubated with the acceptor cell lines. After washing, the amount of HLA antigen on the cell surface was quantitated by double antibody radioimmunoassay. Uptake and surface expression was shown to be dependent upon vesicle phospholipid composition, vesicle:cell ratio, acceptor cell line, and stage of cell growth. These studies indicate that by using vesicles composed of 50% phosphatidylethanolamine/50% phosphatidylserine it is possible to introduce into the mouse EL4 cell surface about 70% of the amount of HLA expressed normally on the human lymphoblastoid line JY. The antigen is stably expressed on the surface for several hours and appears to be integrated into the cell membrane, as assessed by both fluorescence microscopy and susceptibility to lysis by using anti-HLA antibody and complement. The method used has the advantage over previously described procedures of not requiring the use of fusogenic proteins or agents such as polyethylene glycol or lysophospholipids, which might perturb overall membrane structure or properties.