Cystatin C reduces the in vitro formation of soluble Abeta1-42 oligomers and protofibrils.

There are an increasing number of genetic and neuropathological observations to suggest that cystatin C, an extracellular protein produced by all nucleated cells, might play a role in the pathophysiology of sporadic Alzheimer's disease (AD). Recent observations indicate that small and large soluble oligomers of the beta-amyloid protein (Abeta) impair synaptic plasticity and induce neurotoxicity in AD. The objective of the present study was to investigate the influence of cystatin C on the production of such oligomers in vitro. Co-incubation of cystatin C with monomeric Abeta1-42 significantly attenuated the in vitro formation of Abeta oligomers and protofibrils, as determined using electron microscopy (EM), dodecyl sulphate polyacrylamide gel electrophoresis (SDS-PAGE), immunoblotting, thioflavin T (ThT) spectrofluorimetry and gel chromatography. However, cystatin C did not dissolve preformed Abeta oligomers. Direct binding of cystatin C to Abeta was demonstrated with the formation of an initial 1:1 molar high-affinity complex. These observations suggest that cystatin C might be a regulating element in the transformation of monomeric Abeta to larger and perhaps more toxic molecular species in vivo.

Efficient assembly of recombinant major histocompatibility complex class I molecules with preformed disulfide bonds.

The expression of major histocompatibility class I (MHC-I) crucially depends upon the binding of appropriate peptides. MHC-I from natural sources are therefore always preoccupied with peptides complicating their purification and analysis. Here, we present an efficient solution to this problem. Recombinant MHC-I heavy chains were produced in Escherichia coli and subsequently purified under denaturing conditions. In contrast to common practice, the molecules were not reduced during the purification. The oxidized MHC-I heavy chain isoforms were highly active with respect to peptide binding. This suggests that de novo folding of denatured MHC-I molecules proceed efficiently if directed by preformed disulfide bond(s). Importantly, these molecules express serological epitopes and stain specific T cells; and they bind peptides specifically. Several denatured MHC-I heavy chains were analyzed and shown to be of a quality, which allowed quantitative analysis of peptide binding. The analysis of the specificity of the several hundred human MHC haplotypes, should benefit considerably from the availability of pre-oxidized recombinant MHC-I.

Quantitative predictions of peptide binding to MHC class I molecules using specificity matrices and anchor-stratified calibrations.

Peptides are key immune targets. They are generated by fragmentation of antigenic proteins, selected by major histocompatibility complex (MHC) molecules and subsequently presented to T cells. One of the most selective requirements is that of peptide binding to MHC. Accurate descriptions and predictions of peptide-MHC interactions are therefore important. Quantitative matrices representing MHC class I specificity can be used to search any query protein for the presence of MHC binding peptides. Assuming that each peptide residue contributes to binding in an additive and sequence independent manner, such "crude" matrix-driven predictions can be expressed as a quantitative estimates of binding strength. Crude matrix-driven predictions are reasonably uniform (i.e. precise), however, there is a general tendency towards overestimating binding (i.e. being inaccurate). To evaluate and possibly improve predictions, we have measured the MHC class I binding of a large number of peptides. In an attempt to further improve predictions and to include sequence dependency, we subdivided the panel of peptides according to whether the peptides had zero, one or two primary anchor residues. This allowed us to define unique anchor-stratified calibrations, which led to predictions of improved precision and accuracy.

Phage display of peptide / major histocompatibility class I complexes.

Major histocompatibility complex class I (MHC-I) molecules sample peptides from the intracellular environment and present them to cytotoxic T cells (CTL). To establish a selection system, and, thereby, enable a library approach to identify the specificities involved (that of the MHC-I for peptides and subsequently that ot the T cell receptor for peptide-MHC-I complex), we have fused a single chain peptide-MHC-I complex to the phage minor coat protein, gpIII, and displayed it on filamentous phage. Expression of peptide-MHC-I complexes was shown with relevant conformation-specific monoclonal antibodies and, more importantly, with a unique "T cell receptor-like" (i. e. peptide-specific, MHC-I-restricted) antibody. Thus, properly assembled and folded peptide-MHC-I complexes can be displayed on filamentous phage. Despite the successful display, interaction with T cells could not be demonstrated.

Measurement of peptide-MHC interactions in solution using the spin column filtration assay.

This unit describes how peptide-MHC complexes can be generated in vitro using affinity-purified MHC and synthetic peptide. The unit first describes how the interaction between peptide and MHC interaction can be measured in an accurate, quantitative biochemical assay. This procedure has been optimized for efficient separation of free peptide and MHC-bound peptide through a novel principle, termed "gradient centrifugation." The first two support protocols describe how to set up a biochemical fluid-phase binding reaction between peptide and MHC class I and class II, respectively. Also, an alternative procedure for setting up a biochemical fluid phase binding reaction between beta(2)m and MHC class I is included. Finally a more versatile inhibition assay is described. The assay is simple and robust, and has several advantages compared to the classical gel-filtration assay, including increased sensitivity and throughput. It also demands fewer resources both in terms of unique reagents and labor, and it generates less hazardous waste. Thus, the spin column gel-filtration assay is ideal for routine work.