MHC class I peptide

Leinders-Zufall, T., Brennan, P., Widmayer, P., Chandramani, P., Maul-Pavicic, A., Jager, M., Li, X.H., Breer, H., Zufall, F. and Boehm, T. (2004) MHC class I peptides as chemosensory signals in the vomeronasal organ. Science 306, 1033-1037. 

The path that leads from full sized protein to epitopes at the cell surface is complex, consisting of the generation of small peptides, translocation of the peptides to the endoplasmic reticulum by a transporter complex, loading of the peptides onto MHC class-I molecules and relocation of the MHC class-I-peptide complex to the cell surface (Rock and Goldberg, 1999). Since many of these steps are known to be prime targets for viral evasion strategies, the next step was to identify at what point the GAr interferes with the presentation of EBNAl. 

P. Cresswell, N. Bangia, T. Dick, and G. Diedrich. 1999. The nature of the MHC class I peptide loading complex Immunol. Rev. 172 21-28. (PubMed)... 

SEQUENCING MHC CLASS I PEPTIDES USING MEMBRANE PRECONCENTRATION-CAPILLARY ELECTROPHORESIS TANDEM MASS SPECTROMETRY (mPC-CE-MS/MS)... 

Major histocompatibility complex (MHC) proteins are essential components of the immune system (1). One speeific role is for them to bind and present cellularly derived peptides (-8-10 amino acids - MHC Class I peptides) at the cell surface. These peptides are subsequently challenged by cytolytic T-lymphocytes (CTL s) which are programmed to differentiate between self and exogenous peptides. T-cell recognition of these latter peptides initiates a response that ultimately results in cell lysis and death of the infected cell. Hence, structural characterization of such peptides could potentially result in the development of therapeutie treatments of a number of infectious disease states such as viral cancers, AIDS, and autoimmune disease. However, the task of sequencing such peptides is difficult since MHC class I proteins can bind and present 10,000-15,000 different cellularly derived peptides present at the sub-pieo-femtomole level (2,3). 

HPLC followed by on-line membrane preconcentration capillary electrophoresis-MS/MS (mPC-CE-MS/MS) to separate and sequence MHC class I peptides. 

EL-4 cells (3 x 10 ) were lysed with N,N-dimethyl-N-(3-sulfopropyl)-3-[[(3a,5 P,7a, 12a)-3,7,12-trihydroxy-24-oxocholan-24-y 1]-amino]-l-propanaminium hydroxide (CHAPS). The nuclei and membranes were pelleted and the supematent lysate filtered to remove lipids. The lysate was sequentially passed over sepharose columns containing a) normal mouse serum b) Y-3 which is an anti-K monoclonal antibody. Both columns were washed with 45 coliunn volumes of progressively lower molarity salt solutions. The beads were then treated with acetic acid to release antigen-antibody complexes and the complex was denatured by boiling in 10% acetic acid. The mixture was filtered through a 3 kDa pore-size membrane and the filtrate containing MHC class I peptides subjected to reversed phase HPLC. 

Figure 2 Schematic of tITP conditions for use with mPC-CE-MS analysis after MHC class I peptides have been eluted from the adsorptive SDB membrane into the CE capillary. Application of the CE voltage results in rapid migration of H and OH ions with concomitant focusing of the peptides in the high organic solvent zone.

A prerequisite for CTL-mediated immune response is the formation of the MHC-class I-peptide complex and subsequent recognition by the T-cell repertoire, which can be analyzed in cell lysis assays with MCr-loaded target cells. Chromium release is a measure for peptide-induced cell lysis by CTL, and indicates the potency of the peptide to serve as an allele-specific epitope. A synthetic epitope has been identified with the peptide library approach to elucidate the molecular basis for the observed cross-recognition of two ligands by a single receptor [53]. 

Stevens J, Wiesmiiller K-H, Barker PJ, Walden P, Butcher G, Joly E (1998) Efficient generation of MHC class I-peptide complexes using synthetic peptide libraries. J Biol Chem 274 2874-2884. 

Hughes, E. A., and Cresswell, P. (1998). The thiol oxidoreductase ERp57 is a component of the MHC class I peptide-loading complex. Curr. Biol. 8, 709-712. 

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