Identification of a dominant self-ligand bound to three HLA B44 alleles and the preliminary crystallographic analysis of recombinant forms of each complex.
A naturally processed and presented ligand that is shared by human leukocyte antigen (HLA) B*4402, B*4403 and B*4405 molecules has been identified in peptides isolated from immunoaffinity purified HLA B44 complexes. This peptide derived from HLA DPalpha residues 46-54, an endogenous product of HLA DP expressed in the cell line Hmy2.C1R, is a prominent peptide in the mass spectra of species isolated as bound peptides from each allele when the three HLA B44 subtypes were introduced as transfected gene products. Recombinant truncated forms of HLA B*4405(1-276), HLA B*4403(1-276), HLA B*4402(1-276) and beta(2)-microglobulin have been prepared as inclusion bodies in Escherichia coli and refolded in the presence of the DPalpha(46-54) peptide and purified by a combination of size exclusion and anion exchange chromatography. This material was determined to be correctly folded based on detection of a conformational epitope recognized by the W6/32 monoclonal antibody. Large, plate-like crystals of the three complexes were produced using polyethylene glycol as the precipitant. All the crystals belong to the space group P2(1)2(1)2(1) with unit cell dimensions of approximately a=51, b=82, c=110 A. The crystals of three B44/DPalpha complexes diffracted to a resolution of 1.9 A or better. For the first time, using this natural, high abundance ligand of the HLA B44 molecules we have successfully expressed and refolded the three HLA B44 molecules and produced crystals amenable to structural studies.
Macdonald
Whitney
W
Department of Microbiology and Immunology, University of Melbourne, 3010, Melbourne, Vic., Australia.
Williams
David S
DS
Clements
Craig S
CS
Gorman
Jeffery J
JJ
Kjer-Nielsen
Lars
L
Brooks
Andrew G
AG
McCluskey
James
J
Rossjohn
Jamie
J
Purcell
Anthony W
AW
D000483:Alleles; D018360:Crystallography, X-Ray; D000939:Epitopes; D004926:Escherichia coli / Q000235:genetics; D015235:HLA-B Antigens / Q000737:chemistry* / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism*; D059948:HLA-B44 Antigen; D006682:HLA-DP Antigens / Q000276:immunology / Q000378:metabolism*; D006801:Humans; D008024:Ligands; D010455:Peptides / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism; D017510:Protein Folding; D011994:Recombinant Proteins / Q000737:chemistry / Q000235:genetics / Q000276:immunology; D014407:Tumor Cells, Cultured; D001613:beta 2-Microglobulin / Q000737:chemistry / Q000235:genetics / Q000378:metabolism
D000939:Epitopes; D015235:HLA-B Antigens; D059948:HLA-B44 Antigen; D006682:HLA-DP Antigens; D008024:Ligands; D010455:Peptides; D011994:Recombinant Proteins; D001613:beta 2-Microglobulin