No abstract

Objective. Although the products of the HLA subtypes B*2705 and B*2709 differ only in residue 116 (Asp versus His) within their peptide-binding grooves, they are differentially associated with inflammatory rheumatic diseases such as ankylosing spondylitis (AS): B*2705 occurs in AS patients, whereas B*2709 is only rarely encountered. The reasons for this distinct association are still unclear but could include subtypespecific conformational and dynamic properties of these antigens. The present study was undertaken to investigate structural and dynamic differences between B*2705 and B*2709 and their possible relationship to subtypespecific disease association.Methods. The membrane-distal segments of the B*2705 and B*2709 heavy chains were expressed in vitro and reconstituted together with ␤ 2 -microglobulin and a peptide. HLA-B27 complexes loaded with 2 self peptides (TIS [RRLPIFSRL] and pVIPR [RRKWRRWHL]) and a sequence-related viral peptide (pLMP2 [RRRWRRLTV]) were studied by isotope-edited infrared spectroscopy to detect differences in their structure and flexibility at physiologic temperature.Results. Our analyses revealed the existence of subtype-specific conformational differences between the 2 HLA-B27 heavy chains at physiologic temperature, which are undetectable using x-ray crystallography. Irrespective of the bound peptide, the heavy chain of the B*2705 complex exhibited higher conformational flexibility than the B*2709 heavy chain.Conclusion. The present study demonstrates the existence of previously undetected systematic conformational and dynamic differences between the heavy chains of the 2 HLA-B27 subtypes. Since effector cell recognition of cells expressing HLA antigens is dependent on the dynamic properties of the interacting cell surface molecules, this HLA-B27 subtype-specific heavy chain flexibility could have a role in the distinct association of HLA-B27 subtypes with spondylarthritides.

Inflammatory processes are accompanied by the post‐translational modification of certain arginine residues to yield citrulline, and a pH decrease in the affected tissue, which might influence the protonation of histidine residues within proteins. We employed isotope‐edited IR spectroscopy to investigate whether conformational features of two human major histocompatibility antigen class I subtypes, HLA‐B*2705 and HLA‐B*2709, are affected by these changes. Both differ only in residue 116 (Asp vs. His) within the peptide‐binding grooves, but are differentially associated with inflammatory rheumatic disorders. Our analyses of the two HLA‐B27 subtypes in complex with a modified self‐peptide containing a citrulline RRKWURWHL (U = citrulline) revealed that the heavy chain is more flexible in the HLA‐B*2705 subtype than in the HLA‐B*2709 subtype. Together with our previous studies of HLA‐B27 subtypes complexed with the unmodified self‐peptide RRKWRRWHL, these findings support the existence of subtype‐specific conformational features of the heavy chains under physiological conditions, which are undetectable by X‐ray crystallography and exist irrespective of the sequence of the bound peptide and its binding mode. They might thus influence antigenic properties of the respective HLA‐B27 subtype. Furthermore, a decrease in the pH from 7.5 to 5.6 during the analyses had an influence only on HLA‐B*2709 complexed with the unmodified self‐peptide, where His116 is not contacted by any peptide side chain. This permits us to conclude that histidines, and in particular His116, influence the stability of MHC:peptide complexes. The conditions prevailing in inflammatory environments in vivo might thus also exert an impact on selected conformational features of HLA‐B27:peptide complexes. Structured digital abstract http://www.uniprot.org/uniprot/P03989  and http://www.uniprot.org/uniprot/P32241 http://www.ebi.ac.uk/ontology-lookup/?termId=MI:0407 by http://www.ebi.ac.uk/ontology-lookup/?termId=MI:0013 (http://mint.bio.uniroma2.it/mint/search/interaction.do?interactionAc=MINT-8144492).

The circular polarization of luminescence (CPL) emitted by tryptophan residues was used as a sensitive probe for measuring ligand-induced structural changes in a homogeneous type III pneumococcal antibody. A series of oligosaccharide ligands of increasing size derived from type III polysaccharide by partial acid hydrolysis was assayed. Ligand-induced changes in the circular polarization of fluorescence of the antibody were observed for all antigens tested, including tetra-, hexa-, and octasaccharides and a 16-residue oligomer, the largest changes being recorded upon interaction with the intact soluble type III pneumococcal (SIII) polysaccharide. When Fab' or F(ab')2 fragments were used instead of the antibody IgG for binding of SIII polysaccharide, the extent of conformational changes was decreased. This suggests interactions between Fab and Fc portions in the IgG molecule and subsequent conformational changes in Fc part upon antigen binding. Reduction of interchain disulfide bonds abolished the additional spectral changes attributed to the Fc part but not the changes observed in the Fab part, thus suggesting that the presence of the interchain disulfide bond in the hinge region is required for maximal CPL changes to occur. Small monovalent ligands, i.e., the tetra-, hexa-, and octasaccharides, were capable of inducing CPL changes in the Fab part of the antibody molecule as well as CPL changes attributed to the Fc portion. A multivalent ligand containing about 16 sugar residues appears to be the minimal antigenic size required for triggering conformational changes attributed to the Fc part, similar to those seen in the interaction with the whole polysaccharide antigen.