Intrinsic Folding Properties of the HLA-B27 Heavy Chain Revealed by Single Chain Trimer Versions of Peptide-Loaded Class I Major Histocompatibility Complex Molecules.

Peptide-loaded Major Histocompatibility Complex (pMHC) class I molecules can be expressed in a single chain trimeric (SCT) format, composed of a specific peptide fused to the light chain beta-2 microglobulin (ß2m) and MHC class I heavy chain (HC) by flexible linker peptides. pMHC SCTs have been used as effective molecular tools to investigate cellular immunity and represent a promising vaccine platform technology, due to their intracellular folding and assembly which is apparently independent of host cell folding pathways and chaperones. However, certain MHC class I HC molecules, such as the Human Leukocyte Antigen B27 (HLA-B27) allele, present a challenge due to their tendency to form HC aggregates. We constructed a series of single chain trimeric molecules to determine the behaviour of the HLA-B27 HC in a scenario that usually allows for efficient MHC class I molecule folding. When stably expressed, a pMHC SCT incorporating HLA-B27 HC formed chaperone-bound homodimers within the endoplasmic reticulum (ER). A series of HLA-B27 SCT substitution mutations revealed that the F pocket and antigen binding groove regions of the HLA-B27 HC defined the folding and dimerisation of the single chain complex, independently of the peptide sequence. Furthermore, pMHC SCTs can demonstrate variability in their association with the intracellular antigen processing machinery.

Differences in Cellular Clearing Mechanisms of Aggregates of Two Subtypes of HLA-B27.

Ankylosing spondylitis (AS) belongs to a group of diseases, called spondyloarthropathies (SpA), that are strongly associated with the genetic marker HLA-B27. AS is characterized by inflammation of joints and primarily affects the spine. Over 160 subtypes of HLA-B27 are known, owing to high polymorphism. Some are strongly associated with disease (e.g., B*2704), whereas others are not (e.g., B*2709). Misfolding of HLA-B27 molecules [as dimers, or as high-molecular-weight (HMW) oligomers] is one of several hypotheses proposed to explain the link between HLA-B27 and AS. Our group has previously established the existence of HMW species of HLA-B27 in AS patients. Still, very little is known about the mechanisms underlying differences in pathogenic outcomes of different HLA-B27 subtypes. We conducted a proteomics-based evaluation of the differential disease association of HLA B*2704 and B*2709, using stable transfectants of genes encoding the two proteins. A clear difference was observed in protein clearance mechanisms: whereas unfolded protein response (UPR), autophagy, and aggresomes were involved in the degradation of B*2704, the endosome-lysosome machinery was primarily involved in B*2709 degradation. These differences offer insights into the differential disease association of B*2704 and B*2709.

Ankylosing Spondylitis: A Trade Off of HLA-B27, ERAP, and Pathogen Interconnections? Focus on Sardinia.

The frequency of HLA-B27 in patients with Ankylosing Spondylitis (AS) is over 85%. There are more than 170 recognized HLA-B27 alleles but the majority of them is not sufficiently represented for genetic association studies. So far only two alleles, the HLA-B*2706 in Asia and the HLA-B*2709 in Sardinia, have not been found to be associated with AS. The highly homogenous genetic structure of the Sardinian population has favored the search of relevant variants for disease-association studies. Moreover, malaria, once endemic in the island, has been shown to have contributed to shape the native population genome affecting the relative allele frequency of relevant genes. In Sardinia, the prevalence of HLA-B*2709, which differs from the strongly AS-associated B*2705 prototype for one amino acid (His/Asp116) in the F pocket of the peptide binding groove, is around 20% of all HLA-B27 alleles. We have previously hypothesized that malaria could have contributed to the establishment of this allele in Sardinia. Based on our recent findings, in this perspective article we speculate that the Endoplasmic Reticulum Amino Peptidases, ERAP1 and 2, associated with AS and involved in antigen presentation, underwent co-selection by malaria. These genes, besides shaping the immunopeptidome of HLA-class I molecules, have other biological functions that could also be involved in the immunosurveillance against malaria.