Barbara Uchanska-Ziegler

HLA-B27 Misfolding Is Associated with Aberrant Intermolecular Disulfide Bond Formation (Dimerization) in the Endoplasmic Reticulum

The class I protein HLA-B27 confers susceptibility to inflammatory arthritis in humans and when overexpressed in rodents for reasons that remain unclear. We demonstrated previously that HLA-B27 heavy chains (HC) undergo endoplasmic reticulum (ER)-associated degradation. We report here that HLA-B27 HC also forms two types of aberrant disulfide-linked complexes (dimers) during the folding and assembly process that can be distinguished by conformation-sensitive antibodies W6/32 and HC10. HC10-reactive dimers form immediately after HC synthesis in the ER and constitute at least 25% of the HC pool, whereas W6/32-reactive dimers appear several hours later and represent less than 10% of the folded HC. HC10-reactive dimers accumulate in the absence of tapasin or β2-microglobulin, whereas W6/32-reactive dimers are not detected. Efficient formation of W6/32-reactive dimers appears to depend on the transporter associated with antigen processing, tapasin, and β2-microglobulin. The unpaired Cys67 and residues at the base of the B pocket that dramatically impair HLA-B27 HC folding are critical for the formation of HC10-reactive ER dimers. Although certain other alleles also form dimers late in the assembly pathway, ER dimerization of HLA-B27 may be unique. These results demonstrate that residues comprising the HLA-B27 B pocket result in aberrant HC folding and disulfide bond formation, and thus confer unusual properties on this molecule that are unrelated to peptide selection per se, yet may be important in disease pathogenesis.

Amnion Epithelial Cells, in Contrast to Trophoblast Cells, Express All Classical HLA Class I Molecules Together With HLA-G

PROBLEM: The expression of the non‐classical HLA‐G gene has been shown at the protein level on trophoblast‐derived embryonic tissue, like the extravillous cytotrophoblast. However, the presence of HLA‐G on embryoblast‐derived cells is currently controversial. The amnion epithelium is an embryoblast‐derived cell layer covering the amnion cavity and is the main source for the amnion fluid.

Expression of HLA class I molecules in human first trimester and term placenta trophoblast

Abstract.Expression of HLA class I molecules in trophoblast cells from various locations in normal human first trimester and term placenta was investigated by immunohistochemistry with a panel of monoclonal antibodies against the heavy chains or complete HLA class I molecules complexed with β2-microglobulin. These reagents were also employed to distinguish between the products of different HLA class I loci. In addition to previously characterized reagents, a novel monoclonal antibody against HLA-A molecules (TÜ155) was used. Various choriocarcinoma and transfected cell lines served as controls for the specificities of the monoclonal antibodies. Cells in close contact with maternal cells, such as invading trophoblast cells and cells of the basal plate, expressed β2-m micro globulin in association with HLA-G and HLA-C heavy chains. These class I heavy chains may also have been present as isolated molecules, although not in each of the cells. In contrast, cells of the chorion laeve exclusively expressed HLA-G, and not HLA-A, -B, or -C antigens. Our data support the often discussed immune protective function and the regulatory function of the HLA-G molecule, during invasion. In addition, by using monoclonal antibodies HCA2 (anti-HLA-A and -G), HC10 (anti-HLA-B and -C), TÜ149 (anti-HLA-B, -C, and some -A alleles), SFR8-B6 (anti-HLA-Bw6 and some -C), LA45 (some HLA-A and -B), TÜ48 (anti-HLA-Bw4 and some -A), and TÜ155 (anti-HLA-A), we show the presence of HLA-C molecules in all extravillous trophoblast cells of the cell columns and in the basal plate; the trophoblast cells of the chorion laeve lack this antigen. The function of this molecule is not clear, although a protective function against natural killer cell activity in the endometrium is postulated.

Differential Peptide Dynamics Is Linked to Major Histocompatibility Complex Polymorphism

Peptide presentation by major histocompatibility complex (MHC) molecules is of central importance for immune responses, which are triggered through recognition of peptide-loaded MHC molecules (pMHC) by cellular ligands such as T-cell receptors (TCR). However, a unifying link between structural features of pMHC and cellular responses has not been established. Instead, pMHC/TCR binding studies suggest conformational and/or flexibility changes of the binding partners as a possible cause of differential T-cell stimulation, but information on real-time dynamics is lacking. We therefore probed the real-time dynamics of a MHC-bound nonapeptide (m9), by combining time-resolved fluorescence depolarization and molecular dynamics simulations. Here we show that the nanosecond dynamics of this peptide presented by two human MHC class I subtypes (HLA-B*2705 and HLA-B*2709) with differential autoimmune disease association varies dramatically, despite virtually identical crystal structures. The peptide dynamics is linked to the single, buried polymorphic residue 116 in the peptide binding groove. Pronounced peptide flexibility is seen only for the non-disease-associated subtype HLA-B*2709, suggesting an entropic control of peptide recognition. Thermodynamic data obtained for two additional peptides support this hypothesis.

Possible Roles for Products of Polymorphic MHC and Linked Olfactory Receptor Genes during Selection Processes in Reproduction

PROBLEM:u2002Polymorphic genes of the human major histocompatibility complex [MHC; human leukocyte antigen (HLA)] are probably important in determining resistance to parasites and avoidance of inbreeding. We investigated whether HLA‐associated sexual selection could also involve HLA‐linked olfactory receptor (OR) genes, which might not only participate in olfaction‐guided mate choice, but also in selection processes within the testis.

Thermodynamic and Structural Analysis of Peptide- and Allele-dependent Properties of Two HLA-B27 Subtypes Exhibiting Differential Disease Association

Selected HLA-B27 subtypes are associated with spondyloarthropathies, but the underlying mechanism is not understood. To explain this association in molecular terms, a comparison of peptide-dependent dynamic and structural properties of the differentially disease-associated subtypes HLA-B*2705 and HLA-B*2709 was carried out. These molecules differ only by a single amino acid at the floor of the peptide binding groove. The thermostabilities of a series of HLA-B27 molecules complexed with nonameric and decameric peptides were determined and revealed substantial differences depending on the subtype as well as the residues at the termini of the peptides. In addition we present the crystal structure of the B*2709 subtype complexed with a decameric peptide. This structure provides an explanation for the preference of HLA-B27 for a peptide with an N-terminal arginine as secondary anchor and the lack of preference for tyrosine as peptide C terminus in B*2709. The data show that differences in thermodynamic properties between peptide-complexed HLA-B27 subtypes are correlated with a variety of structural properties.

THE MONOCLONAL ANTIBODY HCA2 RECOGNISES A BROADLY SHARED EPITOPE ON SELECTED CLASSICAL AS WELL AS SEVERAL NON-CLASSICAL HLA CLASS I MOLECULES

HCA2 is a widely used monoclonal antibody, thought to be highly selective for HLA-A and -G heavy chains. We demonstrate here that it also shows affinity to HLA-B73 and HLA-E molecules on intact cells. By comparing the differences in the amino acid (AA) sequences of several HLA class I alleles that are either recognised or not recognised by HCA2, a likely epitope of HCA2 has been deduced. It extends from position 76 to position 83 of the alpha1-domain. In intact cells, the solvent-exposed AA in positions 76 (Ala, Val, or Met), 80 (Asn or Thr) and 83 (Gly) are likely to constitute the recognition region. Inhibition experiments with peptides spanning the region of the alpha1-domain from position 74 to 85 of various HLA class I heavy chains prove that HCA2 recognizes a broadly shared epitope on HLA-E, -F and -G molecules as well as selected HLA-A, -B and -C antigens.

Ankylosing spondylitis: a β2m–deposition disease?

Abstract To explain the strong association between HLA-B27 and ankylosing spondylitis, we suggest that the release of β 2 -microglobulin (β 2 m) from a subpopulation of cell surface-expressed HLA-B27 molecules leads to β 2 m-deposition within synovia and to the initiation of an inflammatory process, which culminates in destructive spondyloarthropathy.

Differential expression of major histocompatibility complex class I molecules in the brain of a New World monkey, the common marmoset (Callithrix jacchus)

It has been supposed that central nervous neurons do not express MHC class I molecules. However, recent studies clearly demonstrated functional MHC class I expression in the rodent brain. In the present study, we have extended these studies and investigated the presence of MHC class I transcripts and proteins in the brain of a non-human primate species, the common marmoset monkey (Callithrix jacchus). Using in-situ hybridization, we found strong expression of MHC class I transcripts in neocortex, hippocampal formation, substantia nigra and nucleus ruber. In-situ hybridization with emulsion autoradiography demonstrated MHC class I mRNA in distinct pyramidal neurons of cortex and hippocampus, in granule neurons of the dentate gyrus, in dopaminergic neurons of substantia nigra and in motor neurons of nucleus ruber. Immunocytochemistry confirmed MHC class I protein expression in these neurons. Two monoclonal antibodies, MRC-Ox18 and HB115, reacted differentially with MHC class I proteins on neuronal and non-neuronal cells, respectively. Interestingly, in marmoset monkeys that were immunosuppressed with FK506 (tacrolimus), expression of neuronal MHC class I proteins, which could be detected with MRC-Ox18, was either very low (neocortex, nucleus ruber, substantia nigra) or absent (hippocampus). In contrast, class I expression in endothelial cells, which was detected by HB115, was not affected by immunosuppression. Our data show that selected neurons in the brain of a non-human primate express MHC class I molecules and that this expression can be modulated by immunosuppression.

Polymorphic olfactory receptor genes and HLA loci constitute extended haplotypes

Olfactory receptor (OR) genes are often clustered and are known to be located on most human chromosomes. In the largest OR gene cluster so far analyzed and sequenced in any organism, we have identified 27 OR genes between HLA-F and HFE, of which so far 23 are located within about 650 kilobasepairs between HLA-F and RFP. Their products could be involved in the recognition of individual-specific, MHC-dependent odours and be tailored to perform this function. In this first systematic OR gene polymorphism study, twelve HLA-linked OR genes were analyzed on ten HLA-homozygous or — hemizygous cell lines with different HLA haplotypes. All potentially functional HLA-linked OR genes were found to exhibit polymorphism, although the degree differed considerably. The nucleotide changes within a given gene occur at defined positions, and may be part of putative transmembrane domains or occur at other residues. It has been possible so far to define 12 haplotypes for the HLAlinked OR genes on the 18 chromosomes 6 analyzed. The strong linkage disequilibrium, which is known to extend from HLA-A to HFE, is expected to conserve these extended HLA/OR haplotypes. If HLA and HLA-linked polymorphic OR genes also turn out to be functionally connected, a view supported by our finding of polymorphism of these OR genes, the entire region might be considered to constitute an extended gene complex which could, as we have already suggested, be designated “Immuno-Olfactory Supercomplex” (IOS).