Low genetic variation in the MHC class II DRB gene and MHC-linked microsatellites in endangered island populations of the leopard cat (Prionailurus bengalensis) in Japan.

Isolated populations of the leopard cat (Prionailurus bengalensis) on Tsushima and Iriomote islands in Japan are classified as subspecies P. b. euptilurus and P. b. iriomotensis, respectively. Because both populations have decreased to roughly 100, an understanding of their genetic diversity is essential for conservation. We genotyped MHC class II DRB exon 2 and MHC-linked microsatellite loci to evaluate the diversity of MHC genes in the Tsushima and Iriomote cat populations. We detected ten and four DRB alleles in these populations, respectively. A phylogenetic analysis showed DRB alleles from both populations to be closely related to those in other felid DRB lineages, indicating trans-species polymorphism. The MHC-linked microsatellites were more polymorphic in the Tsushima than in the Iriomote population. The MHC diversity of both leopard cat populations is much lower than in the domestic cat populations on these islands, probably due to inbreeding associated with founder effects, geographical isolation, or genetic drift. Our results predict low resistance of the two endangered populations to new pathogens introduced to the islands.

In silico analysis of candidate proteins sharing homology with Streptococcus agalactiae proteins and their role in male infertility.

Leukocytospermia is a physiologic condition defined as human semen with a leukocyte count of >1 x 106 cells/ml that is often correlated with male infertility. Moreover, bacteriospermia has been associated with leukocytospermia ultimately leading to male infertility. We have found that semen samples with >1 x 106/ml leukocytes and/or bacteriospermia have oxidative predominance as evidenced by augmented protein carbonyl and lipid peroxidation status of the semen which is implicated in sperm dysfunction. It has been reported that Streptococcus agalactiae is present in bacteriospermic samples. Previous research has shown that human leukocyte antigen beta chain paralog (HLA-DRB) alleles interact best with the infected sperm cells rather than the non-infected cells. Little is known about the interaction of major histocompatibility complex (MHC) present on leukocytes with the sperm upon bacterial infection and how it induces an immunological response which we have addressed by epitope mapping. Therefore, we examined MHC class II derived bacterial peptides which might have human sperm-related functional aspects. Twenty-two S. agalactiae proteins were obtained from PUBMED protein database for our study. Protein sequences with more than two accession numbers were aligned using CLUSTAL Omega to check their conservation pattern. Each protein sequence was then analyzed for T-cell epitope prediction against HLA-DRB alleles using the immune epitope database (IEDB) analysis tool. Out of a plethora of peptides obtained from this analysis, peptides corresponding to proteins of interest such as DNA binding response regulator, hyaluronate lyase and laminin binding protein were screened against the human proteome using Blastp. Interestingly, we have found bacterial peptides sharing homology with human peptides deciphering some of the important sperm functions. Antibodies raised against these probable bacterial antigens of fertility will not only help us understand the mechanism of leukocytospermia/bacteriospermia induced male factor infertility but also open new avenues for immunocontraception. ABBREVIATIONS: AA: amino acid; ASA: antisperm antibodies; GBS: group B streptococcus; HLA: human leukocyte antigen; HAS3: hyaluronan synthase 3: IEDB: immune epitope database; MAPO2: O6-methylguanine-induced apoptosis 2; MHC: major histocompatibility complex; ROS: reactive oxygen species; Rosbin1: round spermatid basic protein 1; S. agalactiae: Streptococcus agalactiae;SA: sperm antigen; SPATA17: spermatogenesis associated protein17; SPNR: spermatid perinuclear RNA binding protein; TEX15: testis-expressed sequence 15 protein; TOPAZ: testis- and ovary-specific PAZ domain-containing protein; TPABP: testis-specific poly-A binding protein; TPAP: testis-specific poly(A) polymerase; WHO: World Health Organization.

A novel family of human leukocyte antigen class II receptors may have its origin in archaic human species.

HLA class II α and ß chains form receptors for antigen presentation to CD4(+) T cells. Numerous pairings of class II α and ß subunits from the wide range of haplotypes and isotypes may form, but most of these combinations, in particular those produced by isotype mixing, yielded mismatched dimers. It is unclear how selection of functional receptors is achieved. At the atomic level, it is not known which interactions of class II residues regulate selection of matched αß heterodimers and the evolutionary origin of matched isotype mixed dimer formation. In this study we investigated assembly of isotype-mixed HLA class II α and ß heterodimers. Assembly and carbohydrate maturation of various HLA-class II isotype-mixed α and ß subunits was dependent on the groove binding section of the invariant chain (Ii). By mutation of polymorphic DPß sequences, we identified two motifs, Lys-69 and GGPM-(84-87), that are engaged in Ii-dependent assembly of DPß with DRα. We identified five members of a family of DPß chains containing Lys-69 and GGPM 84-87, which assemble with DRα. The Lys/GGPM motif is present in the DPß sequence of the Neanderthal genome, and this ancient sequence is related to the human allele DPB1*0401. By site-directed mutagenesis, we inspected Neanderthal amino acid residues that differ from the DPB1*0401 allele and aimed to determine whether matched heterodimers are formed by assembly of DPß mutants with DRα. Because the *0401 allele is rare in the sub-Saharan population but frequent in the European population, it may have arisen in modern humans by admixture with Neanderthals in Europe.

A molecular basis for the association of the HLA-DRB1 locus, citrullination, and rheumatoid arthritis.

Rheumatoid arthritis (RA) is strongly associated with the human leukocyte antigen (HLA)-DRB1 locus that possesses the shared susceptibility epitope (SE) and the citrullination of self-antigens. We show how citrullinated aggrecan and vimentin epitopes bind to HLA-DRB1*04:01/04. Citrulline was accommodated within the electropositive P4 pocket of HLA-DRB1*04:01/04, whereas the electronegative P4 pocket of the RA-resistant HLA-DRB1*04:02 allomorph interacted with arginine or citrulline-containing epitopes. Peptide elution studies revealed P4 arginine-containing peptides from HLA-DRB1*04:02, but not from HLA-DRB1*04:01/04. Citrullination altered protease susceptibility of vimentin, thereby generating self-epitopes that are presented to T cells in HLA-DRB1*04:01(+) individuals. Using HLA-II tetramers, we observed citrullinated vimentin- and aggrecan-specific CD4(+) T cells in the peripheral blood of HLA-DRB1*04:01(+) RA-affected and healthy individuals. In RA patients, autoreactive T cell numbers correlated with disease activity and were deficient in regulatory T cells relative to healthy individuals. These findings reshape our understanding of the association between citrullination, the HLA-DRB1 locus, and T cell autoreactivity in RA.

Sulfamethoxazole induces a switch mechanism in T cell receptors containing TCRVß20-1, altering pHLA recognition.

T cell receptors (TCR) containing Vß20-1 have been implicated in a wide range of T cell mediated disease and allergic reactions, making it a target for understanding these. Mechanics of T cell receptors are largely unexplained by static structures available from x-ray crystallographic studies. A small number of molecular dynamic simulations have been conducted on TCR, however are currently lacking either portions of the receptor or explanations for differences between binding and non-binding TCR recognition of respective peptide-HLA. We performed molecular dynamic simulations of a TCR containing variable domain Vß20-1, sequenced from drug responsive T cells. These were initially from a patient showing maculopapular eruptions in response to the sulfanilamide-antibiotic sulfamethoxazole (SMX). The CDR2ß domain of this TCR was found to dock SMX with high affinity. Using this compound as a perturbation, overall mechanisms involved in responses mediated by this receptor were explored, showing a chemical action on the TCR free from HLA or peptide interaction. Our simulations show two completely separate modes of binding cognate peptide-HLA complexes, with an increased affinity induced by SMX bound to the Vß20-1. Overall binding of the TCR is mediated through a primary recognition by either the variable ß or α domain, and a switch in recognition within these across TCR loops contacting the peptide and HLA occurs when SMX is present in the CDR2ß loop. Large binding affinity differences are induced by summed small amino acid changes primarily by SMX modifying only three critical CDR2ß loop amino acid positions. These residues, TYRß57, ASPß64, and LYSß65 initially hold hydrogen bonds from the CDR2ß to adjacent CDR loops. Effects from SMX binding are amplified and traverse longer distances through internal TCR hydrogen bonding networks, controlling the overall TCR conformation. Thus, the CDR2ß of Vß20-1 acts as a ligand controlled switch affecting overall TCR binding affinity.

Co-regulated expression of alpha and beta mRNAs encoding HLA-DR surface heterodimers is mediated by the MHCII RNA operon.

Major histocompatibility complex class II (MHCII) molecules are heterodimeric surface proteins involved in the presentation of exogenous antigens during the adaptive immune response. We demonstrate the existence of a fine level of regulation, coupling the transcription and processing of mRNAs encoding α and ß chains of MHCII molecules, mediated through binding of their Untraslated Regions (UTRs) to the same ribonucleoproteic complex (RNP). We propose a dynamic model, in the context of the 'MHCII RNA operon' in which the increasing levels of DRA and DRB mRNAs are docked by the RNP acting as a bridge between 5'- and 3'-UTR of the same messenger, building a loop structure and, at the same time, joining the two chains, thanks to shared common predicted secondary structure motifs. According to cell needs, as during immune surveillance, this RNP machinery guarantees a balanced synthesis of DRA and DRB mRNAs and a consequent balanced surface expression of the heterodimer.

Structural requirements for recognition of major histocompatibility complex class II by membrane-associated RING-CH (MARCH) protein E3 ligases.

MARCH E3 ligases play a key role in controlling MHC class II surface expression by regulated ubiquitination of a lysine residue in the ß-chain. Little is known concerning how these enzymes target their specific substrates. Here we show that recognition of HLA-DR by MARCH proteins is complex. Several features associated with the transmembrane domain and bordering regions influence the overall efficiency of receptor internalization. A cluster of residues at the interface of the lipid bilayer and the cytosol plays the most important role in MARCH8 recognition of HLA-DRß. Variation in this sequence also determines specificity of MARCH9 for HLA-DQ. Residues located in helical face four of HLA-DRß together with a charged residue at the boundary with the stalk region also contribute significantly to recognition. Truncation analysis suggested that a dileucine-like motif in the DRß cytoplasmic tail influences the efficiency of co-localization of HLA-DR with MARCH8. The DRß-encoded acceptor lysine functioned optimally when placed in its natural location relative to the bilayer. In the DRα/DRß dimer most other amino acids in the cytoplasmic tail could be substituted for alanine with minimal influence on function. Our data support a model whereby multiple features of HLA-DR are involved in substrate recognition by MARCH8. The single most important region is located at the interface between the transmembrane domain and the cytosol. Variation in sequence in this location between different class II isotypes controls efficiency of recognition by different MARCH E3 ligases.

Association of HLA class II alleles with sensitization to cow dander Bos d 2, an important occupational allergen.

Allergic sensitization results from a complex interaction between genetic and environmental factors. Earlier studies have shown that highly polymorphic HLA genes are associated with a variety of allergies. Several important respiratory allergens belong to the family of lipocalin proteins. These include occupational sensitizers, such as cow Bos d 2 or rat Rat n 1, and prevalent indoor sensitizers, such as dog Can f 1 or cockroach Bla g 4. HLA associations with sensitization to lipocalin allergens are incompletely known. In the present study we have investigated an association between HLA alleles and sensitization to the major cow allergen Bos d 2. The HLA-DR/DQ genotypes of 40 Bos d 2-sensitized subjects having occupational asthma were determined by polymerase chain reaction (PCR) and the results were compared with the genotypes of 151 unrelated Finnish subjects. The frequencies of HLA class II alleles DRB1*0101, DRB1*0404, DQB1*0302, and DQB1*0501 were significantly higher among Bos d 2-sensitized than among control subjects. In addition, the allergic subjects expressed significantly lower frequencies of HLA-DRB1*0301 and DQB1*0201 alleles than did the control subjects. These data suggest that the HLA class II alleles DRB1*0101, DRB1*0404, DQB1*0302, and DQB1*0501, and the haplotypes that include them, are associated with sensitization to the major cow allergen Bos d 2, whereas HLA-DRB1*0301 and DQB1*0201 are dissociated with it. Amino acid analysis provides a biologically plausible explanation for the HLA associations.