Jan Salomonsen

Peptide motifs of the single dominantly expressed class I molecule explain the striking MHC-determined response to Rous sarcoma virus in chickens

Compared with the MHC of typical mammals, the chicken MHC is smaller and simpler, with only two class I genes found in the B12 haplotype. We make five points to show that there is a single-dominantly expressed class I molecule that can have a strong effect on MHC function. First, we find only one cDNA for two MHC haplotypes (B14 and B15) and cDNAs corresponding to two genes for the other six (B2, B4, B6, B12, B19, and B21). Second, we find, for the B4, B12, and B15 haplotypes, that one cDNA is at least 10-fold more abundant than the other. Third, we use 2D gel electrophoresis of class I molecules from pulse-labeled cells to show that there is only one heavy chain spot for the B4 and B15 haplotypes, and one major spot for the B12 haplotype. Fourth, we determine the peptide motifs for B4, B12, and B15 cells in detail, including pool sequences and individual peptides, and show that the motifs are consistent with the peptides binding to models of the class I molecule encoded by the abundant cDNA. Finally, having shown for three haplotypes that there is a single dominantly expressed class I molecule at the level of RNA, protein, and antigenic peptide, we show that the motifs can explain the striking MHC-determined resistance and susceptibility to Rous sarcoma virus. These results are consistent with the concept of a “minimal essential MHC” for chickens, in strong contrast to typical mammals.

Gene organisation determines evolution of function in the chicken MHC

Summary: Some years ago, we used our data for class I genes, proteins and peptide‐binding specificities to develop the hypothesis that the chicken B‐F/B‐L region represents a “minimal essential MHC”, In this view, the B locus contains the classical (highly expressed and polymorphic) class I α and class II β multigene families, which are reduced to one or two members, with many other genes moved away or deleted from the chicken genome altogether. We found that a single dominantly expressed class I gene determines the immune response to certain infectious pathogens, due to peptide‐binding specificity and cell‐surface expression level. This stands in stark contrast to well‐studied mammals like humans and mice, in which every haplotype is more‐or‐less responsive to every pathogen and vaccine, presumably due to the multigene family of MHC molecules present. In order to approach the basis for a single dominantly expressed class I molecule, we have sequenced a portion of the B complex and examined the location and polymorphism of the class I (B‐F)α, TAP and class II (B‐L) β genes. The region is remarkably compact and simple, with many of the genes expected from the MHC of mammals absent, including LMP, class II α and DO genes as well as most class III region genes. However, unexpected genes were present, including tapasin and putative natural killer receptor genes. The region is also organised differently from mammals, with the TAPs in between the class I genes, the tapasin gene in between the class II (B‐L) β genes, and the C4 gene outside of the class I α and class II β genes. The close proximity of TAP and class I α genes leads to the possibility of co‐evolution, which can drive the use of a single dominantly expressed class I molecule with peptide‐binding specificity like the TAP molecule. There is also a single dominantly expressed class II β gene, but the reason for this is not yet clear. Finally, the presence of the C4 gene outside of the classical class I α and class II β genes suggests the possibility that this organisation was ancestral, although a number of models of organization and evolution are still possible, given the presence of the Rfp‐γ region with non‐classical class I α and class II β genes as well as the presence of multigene families of B‐G and rRNA genes.

The homologue of mannose-binding lectin in the carp family Cyprinidae is expressed at high level in spleen, and the deduced primary structure predicts affinity for galactose

Abstract. Mannose-binding lectin (MBL) participates in the innate immune system as an activator of the complement system and as an opsonin after binding to certain carbohydrate structures on microorganisms. We isolated and characterized cDNA transcripts encoding an MBL homologue from three members of the carp family Cyprinidae, the zebrafish Danio rerio, the goldfish Carassius auratus, and the carp Cyprinus carpio. The carp and zebrafish transcripts contain two polyadenylation sites and RT-PCR on mRNA from carp tissues revealed the carp transcript to be most prominently expressed in the spleen. The deduced mature proteins contain 228 or 233 amino acids with a short N-terminal segment containing a single conserved cysteine expected to form interchain disulfide bridges, a collagen domain interrupted by four amino acids between two glycine residues, a neck region predicted to form an α-helical coiled-coil structure, and a C-terminal carbohydrate recognition domain (CRD). Several of the structurally important residues in the CRD are conserved, but the residues known to interact with the calcium ion and hydroxyl groups of the carbohydrate ligand are different. The amino acid motif EPN, important for mannose specificity, was QPD in the Cyprinidae homologue, suggesting specificity for galactose instead. The identity between the deduced amino acid sequences is more than 90% between the carp and the goldfish and 68% and 65% between these two species, respectively, and the zebrafish. The identity with bird and mammalian MBLs ranges from 28 to 33%.

The MHC Molecules of Nonmammalian Vertebrates

There is very little known about the long-term evolution of the MHC and MHC-like molecules. This is because both the theory (the evolutionary questions and models) and the practice (the animals systems, functional assays and reagents to identify and characterize these molecules) have been difficult to develop. There is no molecular evidence yet to decide whether vertebrate immune systems (and particularly the MHC molecules) are evolutionarily related to invertebrate allorecognition systems, and the functional evidence can be interpreted either way. Even among the vertebrates, there is great heterogeneity in the quality and quantity of the immune response. The functional evidence for T-lymphocyte function in jawless and cartilagenous fish is poor, while the bony fish seem to have many characteristics of a mammalian immune system. The organization and sequence of fish Ig genes also indicate that important events in the evolution of the immune system and the MHC occurred in the fish, but thus far there is no molecular evidence for recognizable MHC-like molecules in any fish. There is clearly an MHC in amphibians and birds with many characteristics like the MHC of mammals (a single genetic region encoding polymorphic class I and class II molecules) and evidence for polymorphic class I and class II molecules in reptiles. However, many details differ from the mammals, and it is not clear whether these reflect historical accident or selection for different lifestyles or environment. For example, the adult frog Xenopus has a vigorous immune system with many similarities to mammals, a ubiquitous class I molecule, but a much wider class II tissue distribution than human, mouse and chicken. The Xenopus tadpole has a much more restricted immune response, no cell surface class I molecules and a mammalian class II distribution. The axolotl has a very poor immune response (as though there are no helper T cells), a wide class II distribution and, for most animals, no cell surface class I molecule. It would be enlightening to understand both the mechanisms for the regulation of the MHC molecules during ontogeny and the consequences for the immune system and survival of the animals. These animals also differ markedly in the level of MHC polymorphism. Another difference from mammals is the presence of previously uncharacterized molecules. In Xenopus and reptiles, there are two populations of class I alpha chain on the surface of erythrocytes, those in association with beta 2m and those in association with a disulfide-linked homodimer.(ABSTRACT TRUNCATED AT 400 WORDS)

The chicken erythrocyte-specific MHC antigen. Characterization and purification of the B-G antigen by monoclonal antibodies

Mouse monoclonal antibodies with B-G antigen (major histocompatibility complex class IV) specificity were obtained after immunization with erythrocytes or partially purified B-G antigen. The specificities of the hybridoma antibodies were determined by precipitation of B-G antigens from 125I-labeled chicken erythrocyte membranes (CEM) followed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and autoradiography. The B-G antigen had an approximate molecular mass of 46–48 kd in reduced samples, depending on the haplotype, and in unreduced samples contained either dimers (85 kd), when labeled erythrocytes were the antigen source, or trimers (130 kd), when B-G was purified and precipitated from CEM. The B-G antigen was unglycosylated as studied by (1) in vitro synthesis in the presence or absence of tunicamycin, (2) binding experiments with lectin from Phaseolus limensis, and (3) treatment of purified B-G antigen with Endoglycosidase-F or trifluoromethanesulfonic acid. Two-way sequential immunoprecipitation studies of erythrocyte membrane extracts with anti-B-G alloantisera and monoclonal antibodies revealed only one population of B-G molecules. Pulse-chase experiments have shown B-G to be synthesized as a monomer, with dimerization taking place after 20–30 min. No change in the monomers molecular mass due to posttranslational modifications was revealed. The antigen was purified from detergent extract of CEM by affinity chromatography with a monoclonal antibody, and then reduced and alkylated and affinity-purified once more. Finally, reverse-phase chromatography resulted in a pure product. The B-G antigen was identified in the various fractions by rocket immunoelectrophoresis. The final product was more than 99% pure, as estimated by SDSPAGE analysis followed by silver stain of proteins. The yield from the affinity chromatography step was 3–4 μg B-G/ml blood, calculated from Coomassie-stained SDS-PAGE of B-G using ovalbumin standards. The monoclonal antibodies were also used to identify the B-G (class IV) precipitation arc in crossed immunoelectrophoresis. No common precipitate with the B-F (class I) antigen was observed.

BG: we know what it is, but what does it do?☆

B-G molecules are polymorphic cell surface proteins that are encoded by the chicken MHC. Here, Jim Kaufman and Jan Salomonsen briefly summarize developments in the molecular genetics, the structure and the tissue distribution of B-G molecules, and discuss possible functions of this intriguing multigene family.

B-G cDNA clones have multiple small repeats and hybridize to both chicken MHC regions.

We used rabbit antisera to the chicken MHC erythrocyte molecule B-G and to the class I α chain (B-F) to screen λgt11 cDNA expression libraries made with RNA selected by oligo-dT from bone marrow cells of anemicB19 homozygous chickens. Eight clones were found to encode B-G molecules which hybridize with sequences in the chicken MHC as defined by congenic strains; the fusion proteins react with multiple immune but not preimmune sera, they select antibodies from the antisera to B-G, which then react with distinct erythrocyte B-G protein patterns, and they elicit antibodies from mice which in turn react with authentic B-G proteins. None of the clones represent a complete message, some — if not all — bear introns, and none of them match with any sequences presently stored in the data banks. The following new information did, however, emerge. At least two homologous transcripts are present in this homozygous chicken, thereby formally proving the existence of an expressed multigene family. The 3′ ends (3′UT) are simple sequences with 80% nucleotide identity between clones, while the 5′ ends (either coding or noncoding) are composed of multiple short repeats which are far less similar. These repeats could explain the bewildering variation in size of B-G proteins within and between haplotypes. Southern blots of genomic chicken DNA gave complex patterns for most probes, with many bands in common using different probes, but few bands in common between haplotypes. The sequences detected are all present in the MHC, based on the congenic lines CB and CC. Most of these sequences map into theB-G region, but some map into theB-F/B-L region as defined by the haplotypesB15, B21, and their apparently reciprocal recombinantsB21r3 andB15r1.

Two mannose-binding lectin homologues and an MBL-associated serine protease are expressed in the gut epithelia of the urochordate species Ciona intestinalis.

The lectin complement pathway has important functions in vertebrate host defence and accumulating evidence of primordial complement components trace its emergence to invertebrate phyla. We introduce two putative mannose-binding lectin homologues (CioMBLs) from the urochordate species Ciona intestinalis. The CioMBLs display similarities with vertebrate MBLs and comprise a collagen-like region, alpha-helical coiled-coils and a carbohydrate recognition domain (CRD) with conserved residues involved in calcium and carbohydrate binding. Structural analysis revealed an oligomerization through interchain disulphide bridges between N-terminal cysteine residues and cysteines located between the neck region and the CRD. RT-PCR showed a tissue specific expression of CioMBL in the gut and by immunohistochemistry analysis we also demonstrated that CioMBL co-localize with an MBL-associated serine protease in the epithelia cells lining the stomach and intestine. In conclusion we present two urochordate MBLs and identify an associated serine protease, which support the concept of an evolutionary ancient origin of the lectin complement pathway.

Isolation and characterization of chicken and turkey beta2-microglobulin

Abstract Chicken and turkey beta2-m were isolated from citrated plasma in sequential use of three Chromatographie steps: affinity chromatography, gel filtration chromatography and anion-exchange chromatography. The purified protein was identified as beta2-m by reaction with a beta2-m specific monoclonal antibody and by the ability to recombine with the chicken MHC class I heavy chain. The purity was estimated by SDS-PAGE and IEF. The pI was between 5.1 and 5.3 for chicken beta2-m and 4.7 and 4.8 for turkey beta2-m, which fact is reflected in their different electrophoretic mobilities in agarose gel (turkey migrates in the alpha and chicken migrates in the beta region). The mol. wt of both chicken and turkey beta2-m was 14,500 estimated by SDS-PAGE whereas calculations based on the amino acid compositions gave mol. wts of 11,000. E M 280 was 15.9 for chicken beta2-m and 16.4 for turkey beta2-m. The amino acid compositions and sequences of the two avian beta2-m molecules have been compared with earlier data from the literature. The sequence of the 23 N -terminal amino acids was found to be identical in our preparations from both chicken and turkey, namely DLTPKVQVYSRFPASAGTKNVLN, and is incompatible with a previously published sequence also thought to be from turkey beta2-m. Reasons for our opinion that the molecules isolated and sequenced in this paper are the correct ones are given.

Sequence of a complete chicken BG haplotype shows dynamic expansion and contraction of two gene lineages with particular expression patterns.

Many genes important in immunity are found as multigene families. The butyrophilin genes are members of the B7 family, playing diverse roles in co-regulation and perhaps in antigen presentation. In humans, a fixed number of butyrophilin genes are found in and around the major histocompatibility complex (MHC), and show striking association with particular autoimmune diseases. In chickens, BG genes encode homologues with somewhat different domain organisation. Only a few BG genes have been characterised, one involved in actin-myosin interaction in the intestinal brush border, and another implicated in resistance to viral diseases. We characterise all BG genes in B12 chickens, finding a multigene family organised as tandem repeats in the BG region outside the MHC, a single gene in the MHC (the BF-BL region), and another single gene on a different chromosome. There is a precise cell and tissue expression for each gene, but overall there are two kinds, those expressed by haemopoietic cells and those expressed in tissues (presumably non-haemopoietic cells), correlating with two different kinds of promoters and 5′ untranslated regions (5′UTR). However, the multigene family in the BG region contains many hybrid genes, suggesting recombination and/or deletion as major evolutionary forces. We identify BG genes in the chicken whole genome shotgun sequence, as well as by comparison to other haplotypes by fibre fluorescence in situ hybridisation, confirming dynamic expansion and contraction within the BG region. Thus, the BG genes in chickens are undergoing much more rapid evolution compared to their homologues in mammals, for reasons yet to be understood.