Robert N. Haire

Identification of diversified genes that contain immunoglobulin-like variable regions in a protochordate

The evolutionary origin of adaptive immune receptors is not understood below the phylogenetic level of the jawed vertebrates. We describe here a strategy for the selective cloning of cDNAs encoding secreted or transmembrane proteins that uses a bacterial plasmid (Amptrap) with a defective β-lactamase gene. This method requires knowledge of only a single target motif that corresponds to as few as three amino acids; it was validated with major histocompatibility complex genes from a cartilaginous fish. Using this approach, we identified families of genes encoding secreted proteins with two diversified immunoglobulin-like variable (V) domains and a chitin-binding domain in amphioxus, a protochordate. Thus, multigenic families encoding diversified V regions exist in a species lacking an adaptive immune response.

The phylogenetic origins of the antigen-binding receptors and somatic diversification mechanisms

Summary:  The adaptive immune system arose in ancestors of the jawed vertebrates approximately 500 million years ago. Homologs of immunoglobulins (Igs), T‐cell antigen receptors (TCRs), major histocompatibility complex I (MHC I) and MHC II, and the recombination‐activating genes (RAGs) have been identified in all extant classes of jawed vertebrates; however, no definitive homolog of any of these genes has been identified in jawless vertebrates or invertebrates. RAG‐mediated recombination and associated junctional diversification of both Ig and TCR genes occurs in all jawed vertebrates. In the case of Igs, somatic variation is expanded further through class switching, gene conversion, and somatic hypermutation. Although the identity of the ‘primordial’ receptor that was interrupted by the recombination mechanism in jawed vertebrates may never be established, many different families of genes that exhibit predicted characteristics of such a receptor have been described both within and outside the jawed vertebrates. Recent data from various model systems point toward a continuum of immune receptor diversity, encompassing many different families of recognition molecules whose functions are integrated in an organisms response to pathogenic invasion. Various approaches, including both genomic and protein‐functional analyses, currently are being applied in jawless vertebrates, protochordates, and other invertebrate deuterostome systems and may yield definitive evidence regarding the presence or absence of adaptive immune homologs in species lacking adaptive immune systems. Such studies have the potential for uncovering previously unknown mechanisms of generating receptor diversity.

Characterization of three isotypes of immunoglobulin light chains and T-cell antigen receptor α in zebrafish.

Abstract. The zebrafish (Danio rerio) has become a significant model for understanding the developmental regulation of gene expression and holds considerable potential for characterizing the development of the immune system. Using a number of different approaches, including heterologous hybridization and short-primer PCR, cDNAs for three different classes of light-chain genes were identified and characterized. The zebrafish light chains are similar to trout type 1, trout type 2, and catfish type F, respectively. T-cell antigen receptor α (TCRα) was also identified and characterized. A high proportion of unusual transcripts including sterile transcripts, germline VJC transcripts, aberrant splice forms, and V-V transcripts were encountered in the immunoglobulin and TCR cDNAs examined. The light-chain and TCRα loci each consist of multiple families of V gene segments, apparent even from the small numbers of cDNAs of each isotype sequenced. The gene sequences reported provide an essential set of markers of both B- and T-cell lineages that will facilitate investigations of immune system development.

Extraordinary variation in a diversified family of immune-type receptor genes

Immune inhibitory receptor genes that encode a variable (V) region, a unique V-like C2 (V/C2) domain, a transmembrane region, and a cytoplasmic tail containing immunoreceptor tyrosine-based inhibition motifs (ITIMs) have been described previously in two lineages of bony fish. In the present study, eleven related genes encoding distinct structural forms have been identified in Ictalurus punctatus (channel catfish), a well characterized immunological model system that represents a third independent bony fish lineage. Each of the different genes encodes an N-terminal V region but differs in the number of extracellular Ig domains, number and location of joining (J) region-like motifs, presence of transmembrane regions, presence of charged residues in transmembrane regions, presence of cytoplasmic tails, and/or distribution of ITIM(s) within the cytoplasmic tails. Variation in the numbers of genomic copies of the different gene types, their patterns of expression, and relative levels of expression in mixed leukocyte cultures (MLC) is reported. V region-containing immune-type genes constitute a far more complex family than recognized originally and include individual members that might function in inhibitory or, potentially activatory manners.

Identification and characterization of T-cell antigen receptor-related genes in phylogenetically diverse vertebrate species.

Characterization of the structure, multiplicity, organization, and cell lineage-specific expression of T-cell receptor (TCR) genes of nonmammalian vertebrate species is central to the understanding of the evolutionary origins of rearranging genes of the vertebrate immune system. We recently described a polymerase chain reaction (PCR) strategy that relies on short sequence similarities shared by nearly all vertebrate TCR and immunoglobulin (Ig) variable (V) regions and have used this approach to isolate a TCR beta (TCRB) homolog from a cartilaginous fish. Using these short PCR products as probes in spleen cDNA and genomic libraries, we were able to isolate a variety of unique TCR and TCR-like genes. Here we report the identification and characterization of a chicken TCR gamma (TCRG) homolog, apparent Xenopus and pufferfish TCR alpha (TCRA) homologs, and two horned shark TCR delta (TCRD)-like genes. In addition, we have identified what could be a novel representative of the Ig gene super-family in the pufferfish. This method of using short, minimally degenerate PCR primers should speed progress in the phylogenetic investigations of the TCR and related genes and lend important insights into both the origins and functions of these unique gene systems.

Individual protochordates have unique immune-type receptor repertoires

Innate immunity is mediated by a variety of different, non-rearranging receptors and soluble molecules that recognize and facilitate the elimination of a wide range of pathogens [1]. Immunoglobulin (Ig)-type variable (V) region-containing chitin-binding proteins (VCBPs) found in the protochordate amphioxus, which diverged from the vertebrate lineage before the emergence of adaptive immunity, show structural characteristics of innate immune receptors [2]. Here we describe a very high degree of regionalized hypervariability in one family of VCBPs at the level of the individual germline, producing a unique repertoire of proteins in every animal so far analyzed. In species with large populations, such as amphioxus, extensive polymorphisms may compensate for the absence of somatic modification in the maintenance of immune receptor diversity. The diversified VCBPs in the amphioxus Branchiostoma floridae, a cephalochordate, are soluble proteins consisting of two Ig-type V regions joined to a carboxy-terminal chitin-binding domain [2]. Although their ligands are unknown, VCBPs are likely candidates for innate immune receptors and represent the only example of an innate receptor in which the functional unit is a hyperdiversified Ig-type V region. VCBPs are distributed in at least five families and are expressed specifically and abundantly in the gut. Regionalized peptide sequence hypervariability was noted in the V regions of pooled amphioxus VCBP2 cDNAs [2]. The hypervariable region is centered ~18 residues amino-terminal to the first intradomain cysteine and does not correspond to any of the known complementarity-determining regions of the rearranging antigen-binding receptors found in jawed vertebrates. However, the basis for the hypervariation is not clear. The hypervariable region of the amino-terminal V region of VCBP2 has been characterized in genomic DNA from individual animals collected in the same local geographical area. The germline of every animal encodes a unique VCBP2 receptor repertoire. In a parallel investigation, a 17-fold representative bacterial artificial chromosome library (CHORI-302) constructed from a single reference animal was screened and six VCBP2 family genes were identified, consistent with previous Southern blot analyses [2]. In sum, a total of 43 different peptides are encoded across the amplified region of VCBP2 by only 13 different animals. The most pronounced differences occur across a segment of ~12–15 residues within a 23–30 residue-encoding amplicon (Figure 1). In the majority of cases, any two animals share no more than two specific VCBP2 hypervariable sequences; however, four pairs of animals share three specific hypervariable sequences. Amplicons encoding more conserved regions of the amino-terminal V region …

Members of the Ikaros Gene Family Are Present in Early Representative Vertebrates

Members of the Ikaros multigene family of zinc finger proteins are expressed in a tissue-specific manner and most are critical determinants in the development of both the B and T lymphocytes as well as NK and dendritic APC lineages. A PCR amplification strategy that is based on regions of shared sequence identity in Ikaros multigene family members found in mammals and several other vertebrates has led to the recovery of cDNAs that represent the orthologues of Ikaros, Aiolos, Helios, and Eos in Raja eglanteria (clearnose skate), a cartilaginous fish that is representative of an early divergence event in the phylogenetic diversification of the vertebrates. The tissue-specific patterns of expression for at least two of the four Ikaros family members in skate resemble the patterns observed in mammals, i.e., in hematopoietic tissues. Prominent expression of Ikaros in skate also is found in the lymphoid Leydig organ and epigonal tissues, which are unique to cartilaginous fish. An Ikaros-related gene has been identified in Petromyzon marinus (sea lamprey), a jawless vertebrate species, in which neither Ig nor TCRs have been identified. In addition to establishing a high degree of evolutionary conservation of the Ikaros multigene family from cartilaginous fish through mammals, these studies define a possible link between factors that regulate the differentiation of immune-type cells in the jawed vertebrates and related factors of unknown function in jawless vertebrates.

A role for variable region-containing chitin-binding proteins (VCBPs) in host gut-bacteria interactions.

A number of different classes of molecules function as structural matrices for effecting innate and adaptive immunity. The most extensively characterized mediators of adaptive immunity are the immunoglobulins and T-cell antigen receptors found in jawed vertebrates. In both classes of molecules, unique receptor specificity is effected through somatic variation in the variable (V) structural domain. V region-containing chitin-binding proteins (VCBPs) consist of two tandem Ig V domains as well as a chitin-binding domain. VCBPs are encoded at four loci (i.e., VCBPA–VCBPD) in Ciona, a urochordate, and are expressed by distinct epithelial cells of the stomach and intestine, as well as by granular amoebocytes present in the lamina propria of the gut and in circulating blood. VCBPs are secreted into the gut lumen, and direct binding to bacterial surfaces can be detected by immunogold analysis. Affinity-purified native and recombinant VCBP-C, as well as a construct consisting only of the tandem V domains, enhance bacterial phagocytosis by granular amoebocytes in vitro. Various aspects of VCBP expression and function suggest an early origin for the key elements that are central to the dialogue between the immune system of the host and gut microflora.

Extreme variation in X-linked agammaglobulinemia phenotype in a three-generation family

BACKGROUND X-linked agammaglobulinemia is typically a severe life-threatening disease characterized by the failure of B-cell differentiation and antibody production, which manifests in infancy and early childhood. Recently, we reported a novel mutation (Cys145-->STOP) in Brutons tyrosine kinase in a 51-year-old man who was referred for evaluation because of chronic nasal congestion, recurrent sinusitis, sporadic pneumonia, and a family history suggestive of an X-linked immunodeficiency disease. He had not been treated with gammaglobulin. OBJECTIVE This study was performed to investigate the clinical and immunologic phenotypes of this patients other affected male family members. METHODS A detailed family history and comprehensive review of medical records was carried out. Genetic mutation analysis of the gene encoding Brutons tyrosine kinase was carried out in the probands brother and nephew. RESULTS Clinically affected male family members exhibit marked phenotypic variation with manifestations ranging from extremely mild to severe recurrent infections. Immunologic evaluation revealed extreme variation in immunoglobulin levels, B-cell numbers, and functional antibody titers. Genetic analysis documented a novel mutation in the gene encoding Brutons tyrosine kinase in the proband, his brother, and his nephew. CONCLUSIONS Despite their sharing the same genetic abnormality, extreme variation was noted in the immunologic findings and phenotypic expression of affected family members. This family study is extraordinary in that clinically affected male members who did not receive aggressive medical treatment died of the disease in childhood or survived into late adulthood.

Ancient evolutionary origin of diversified variable regions demonstrated by crystal structures of an immune-type receptor in amphioxus

Although the origins of genes encoding the rearranging binding receptors remain obscure, it is predicted that their ancestral forms were nonrearranging immunoglobulin-type domains. Variable region–containing chitin-binding proteins (VCBPs) are diversified immune-type molecules found in amphioxus (Branchiostoma floridae), an invertebrate that diverged early in deuterostome phylogeny. To study the potential evolutionary relationships between VCBPs and vertebrate adaptive immune receptors, we solved the structures of both a single V-type domain (to 1.15 Å) and a pair of V-type domains (to 1.85 Å) from VCBP3. The deduced structures show integral features of the ancestral variable-region fold as well as unique features of variable-region pairing in molecules that may reflect characteristics of ancestral forms of diversified immune receptors found in modern-day vertebrates.