Masanori Kasahara

Origin and evolution of the adaptive immune system: genetic events and selective pressures

The adaptive immune system (AIS) in mammals, which is centred on lymphocytes bearing antigen receptors that are generated by somatic recombination, arose approximately 500 million years ago in jawed fish. This intricate defence system consists of many molecules, mechanisms and tissues that are not present in jawless vertebrates. Two macroevolutionary events are believed to have contributed to the genesis of the AIS: the emergence of the recombination-activating gene (RAG) transposon, and two rounds of whole-genome duplication. It has recently been discovered that a non-RAG-based AIS with similarities to the jawed vertebrate AIS — including two lymphoid cell lineages — arose in jawless fish by convergent evolution. We offer insights into the latest advances in this field and speculate on the selective pressures that led to the emergence and maintenance of the AIS.

The amphioxus genome illuminates vertebrate origins and cephalochordate biology

Cephalochordates, urochordates, and vertebrates evolved from a common ancestor over 520 million years ago. To improve our understanding of chordate evolution and the origin of vertebrates, we intensively searched for particular genes, gene families, and conserved noncoding elements in the sequenced genome of the cephalochordate Branchiostoma floridae, commonly called amphioxus or lancelets. Special attention was given to homeobox genes, opsin genes, genes involved in neural crest development, nuclear receptor genes, genes encoding components of the endocrine and immune systems, and conserved cis-regulatory enhancers. The amphioxus genome contains a basic set of chordate genes involved in development and cell signaling, including a fifteenth Hox gene. This set includes many genes that were co-opted in vertebrates for new roles in neural crest development and adaptive immunity. However, where amphioxus has a single gene, vertebrates often have two, three, or four paralogs derived from two whole-genome duplication events. In addition, several transcriptional enhancers are conserved between amphioxus and vertebrates--a very wide phylogenetic distance. In contrast, urochordate genomes have lost many genes, including a diversity of homeobox families and genes involved in steroid hormone function. The amphioxus genome also exhibits derived features, including duplications of opsins and genes proposed to function in innate immunity and endocrine systems. Our results indicate that the amphioxus genome is elemental to an understanding of the biology and evolution of nonchordate deuterostomes, invertebrate chordates, and vertebrates.

The MHC class I ligand-generating system: roles of immunoproteasomes and the interferon-4gMY-inducible proteasome activator PA28

Summary: Production of antigenic peptides that serve as MHC class I ligands is essential for initiation of cell‐mediated immunity. Accumulating evidence indicates that the proteasome, a large multisubunit protein degradative machine in eukaryotes, functions as a processing enzyme responsible for the generation of MHC class I ligands. This processing system is elaborately regulated by various immunomodulatory cytokines. In particular, incerferon‐γ induces the formation of immunoproteasomes and a recently identified proteasomal regulatory factor, PA28, which in concert contribute co efficient production of MHC class I ligands. Many of the MHC‐encoded genes including LMP appear to have emerged by an ancient chromosomal duplication, suggesting that modifications and renewal of pre‐existing non‐immune genes were instrumental in the emergence of adaptive immunity.

Cloning and mapping of a testis-specific gene with sequence similarity to a sperm-coating glycoprotein gene ☆

A testis-specific gene Tpx-1, located between Pgk-2 and Mep-1 on mouse chromosome 17, was isolated from a cosmid clone, and its cDNA sequences were determined. The predicted coding sequence of Tpx-1 isolated from BALB/c mice showed 64.2% nucleotide and 55.1% amino acid sequence similarity with that of a rat sperm-coating glycoprotein gene, the protein product of which is secreted by the epididymis. To examine the evolutionary relationship between Tpx-1 and a sperm-coating glycoprotein gene, the cDNA sequence of TPX1, the human counterpart of Tpx-1, was determined. The comparison of the predicted coding sequences of Tpx-1 and TPX1 showed 77.8% nucleotide and 70% amino acid sequence similarity. Since Tpx-1 (from mouse) is more similar to TPX1 (from man) than it is to a rat sperm-coating glycoprotein gene, we conclude that Tpx-1 (TPX1) and a sperm-coating glycoprotein gene are closely related, but distinct, genes belonging to the same gene family. The predicted Tpx-1 protein of a t mutant mouse CRO437 differs from that of BALB/c mice by one amino acid insertion in the putative signal peptide. TPX1 was mapped to 6p21-qter by Southern blot analysis of interspecies somatic hybrid cell lines.

Growth Retardation in Mice Lacking the Proteasome Activator PA28γ

The proteasome activator PA28 binds to both ends of the central catalytic machine, known as the 20 S proteasome, in opposite orientations to form the enzymatically active proteasome. The PA28 family is composed of three members designated α, β, and γ; PA28α and PA28β form the heteropolymer mainly located in the cytoplasm, whereas PA28γ forms a homopolymer that predominantly occurs in the nucleus. Available evidence indicates that the heteropolymer of PA28α and PA28β is involved in the processing of intracellular antigens, but the function of PA28γ remains elusive. To investigate the role of PA28γ in vivo, we generated mice deficient in the PA28γ gene. The PA28γ-deficient mice were born without appreciable abnormalities in all tissues examined, but their growth after birth was retarded compared with that of PA28γ+/− or PA28γ+/+ mice. We also investigated the effects of the PA28γ deficiency using cultured embryonic fibroblasts; cells lacking PA28γ were larger and displayed a lower saturation density than their wild-type counterparts. Neither the expression of PA28α/β nor the subcellular localization of PA28α was affected in PA28γ−/− cells. These results indicate that PA28γ functions as a regulator of cell proliferation and body growth in mice and suggest that neither PA28α nor PA28β compensates for the PA28γ deficiency.

VLR-Based Adaptive Immunity

Lampreys and hagfish are primitive jawless vertebrates capable of mounting specific immune responses. Lampreys possess different types of lymphocytes, akin to T and B cells of jawed vertebrates, that clonally express somatically diversified antigen receptors termed variable lymphocyte receptors (VLRs), which are composed of tandem arrays of leucine-rich repeats. The VLRs appear to be diversified by a gene conversion mechanism involving lineage-specific cytosine deaminases. VLRA is expressed on the surface of T-like lymphocytes; B-like lymphocytes express and secrete VLRB as a multivalent protein. VLRC is expressed by a distinct lymphocyte lineage. VLRA-expressing cells appear to develop in a thymus-like tissue at the tip of gill filaments, and VLRB-expressing cells develop in hematopoietic tissues. Reciprocal expression patterns of evolutionarily conserved interleukins and chemokines possibly underlie cell-cell interactions during an immune response. The discovery of VLRs in agnathans illuminates the origins of adaptive immunity in early vertebrates.

A novel type of class I gene organization in vertebrates: a large family of non-MHC-linked class I genes is expressed at the RNA level in the amphibian Xenopus.

A Xenopus class I cDNA clone, isolated from a cDNA expression library using antisera, is a member of a large family of non‐classical class I genes (class Ib) composed of at least nine subfamilies, all of which are expressed at the RNA level. The subfamilies are well conserved in their immunoglobulin‐like alpha 3 domains, but their peptide‐binding regions (PBRs) and cytoplasmic domains are very divergent. In contrast to the great allelic diversity found in the PBR of classical class I genes, the alleles of one of the Xenopus non‐classical subfamilies are extremely well conserved in all regions. Several of the invariant amino acids essential for the anchoring of peptides in the classical class I groove are not conserved in some subfamilies, but the class Ib genes are nevertheless more closely related in the PBR to classical and non‐classical genes linked to the MHC in mammals and birds than to any other described class I genes like CD1 and the neonatal rat intestinal Fc receptor. Comparison with the Xenopus MHC‐linked class Ia protein indicate that amino acids presumed to interact with beta 2‐microglobulin are identical or conservatively changed in the two major class I families. Genomic analyses of Xenopus species suggest that the classical and non‐classical families diverged from a common ancestor before the emergence of the genus Xenopus over 100 million years ago; all of the non‐classical genes appear to be linked on a chromosome distinct from the one harboring the MHC. We hypothesize that this class Ib gene family is under very different selection pressures from the classical MHC genes, and that each subfamily may have evolved for a particular function.

Immunoproteasome assembly and antigen presentation in mice lacking both PA28α and PA28β

Two members of the proteasome activator, PA28α and PA28β, form a heteropolymer that binds to both ends of the 20S proteasome. Evidence in vitro indicates that this interferon‐γ (IFN‐γ)‐inducible heteropolymer is involved in the processing of intracellular antigens, but its functions in vivo remain elusive. To investigate the role of PA28α/β in vivo, we generated mice deficient in both PA28α and PA28β genes. The ATP‐dependent proteolytic activities were decreased in PA28α−/−/β−/− cells, suggesting that ‘hybrid proteasomes’ are involved in protein degradation. Treatment of PA28α−/−/β−/− cells with IFN‐γ resulted in sufficient induction of the ‘immunoproteasome’. Moreover, splenocytes from PA28α−/−/β−/− mice displayed no apparent defects in processing of ovalbumin. These results are in marked contrast to the previous finding that immunoproteasome assembly and immune responses were impaired in PA28β−/− mice. PA28α−/−/β−/− mice also showed apparently normal immune responses against infection with influenza A virus. However, they almost completely lost the ability to process a melanoma antigen TRP2‐derived peptide. Hence, PA28α/β is not a prerequisite for antigen presentation in general, but plays an essential role for the processing of certain antigens.

Structural Diversity of the Hagfish Variable Lymphocyte Receptors

Variable lymphocyte receptors (VLRs) are recently discovered leucine-rich repeat (LRR) family proteins that mediate adaptive immune responses in jawless fish. Phylogenetically it is the oldest adaptive immune receptor and the first one with a non-immunoglobulin fold. We present the crystal structures of one VLR-A and two VLR-B clones from the inshore hagfish. The hagfish VLRs have the characteristic horseshoe-shaped structure of LRR family proteins. The backbone structures of their LRR modules are highly homologous, and the sequence variation is concentrated on the concave surface of the protein. The conservation of key residues suggests that our structures are likely to represent the LRR structures of the entire repertoire of jawless fish VLRs. The analysis of sequence variability, prediction of protein interaction surfaces, amino acid composition analysis, and structural comparison with other LRR proteins suggest that the hypervariable concave surface is the most probable antigen binding site of the VLR.

Which came first, MHC class I or class II?

The topic of this paper, the origins of MHC proteins, seems too difficult to hope to understand. It is our intention, however, to introduce an idea that may influence the way one views the evolution of the MHC. MHC-encoded class I and class II proteins are composed of four extracellular domains, each made up of approximately 90 amino acids. The two membrane-proximal domains are members of the C-1 set of the immunoglobulin (Ig) superfamily (Williams and Barclay 1988). The two two membrane distal domains of class I (and presumably class II) combine to form a peptide-binding cleft composed of a floor of eight /3 strands upon which rest two antiparallel a helices (Bjorkman et al. 1987). The MHC-encoded class I e~ chain is composed of three domains, and is non-covalently associated with the non-MHC encoded /32-microglobulin (Silver and Hood 1974). The peptide-binding domains, both found in the a chain (~-1 and a-2), form an intramolecular dimer (Bjorkman et al. 1987). Both class II proteins are MHCencoded and composed of two domains each; the membrane-distal domains probably form an intermolecular dimer, perhaps stabilized by the peptide (Brown et al. 1988; Mellins et al. 1990). While the membrane-distal domains of MHC molecules are certainly members of the Ig superfamily, the membrane-distal domains that comprise the peptidebinding region are unique and unrelated to any described proteins (Kaufman and Strominger 1982; Bjorkman et al. 1987). Since the peptide-binding domains are approximately the same size as Ig domains, and the intradomain disulfide bond of MHC proteins (in a-2 of class I and/3-1 of class II) and Ig domains is formed by cysteinyl residues spaced approximately the same distance apart, some investigators believe that the peptide-binding domains may be derived from Ig-like domains. In addition, low amino