M. Nonaka

Molecular cloning of rat and mouse membrane cofactor protein (MCP, CD46): preferential expression in testis and close linkage between the mouse Mcp and Cr2 genes on distal chromosome 1

Abstract Human membrane cofactor protein (MCP, CD46) is widely distributed and is one of the plasma membrane complement inhibitors. We isolated cDNA clones encoding genetic homologues of human MCP from a rat testis cDNA library. Northern blot analysis indicated that rat MCP is preferentially expressed in testis, similar to what is found with guinea pig MCP. We identified several different cDNAs, which were presumably generated by alternative splicing from a single-copy gene. The most prevalent isoform corresponded to the Ser/Thr/Pro-rich C type of human MCP. Mouse MCP cDNA was cloned by polymerase chain reaction based on the nucleotide sequence of rat MCP. The deduced amino acid sequence showed 77.8% identity to rat MCP. Mouse MCP was also preferentially expressed in testis. Unique expression in testis in rat and mouse as well as guinea pig suggests that MCPs in these species not only act as complement regulatory proteins but may also have more specialized functions in fertilization or reproduction. Genetic mapping by linkage analysis indicated that the mouse Mcp gene is located on distal chromosome 1, closely linked to the complement receptor 2 (Cr2) gene.

Molecular cloning and linkage analysis of the Japanese medaka fish complementBf/C2 gene

Evolutionary studies of complement factor B (Bf) and C2 in lower vertebrates have revealed the presence of the Bf/C2 common ancestor-like molecule in lamprey (cyclostome) and the Bf molecule encoded by the duplicated genes closely linked to the major histocompatibility complex (MHC) inXenopus (amphibian). To further define whenBf/C2 gene duplication occurred and when linkage between theBf/C2 gene and theMHC was established, we amplified theBf/C2 sequences in teleost, the Japanese medaka (Oryzias latipes), by reverse transcription—polymerase chain reaction with primers corresponding to the common amino acid sequences shared by mammalian Bf and C2. Only a single molecular species has been amplified, and the corresponding cDNA clones were isolated from the liver cDNA library. The longest insert contained 2384 nucleotides with an open reading frame of 754 residues. The deduced amino acid sequence showed 33.6% and 34.1% overall identity with the human Bf and C2 sequences, respectively, hence this clone was named medakaBf/C2. The single-copy medakaBf/C2 gene had exactly the same exon-intron organization as the mammalianBf andC2 genes, and spanned about 8 kilobases. TheBf/C2 locus was mapped to the close proximity (2.9cM) of the superoxide dismutase locus on the linkage group XX by the use of a restriction site polymorphism between two inbred strains of the medaka.

Molecular Cloning of C4 Gene and Identification of the Class III Complement Region in the Shark MHC

To clarify the evolutionary origin of the linkage of the MHC class III complement genes with the MHC class I and II genes, we isolated C4 cDNA from the banded hound shark (Triakis scyllium). Upon phylogenetic tree analysis, shark C4 formed a well-supported cluster with C4 of higher vertebrates, indicating that the C3/C4 gene duplication predated the divergence of cartilaginous fish from the main line of vertebrate evolution. The deduced amino acid sequence predicted the typical C4 three-subunits chain structure, but without the histidine residue catalytic for the thioester bond, suggesting the human C4A-like specificity. The linkage analysis of the complement genes, one C4 and two factor B (Bf) genes, to the shark MHC was performed using 56 siblings from two typing panels of T. scyllium and Ginglymostoma cirratum. The C4 and one of two Bf genes showed a perfect cosegregation with the class I and II genes, whereas two recombinants were identified for the other Bf gene. These results indicate that the linkage between the complement C4 and Bf genes, as well as the linkage between these complement genes and the MHC class I and II genes were established before the emergence of cartilaginous fish >460 million years ago.

Conservation of the modular structure of complement factor I through vertebrate evolution

Mammalian complement factor I plays pivotal roles in the regulation of complement activation and generation of important biological activities from C3. The evolutionary origin of factor I has been unclear except with regard to the molecular cloning of factor I from amphibian Xenopus. Here, we report the identification and characterization of factor I cDNA from the liver of the banded houndshark. The deduced amino acid sequence of shark factor I showed a modular organization that was completely identical to that of mammalian factor I, suggesting the functional conservation of factor I throughout vertebrate evolution. Functionally important amino acid residues such as the basic residues at the processing site and the residues at the active site of the serine protease domain are conserved. Repeated sequences composed of 16 amino acids were inserted at a site between the leader peptide and the factor I/membrane attacking complex module in the shark factor I. This repeat is missing from mammalian and amphibian factor I, and the biological significance of the sequence, if any, is not clear at the moment. There was only one copy of the shark factor I gene, and Northern blotting analysis showed that the shark factor I gene was expressed only in the liver among several organs tested. While the lack of functional data does not exclude the possibility that factor I could have a different function, all these facts, together with the earlier reported data suggest the existence of a well developed complement system in cartilaginous fish.

A Short Consensus Repeat-Containing Complement Regulatory Protein of Lamprey That Participates in Cleavage of Lamprey Complement 3

The prototype of the short consensus repeat (SCR)-containing C regulatory protein is of interest in view of its evolutionary significance with regard to the origin of the C regulatory system. Lamprey is an agnathan fish that belongs to the lowest class of vertebrates. Because it does not possess lymphocytes, it lacks Ig and consequently the classical C pathway. We identified an SCR-containing C regulatory protein from the lamprey. The primary structure predicted from the cDNA sequence showed that this is a secretary protein consisting of eight SCRs. This framework is similar to the α-chain of C4b-binding protein (C4bp). SCR2 and -3 of human C4bp are essential for C4b inactivation, and this region is fairly well conserved in the lamprey protein. However, the other SCRs of this protein are similar to those of other human C regulatory proteins. The lamprey protein binds to the previously reported lamprey C3b/C3bi deposited on yeast and cleaves lamprey C3b-like C3 together with a putative serum protease. The scheme resembles the C regulatory system of mammals, where factor I and its cofactor inactivate C3b. Unlike human cofactors, the lamprey protein requires divalent cations for C3b-like C3 cleavage. Its artificial membrane-anchored form protects host cells from lamprey C attack via the lectin pathway. Thus, the target of this protein appears to be C3b and/or its family. We named this protein Lacrep, the lamprey C regulatory protein. Lacrep is a member of SCR-containing C regulators, the first of its kind identified in the lowest vertebrates.

Retained Orthologous Relationships of the MHC Class I Genes during Euteleost Evolution

Major histocompatibility complex (MHC) class I molecules play a pivotal role in immune defense system, presenting the antigen peptides to cytotoxic CD8+ T lymphocytes. Most vertebrates possess multiple MHC class I loci, but the analysis of their evolutionary relationships between distantly related species has difficulties because genetic events such as gene duplication, deletion, recombination, and/or conversion have occurred frequently in these genes. Human MHC class I genes have been conserved only within the primates for up to 46-66 My. Here, we performed comprehensive analysis of the MHC class I genes of the medaka fish, Oryzias latipes, and found that they could be classified into four groups of ancient origin. In phylogenetic analysis using these genes and the classical and nonclassical class I genes of other teleost fishes, three extracellular domains of the class I genes showed quite different evolutionary histories. The α1 domains generated four deeply diverged lineages corresponding to four medaka class I groups with high bootstrap values. These lineages were shared with salmonid and/or other acanthopterygian class I genes, unveiling the orthologous relationships between the classical MHC class I genes of medaka and salmonids, which diverged approximately 260 Ma. This suggested that the lineages must have diverged in the early days of the euteleost evolution and have been maintained for a long time in their genome. In contrast, the α3 domains clustered by species or fish groups, regardless of classical or nonclassical gene types, suggesting that this domain was homogenized in each species during prolonged evolution, possibly retaining the potential for CD8 binding even in the nonclassical genes. On the other hand, the α2 domains formed no apparent clusters with the α1 lineages or with species, suggesting that they were diversified partly by interlocus gene conversion, and that the α1 and α2 domains evolved separately. Such evolutionary mode is characteristic to the teleost MHC class I genes and might have contributed to the long-term conservation of the α1 domain.

Comparative genomic analysis of the major histocompatibility complex class I region in the teleost genus Oryzias

The major histocompatibility complex (MHC) class I region of teleosts harbors a tight cluster of the class IA genes and several other genes directly involved in class I antigen presentation. Moreover, the dichotomous haplotypic lineages (termed d- and N- lineages) of the proteasome subunit beta genes, PSMB8 and PSMB10, are present in this region of the medaka, Oryzias latipes. To understand the evolution of the Oryzias MHC class I region at the nucleotide sequence level, we analyzed bacterial artificial chromosome clones covering the MHC class I region containing the d- lineage of Oryzias luzonensis and the d- and N- lineages of Oryzias dancena. Comparison among these three elucidated sequences and the published sequences of the d- and N- lineages of O. latipes indicated that the order and orientation of the encoded genes were completely conserved among these five genomic regions, except for the class IA genes, which showed species-specific variation in copy number. The PSMB8 and PSMB10 genes showed trans-species dimorphism. The remaining regions flanking the PSMB10, PSMB8, and class IA genes showed high degrees of sequence conservation at interspecies as well as intraspecies levels. Thus, the three independent evolutionary patterns under apparently distinctive selective pressures are recognized in the Oryzias MHC class I region.

Genetic linkage between the LMP2 and LMP7 genes in the medaka fish, a teleost

Major histocompatibility complex (MHC) class I molecules utilize proteasomes for endogeneous protein degradation. The resulting peptides are then transported into the lumen of the endoplasmic reticulum by a specific peptide transporter, associated with antigen processing (TAP) (Monaco and Nandi 1995). Two 20S proteasome subunit genes, termed low molecular mass polypeptide 2 and7 (LMP2andLMP7) reside together with the TAP1andTAP2genes on a less than 40 kilobase (kb) DNA segment of the human MHC class II region (Beck et al. 1992). Since there are no structural similarities among theLMP2/7, TAP1/2, and class I genes, these genes provide us with an intriguing example of linkage of functionally related, but structurally unrelated genes. The cytokine, gamma interferon (IFNγ) induces transcription of theLMP2andLMP7genes, together with class I, TAP1, and TAP2genes, and LMP2 and LMP7 are incorporated into 20S proteasomes replacing their closest relatives, nonMHCencodedδ (also called Y) and X (also called MB-1), respectively (Fruh et al. 1994; Akiyama et al. 1994). These subunit replacements are reported to induce a change in the specificity of the proteolytic cleavage of the 20S proteasome, which favors the generation of peptides which can be presented by the class I molecules (Driscoll et al. 1993; Gaczynska et al. 1993). Therefore, the gene duplications betweenδ andLMP2, and X andLMP7, as well as the establishment of linkage among the LMP2, LMP7, and class I genes, seem to have been critical events in the history of antigen presentation, providing an opportunity for these genes to co-evolve. Phylogenetic studies have indicated that both theδ/LMP2 and X/LMP7 gene duplications occurred at an early stage of vertebrate evolution, most probably after the divergence of jawless fish and before the appearance of cartilaginous fish (Namikawa et al. 1995; Kandil et al. 1996; Nonaka et al. 1997). Moreover, the LMP2 and LMP7 genes are linked to theMHC in amphibian Xenopus(Namikawa et al. 1995; Nonaka et al. 1997). To help elucidate the phylogenetic origin of the linkage between theLMP2 and LMP7 genes, as well as the linkage between these genes and the MHC, we performed molecular cloning and linkage analysis of the LMP2 andLMP7 genes in a bony fish, Japanese medaka ( Oryzias latipes). Regions of the medaka LMP2 and LMP7 mRNA were amplified by the polymerase chain reaction (PCR) using double-stranded cDNA synthesized from medaka liver as described (Kuroda et al. 1996). The primers employed for semi-nested PCR amplification of LMP2 were CAGAATTCATHATGGCNGTNGARTT (sense primer-1st), ATGGTNGCNGGNTGGGA (sense primer-2nd) and GCRTCNACRTANCCRTADAT (antisense primer) (H = A,C,T; N = A,G,C,T; R = A,G; D = A,G,T) which correspond to amino acid residues 23 to 28, 119 to 124, and 154 to 160, respectively, of the human LMP2 sequence (Kelly et al. 1991). After the second PCR cycle, a DNA band of the expected size [126 base pairs (bp)] was gel purified and inserted into a plasmid using a TA cloning kit (Invitrogen, San Diego CA). Twelve clones were analyzed for nucleotide sequence of the insert, and four of these shared the same sequence which showed 76% identity (22/29) at the amino acid level with the human LMP2 sequence. Using this DNA fragment as a probe, a medaka liver cDNA library (Kuroda et al. 1996) was screened. Only one clone was isolated which contained an 825 bp insert including a 25 bp poly A tail. This clone contained an open reading frame of 199 amino acids, and a comparison with the human LMP2 sequence indicated that several residues of the N-terminal were not represented by this clone. The primers used for LMP7 amplification were the same as previously described (Kandil et al. 1996). PCR amplification products of doublestranded liver cDNA were cloned as described for LMP2. Fourteen clones were analyzed, and 13 shared the same insert which showed 83% amino acid identity (34/41) with The nucleotide sequence data reported in this paper have been submitted to the GenBank, EMBL, and DDBJ nucleotide sequence databases and have been assigned the accession numbers D89724 (LMP2) and D89725 ( LMP7)

Novel Androgen-Dependent Promoters Direct Expression of the C4b-Binding Protein α-Chain Gene in Epididymis

C4b-binding protein (C4BP) is a large plasma protein composed of seven α-chains and one β-chain and is involved in the fluid phase regulation of the classical pathway of the complement system. Complement inhibitory activity is located in the α-chain, and its mRNA has been detected only in liver to date. Here, we have isolated cDNA clones encoding the α-chain of guinea pig C4BP (C4BPα) and have demonstrated significant C4BPα mRNA expression in epididymis as well as liver. The level of C4BPα transcripts increased in the epididymis after birth, while it remained constant in the liver. C4BPα mRNA was also detected in the normal murine epididymis at a significant level, but it decreased drastically after castration, suggesting that epididymal expression of the C4BPα gene is regulated by androgen. Gene analysis of guinea pig C4BPα indicated that liver and epididymis C4BPα mRNA share the coding region and 3′-untranslated region, but are transcribed from independent promoters on a single-copy gene. Two novel epididymis-specific promoters were identified in the region corresponding to the first intron of liver transcripts. The binding motif for hepatocyte NF-1 occurs in the promoter used for transcription of liver C4BPα, whereas androgen-responsive elements occur in both promoters used in the epididymis. These findings present a novel link between complement regulators and reproduction. Furthermore, variation in the 5′-untranslated regions, arising from alternative splicing of the newly identified exons, is demonstrable in the guinea pig C4BPα transcripts.

Evolutionary analysis of two classical MHC class I loci of the medaka fish, Oryzias latipes: haplotype-specific genomic diversity, locus-specific polymorphisms, and interlocus homogenization

The major histocompatibility complex (MHC) region of the teleost medaka (Oryzias latipes) contains two classical class I loci, UAA and UBA, whereas most lower vertebrates possess or express a single locus. To elucidate the allelic diversification and evolutionary relationships of these loci, we compared the BAC-based complete genomic sequences of the MHC class I region of three medaka strains and the PCR-based cDNA sequences of two more strains and two wild individuals, representing nine haplotypes. These were derived from two geographically distinct medaka populations isolated for four to five million years. Comparison of the genomic sequences showed a marked diversity in the region encompassing UAA and UBA even between the strains derived from the same population, and also showed an ancient divergence of these loci. cDNA analysis indicated that the peptide-binding domains of both UAA and UBA are highly polymorphic and that most of the polymorphisms were established in a locus-specific manner before the divergence of the two populations. Interallelic recombination between exons 2 and 3 encoding these domains was observed. The second intron of the UAA genes contains a highly conserved region with a palindromic sequence, suggesting that this region contributed to the recombination events. In contrast, the α3 domain is extremely homogenized not only within each locus but also between UAA and UBA regardless of populations. Two lineages of the transmembrane and cytoplasmic regions are also shared by UAA and UBA, suggesting that these two loci evolved with intimate genetic interaction through gene conversion or unequal crossing over.