The major histocompatibility complex in monotremes: an analysis of the evolution of <Emphasis Type="Italic">Mhc</Emphasis> class I genes across all three mammalian subclasses

We report the isolation and characterization of cDNA clones of expressed, functional major histocompatibility complex class-I ( Mhc-I) genes from two species of monotremes: the duck-billed platypus and the short-beaked echidna. The cDNA clones were isolated from libraries constructed from spleen RNA, clearly establishing their expression in at least this one peripheral lymphoid organ. From the presence of conserved amino acid residues, it appears the expressed sequences encode molecules that likely function as classical Mhc-I. These clones were isolated using monotreme Mhc-I processed pseudogenes as probes. These processed pseudogenes were isolated from genomic DNA and, based on their structure, are likely independently derived in the platypus and echidna. When all the monotreme sequences were included in phylogenetic analyses, we found no apparent orthologous relationships between the platypus and echidna Mhc-I. Analyses that included a large number of Mhc-I sequences from other taxa support a separate monotreme Mhc-I clade, basal to a therian Mhc-I clade that is comprised of sequences from marsupial and placental mammals. The phylogenies also support the hypothesis that Mhc-I genes of placental mammals, marsupials, and monotremes are derived from three separate lineages of Mhc-I genes, best explained by two rounds of duplications and deletions. The first round would have occurred prior to the divergence of monotremes and therians, and the second prior to the divergence of marsupials and placental mammals. The sequences described here represent the first reported functional monotreme Mhc-I, as well as the first processed pseudogenes of any type from monotremes.     

Characterization of the gene encoding mouse mast cell protease 8 (mMCP-8), and a comparative analysis of hematopoietic serine protease genes

Serine proteases are important granule constituents in several of the major hematopoietic cell lineages. We present here the nucleotide sequence of the gene encoding mouse mast cell protease 8 (mMCP-8). mMCP-8 was initially isolated as a cDNA from a mouse mast cell line, but has recently been found to be expressed primarily by mouse basophils. mMCP-8 and its rat homologues, rMCP-8, -9, and -10, form a new group of mast cell/basophil proteases, which are more closely related to the T-cell granzymes and neutrophil cathepsin G than to the mast cell tryptases and chymases. A dot matrix comparison of the mMCP-8 gene with other closely related hematopoietic serine protease genes shows detectable homology only in the exonic regions of the genes. No indication for conservation in the promoter region or introns was observed. This latter finding indicates that the upstream regulatory region has evolved at a relatively high rate. However, despite the low degree of direct sequence conservation, no major differences in the sizes of introns or exons were observed between mMCP-8 and genes for the closest related hematopoietic serine proteases, the mouse T-cell granzymes and cathepsin G, indicating that after evolutionary separation from the T-cell granzymes and cathepsin G, the majority of mutations primarily involved single base pair substitutions or short insertions or deletions.     

Expansion of the mast cell chymase locus over the past 200 million years of mammalian evolution

The acidic granules of natural killer (NK) cells, T cells, mast cells, and neutrophils store large amounts of serine proteases. Functionally, these proteases are involved, e.g., in the induction of apoptosis, the recruitment of inflammatory cells, and the remodeling of extra-cellular matrix. Among the granule proteases are the phylogenetically related mast cell chymases, neutrophil cathepsin G, and T-cell granzymes (Gzm B to H and Gzm N), which share the characteristic absence of a Cys₁₉₁–Cys₂₂₀ bridge. The genes of these proteases are clustered in one locus, the mast cell chymase locus, in all previously investigated mammals. In this paper, we present a detailed analysis of the chymase locus in cattle (Bos taurus) and opossum (Monodelphis domestica). The gained information delineates the evolution of the chymase locus over more than 200 million years. Surprisingly, the cattle chymase locus contains two α-chymase and two cathepsin G genes where all other studied chymase loci have single genes. Moreover, the cattle locus holds at least four genes for duodenases, which are not found in other chymase loci. Interestingly, duodenases seem to have digestive rather than immune functions. In opossum, on the other hand, only two chymase locus-related genes have been identified. These two genes are not arranged in one locus, but appear to have been separated by a marsupial-specific chromosomal rearrangement. Phylogenetic analyses place one of the opossum genes firmly with mast cell α-chymases, which indicates that the α-chymase had already evolved as a separate, clearly identifiable gene before the separation of marsupials and placental mammals. In contrast, the second gene in opossum is positioned phylogenetically between granzymes, cathepsin G, and the duodenases. These genes, therefore, probably evolved as separate subfamilies after the separation of placental mammals from marsupials. In platypus, only one chymase locus-like sequence could be identified. This previously published “granzyme” does not cluster clearly with any of the chymase locus gene families, but shares the absence of the Cys₁₉₁–Cys₂₂₀ bridge with the other chymase locus proteases. These findings indicate that all chymase locus genes are derived from a single ancestor that was present more than 200 million years ago.     

cDNA cloning of a mannose-binding lectin-associated serine protease (MASP) gene from hagfish (Eptatretus burgeri)

Hagfish, agnathan cyclostome, is the most primitive extant vertebrate and its complement (C) system seems to be a primordial system in comparison with a well-developed C system in gnathostome vertebrates. From a phylogenic perspective of defense mechanisms, we have isolated complement C3 from the serum of hagfish (Eptatretus burgeri). In this study, we first attempted to identify a hagfish Bf or C2 as a C3 convertase by RT-PCR using degenerative primers designed on the basis of the conserved amino acid stretches among the several kinds of serine proteases. Contrary to our expectation, homology search of cloned RT-PCR product suggested that there was a partial cDNA encoding the homologue of neither Bf nor C2 but a mannose-binding lectin-associated serine protease (MASP). Analyses of a full-length cDNA clone isolated from a hagfish liver cDNA library by using the partial cDNA as a probe indicated that this cDNA encoded hagfish MASP 1. This evidence strongly suggests that the hagfish defends itself against pathogens at least by the complement system composed of lectin pathway.     

Evidence for the Existence of Granzyme-Like Serine Proteases in Teleost Cytotoxic Cells

Granzymes are granule-associated serine proteases, which are important effector molecules in NK cell and CTL functions. The granzyme family poses a perplexing problem in phylogenetics due to the lack of nonmammalian sequence information. We now report the identification of a cDNA that codes for a granzyme homologue, channel catfish granzyme-1 (CFGR-1), from nonspecific cytotoxic cells (NCC) of a teleost. NCC are the first identified and extensively studied cytotoxic cell population in teleosts. Ictalurus punctatus (channel catfish) granzyme cDNA encodes a protein with ~50% similarity to granzymes A and K. Highly conserved catalytic triad residues of serine proteases and other motifs common to granzymes were also identified. Conserved amino acid sequences, structure–function data available for the serine protease family, and the crystal structure of human granzyme K supported a model of CFGR-1. It suggested an Arg/Lys primary substrate specificity that is shared with granzymes A and K. Furthermore, CFGR-1 has the four conserved disulfide bonds of granzymes A, K, and M. Phylogenetic analysis suggested that this molecule is a member of the granzyme family. Expression of CFGR-1 in NCC was confirmed by RT-PCR analysis. Presence of a granzyme-like molecule that might play an important role in the effector functions of NCC indicates that cell-mediated immunity with granule exocytosis and Fas pathways have been conserved for more than 300 million years.     

Disruption and pseudoautosomal localization of the major histocompatibility complex in monotremes

Background

The monotremes, represented by the duck-billed platypus and the echidnas, are the most divergent species within mammals, featuring a flamboyant mix of reptilian, mammalian and specialized characteristics. To understand the evolution of the mammalian major histocompatibility complex (MHC), the analysis of the monotreme genome is vital.

Results

We characterized several MHC containing bacterial artificial chromosome clones from platypus (Ornithorhynchus anatinus) and the short-beaked echidna (Tachyglossus aculeatus) and mapped them onto chromosomes. We discovered that the MHC of monotremes is not contiguous and locates within pseudoautosomal regions of two pairs of their sex chromosomes. The analysis revealed an MHC core region with class I and class II genes on platypus and echidna X3/Y3. Echidna X4/Y4 and platypus Y4/X5 showed synteny to the human distal class III region and beyond. We discovered an intron-containing class I pseudogene on platypus Y4/X5 at a genomic location equivalent to the human HLA-B,C region, suggesting ancestral synteny of the monotreme MHC. Analysis of male meioses from platypus and echidna showed that MHC chromosomes occupy different positions in the meiotic chains of either species.

Conclusion

Molecular and cytogenetic analyses reveal new insights into the evolution of the mammalian MHC and the multiple sex chromosome system of monotremes. In addition, our data establish the first homology link between chicken microchromosomes and the smallest chromosomes in the monotreme karyotype. Our results further suggest that segments of the monotreme MHC that now reside on separate chromosomes must once have been syntenic and that the complex sex chromosome system of monotremes is dynamic and still evolving.     

Granzymes: a family of lymphocyte granule serine proteases

Granzymes, a family of serine proteases, are expressed exclusively by cytotoxic T lymphocytes and natural killer (NK) cells, components of the immune system that protect higher organisms against viral infection and cellular transformation. Following receptor-mediated conjugate formation between a granzyme-containing cell and an infected or transformed target cell, granzymes enter the target cell via endocytosis and induce apoptosis. Granzyme B is the most powerful pro-apoptotic member of the granzyme family. Like caspases, cysteine proteases that play an important role in apoptosis, it can cleave proteins after acidic residues, especially aspartic acid. Other granzymes may serve additional functions, and some may not induce apoptosis. Granzymes have been well characterized only in human and rodents, and can be grouped into three subfamilies according to substrate specificity: members of the granzyme family that have enzymatic activity similar to the serine protease chymotrypsin are encoded by a gene cluster termed the 'chymase locus'; granzymes with trypsin-like specificities are encoded by the 'tryptase locus'; and a third subfamily cleaves after unbranched hydrophobic residues, especially methionine, and is encoded by the 'Met-ase locus'. All granzymes are synthesized as zymogens and, after clipping of the leader peptide, maximal enzymatic activity is achieved by removal of an amino-terminal dipeptide. They can all be blocked by serine protease inhibitors, and a new group of inhibitors has recently been identified - serpins, some of which are specific for granzymes. Future studies of serpins may bring insights into how cells that synthesize granzymes are protected from inadvertent cell suicide.     

Brain gangliosides in monotremes, marsupials and placentals: phylogenetic and thermoregulatory aspects

The concentration and composition of brain gangliosides of 17 mammalian species belonging to the subclasses of Prototheria (monotremes), Metatheria (marsupials), and Eutheria (placentals) were investigated. The mean concentration of brain gangliosides ranges from 525 to 610 micrograms NeuAc/g wet wt in monotremes, 445-900 micrograms in marsupials and from 630 to 1130 micrograms in the placentals. In the phylogenetic series of mammals, a decrease in the complexity of brain ganglioside composition becomes obvious: a drastic reduction in the number of individual ganglioside fractions particularly those of the c-pathway of biosynthesis, took place from the level of monotremes to that of the marsupials and placentals. In monotremes, marsupials and "lower" placentals (insectivores) the percentage of alkali-labile gangliosides is relatively low (between traces and 5%), whereas in the higher evolved mammals it amounts to about 20% of all gangliosides. The ratio of the contents of the two major mammalian ganglioside fractions GD1a and GT1b is generally in the range of 1.0 and even higher; in the heterothermic platypus from the monotremes and in hibernators among the placental mammals, however, it is much lower (about 0.8). These data support the hypothesis that the brain ganglioside composition not only depends on the phylogenetic level of nervous organization (cephalization) but is additionally correlated with the state of thermal adaptation.     

Identification of Trichinella spiralis early antigens at the pre-adult and adult stages

Three expression cDNA libraries from Trichinella spiralis worms 14 h, 20 h and 48 h post-infection (p.i.) were screened with serum from pigs experimentally infected with 20,000 T. spiralis muscle larvae. Twenty-nine positive clones were isolated from the 14 h p.i. cDNA library, corresponding to 8 different genes. A putative excretory-secretory protein similar to that of T. pseudospiralis was identified. Three clones corresponded to a T. spiralis serine proteinase inhibitor known to be involved in diverse functions such as blood coagulation and modulation of inflammation. Screening of the 20 h p.i. cDNA library selected 167 positive clones representing 12 different sequences. The clone with the highest redundancy encoded a small polypeptide having no sequence identity with any known proteins from Trichinella or other organisms. Fourteen clones displayed sequence identity with the heat shock protein (HSP) 70. HSPs are produced as an adaptive response of the parasite to the hostile environment encountered in the host intestine but their mechanism of action is not yet well defined. From the 48 h p.i. T. spiralis cDNA library, 91 positive clones were identified representing 7 distinct sequences. Most of the positive clones showed high similarity with a member of a putative T. spiralis serine protease family. This result is consistent with a possible major role for serine proteases during invasive stages of Trichinella infection and host-parasite interactions.     

An immune responsive complement factor D/adipsin and kallikrein-like serine protease (PoDAK) from the olive flounder Paralichthys olivaceus

The cDNA encoding of a complement factor D/adipsin and kallikrein-like serine protease, designated PoDAK, was isolated from the olive flounder Paralichthys olivaceus. PoDAK cDNA encodes a polypeptide with 277 amino acids containing conserved catalytic triad residues of serine proteases. The amino acid sequence of PoDAK showed high similarity to the kallikrein-like protein of medaka, mammalian adipsin/complement factor D and tissue kallikrein homolog, KT-14 of trout, complement factor D of zebrafish, and shared 31.6–36.8% homology with complement factor D/adipsin known from other species, including mammals. Phylogenetic analysis revealed that PoDAK clustered with the kallikrein-like protein of medaka and mammalian adipsin/complement factor D and tissue kallikrein homolog KT-14 of trout. The expression of PoDAK mRNA was high in the gills and heart, moderate in muscle, liver, intestine, stomach, kidney, and spleen of healthy flounder, and increased in the kidney, liver, and spleen of flounder challenged by the viral hemorrhagic septicemia virus (VHSV) or Streptococcus iniae. In situ hybridization confirmed that PoDAK mRNA is localized in the kidney and heart of individuals infected with VHSV. Further investigations are needed to clarify the function of PoDAK in vivo and in vitro.     

High degree of conservation of the multigene tryptase locus over the past 150–200 million years of mammalian evolution

Activated mast cells release a number of potent inflammatory mediators including histamine, proteoglycans, cytokines, and serine proteases. The proteases constitute the majority of the mast cell granule proteins, and they belong to either the chymase or the tryptase family. In mammals, these enzymes are encoded by two different loci, the mast cell chymase and the multigene tryptase loci. In mice and humans, a relatively large number of tryptic enzymes are encoded from the latter locus. These enzymes can be grouped into two subfamilies, the group 1 tryptases, with primarily membrane-anchored enzymes, and the group 2 tryptases, consisting of the soluble mast cell tryptases. In order to study the appearance of these enzymes during vertebrate evolution, we have analyzed the dog, cattle, opossum, and platypus genomes and sought orthologues in the genomes of several bird, frog, and fish species as well. Our results show that the overall structure and the number of genes within this locus have been well conserved from marsupial to placental mammals. In addition, two relatively distantly related group 2 tryptase genes and several direct homologues of some of the group 1 genes are present in the genome of the platypus, a monotreme. However, no direct homologues of the individual genes of either group 1 or 2 enzymes were identified in bird, amphibian, or fish genomes. Our results indicate that the individual genes within the multigene tryptase locus, in their present form, are essentially mammal-specific.     

Phylogenetic Analysis of 18S rRNA and the Mitochondrial Genomes of the Wombat, <Emphasis Type="BoldItalic">Vombatus ursinus,</Emphasis> and the Spiny Anteater, <Emphasis Type="BoldItalic">Tachyglossus aculeatus:</Emphasis> Increased Support for the Marsupionta Hypothesis

The monotremes, the duck-billed platypus and the echidnas, are characterized by a number of unique morphological characteristics, which have led to the common belief that they represent the living survivors of an ancestral stock of mammals. Analysis of new data from the complete mitochondrial (mt) genomes of a second monotreme, the spiny anteater, and another marsupial, the wombat, yielded clear support for the Marsupionta hypothesis. According to this hypothesis marsupials are more closely related to monotremes than to eutherians, consistent with a basal split between eutherians and marsupials/monotremes among extant mammals. This finding was also supported by analysis of new sequences from a nuclear gene—18S rRNA. The mt genome of the wombat shares some unique features with previously described marsupial mtDNAs (tRNA rearrangement, a missing tRNA^Lys, and evidence for RNA editing of the tRNA^Asp). Molecular estimates of genetic divergence suggest that the divergence between the platypus and the spiny anteater took place ≈34 million years before present (MYBP), and that between South American and Australian marsupials ≈72 MYBP. Received: 28 October 2000 / Accepted: 23 March 2001     

Characterization of granzymes A and B isolated from granules of cloned human cytotoxic T lymphocytes

A human CD8+ CTL clone with cytolytic potential was shown to express two serine proteases, a 50-kDa homodimer and a 27-kDa monomer, which were purified from cytoplasmic granules. N-terminal sequencing of the purified proteins revealed that the 50-kDa homodimer is the gene product of the human Hanukah factor cDNA clone and that it represents the human homologue to granzyme A. Similarly, the 27-kDa protein was shown to be the serine esterase encoded by the human lymphocyte protease cDNA clone and corresponds to granzyme B. There was no evidence for the presence of other granzymes, in particular for the human homologues to murine granzymes C, D, E, and F. The substrate best cleaved by granzyme A was Gly-Pro-Arg-amido-4-methyl-coumarin after the Arg residue and the pH optimum was 8 to 8.5. Upon triggering of the TCR-CD3 complex with an anti-CD3 mAb, granzyme A was released into the culture medium. Furthermore, a granule-associated hemolytic activity was detected after salt extraction and partial purification of granule proteins. This suggests that hemolytically active human perforin can be obtained from inactive granules.     

Cloning and Molecular Modeling of Duodenase with Respect to Evolution of Substrate Specificity within Mammalian Serine Proteases That Have Lost a Conserved Active-Site Disulfide Bond

Mammalian serine proteases such as the chromosome 14 (Homo sapiens, Mus musculus) located granzymes, chymases, cathepsin G, and related enzymes including duodenase collectively represent a special group within the chymotrypsin family which we refer to here as "granases". Enzymes of this group have lost the ancient active-site disulfide bond Cys191-Cys220 (bovine chymotrypsinogen A numbering) which is strongly conserved in classic serine proteases such as pancreatic, blood coagulation, and fibrinolysis proteases and others (granzymes A, M, K and leukocyte elastases). We sequenced the cDNA encoding bovine (Bos taurus) duodenase, a granase with unusual dual trypsin-like and chymotrypsin-like specificity. The sequence revealed a 17-residue signal peptide and two-residue (GlyLys) activation peptide typical for granases. Production of the mature enzyme is apparently accompanied by further proteolytic processing of the C-terminal pentapeptide extension of duodenase. Similar C-terminal processing is known for another dual-specific granase, human cathepsin G. Using phylogenetic analysis based on 39 granases we retraced the evolution of residues 189 and 226 crucial for serine protease primary specificity. The analysis revealed that while there is no obvious link between mutability of residue 189 and the appearance of novel catalytic properties in granases, the mutability of residue 226 evidently gives rise to different specificity subgroups within this enzyme group. The architecture of the extended substrate-binding site of granases and structural basis of duodenase dual specificity based on molecular dynamic method are discussed. We conclude that the marked selectivity of granases that is crucial to their role as regulatory proteases has evolved through the fine-tuning of specificity at three levels--primary, secondary, and conformational.     

Molecular cloning of cDNA for matriptase, a matrix-degrading serine protease with trypsin-like activity.

A major protease from human breast cancer cells was previously detected by gelatin zymography and proposed to play a role in breast cancer invasion and metastasis. To structurally characterize the enzyme, we isolated a cDNA encoding the protease. Analysis of the cDNA reveals three sequence motifs: a carboxyl-terminal region with similarity to the trypsin-like serine proteases, four tandem cysteine-rich repeats homologous to the low density lipoprotein receptor, and two copies of tandem repeats originally found in the complement subcomponents C1r and C1s. By comparison with other serine proteases, the active-site triad was identified as His-484, Asp-539, and Ser-633. The protease contains a characteristic Arg-Val-Val-Gly-Gly motif that may serve as a proteolytic activation site. The bottom of the substrate specificity pocket was identified to be Asp-627 by comparison with other trypsin-like serine proteases. In addition, this protease exhibits trypsin-like activity as defined by cleavage of synthetic substrates with Arg or Lys as the P1 site. Thus, the protease is a mosaic protein with broad spectrum cleavage activity and two potential regulatory modules. Given its ability to degrade extracellular matrix and its trypsin-like activity, the name matriptase is proposed for the protease.     

Functional Diversity and Evolution of Bitter Taste Receptors in Egg-Laying Mammals

Egg-laying mammals (monotremes) are a sister clade of therians (placental mammals and marsupials) and a key clade to understand mammalian evolution. They are classified into platypus and echidna, which exhibit distinct ecological features such as habitats and diet. Chemosensory genes, which encode sensory receptors for taste and smell, are believed to adapt to the individual habitats and diet of each mammal. In this study, we focused on the molecular evolution of bitter taste receptors (TAS2Rs) in monotremes. The sense of bitter taste is important to detect potentially harmful substances. We comprehensively surveyed agonists of all TAS2Rs in platypus (Ornithorhynchus anatinus) and short-beaked echidna (Tachyglossus aculeatus) and compared their functions with orthologous TAS2Rs of marsupial and placental mammals (i.e., therians). As results, the agonist screening revealed that the deorphanized monotreme receptors were functionally diversified. Platypus TAS2Rs had broader receptive ranges of agonists than those of echidna TAS2Rs. While platypus consumes a variety of aquatic invertebrates, echidna mainly consumes subterranean social insects (ants and termites) as well as other invertebrates. This result indicates that receptive ranges of TAS2Rs could be associated with feeding habits in monotremes. Furthermore, some orthologous receptors in monotremes and therians responded to β-glucosides, which are feeding deterrents in plants and insects. These results suggest that the ability to detect β-glucosides and other substances might be shared and ancestral among mammals.     

Molecular cloning, characterization and expression of a serine protease with clip-domain homologue from scallop Chlamys farreri

Serine proteases play critical roles in a variety of invertebrate immune defense responses, including hemolymph coagulation, antimicrobial peptide synthesis, and melanization. The first mollusk serine protease with clip-domain (designated CFSP1) cDNA was obtained from the scallop Chlamys farreri challenged with Vibrio anguillarum by randomly sequencing a whole tissue cDNA library and rapid amplification of cDNA ends (RACE). The full-length cDNA of the C. farreri serine protease was 1211bp, consisting of a 5'-terminal untranslated region (UTR) of 72bp, a 3'-terminal UTR of 77bp with a canonical polyadenylation signal sequence AATAAA and a poly (A) tail, and an open reading frame of 1062bp. The CFSP1 cDNA encoded a polypeptide of 354 amino acids with a putative signal peptide of 19 amino acids and a mature protein of 335 amino acids. The deduced amino acid sequence of CFSP1 contained an amino-terminal clip domain, a low complexity region, and a carboxyl-terminal serine protease domain. CFSP1 mRNA was mainly expressed constitutively in the hemocytes and was up-regulated and increased 2.9- and 1.9-fold at 16h after injury and injection of bacteria.     

Type I interferon genes from the egg-laying mammal, Tachyglossus aculeatus (short-beaked echidna)

The type I IFN are an important group of multifunctional cytokines that have, for whatever reason, evolved to a high level of complexity in eutherian mammals such as humans and mice. However, until recently, little was known about the type I IFN systems of the other two groups of extant mammals, the marsupials and the egg-laying monotremes. Preliminary partial type I IFN sequences from the short-beaked echidna were previously found to cluster only with the IFN-beta subtype in phylogenetic analyses, but a lack of sequence information made interpretation of these results tenuous. Here, we report cloning of the full-length genes of representatives from the two previously defined groups of echidna type I IFN by genomic walking PCR. Along with analysis of conserved cysteine placement and promoter elements, phylogenetic analysis incorporating these sequences strongly suggest that the two groups of echidna type I IFN genes are in fact homologous to IFN-alpha and IFN-beta, confirming that the duplication leading to these two major classes of type I IFN occurred prior to the divergence of eutherians and monotremes some 180 million years ago. Thus, even though there are major differences in gene copy number and heterogeneity, separate IFN-alpha and IFN-beta gene families are a feature of the cytokine networks of all three groups of living mammals.