Biochemical characteristics and inhibitor selectivity of mouse indoleamine 2,3-dioxygenase-2.

The first step in the kynurenine pathway of tryptophan catabolism is the cleavage of the 2,3-double bond of the indole ring of tryptophan. In mammals, this reaction is performed independently by indoleamine 2,3-dioxygenase-1 (IDO1), tryptophan 2,3-dioxygenase (TDO) and the recently discovered indoleamine 2,3-dioxygenase-2 (IDO2). Here we describe characteristics of a purified recombinant mouse IDO2 enzyme, including its pH stability, thermal stability and structural features. An improved assay system for future studies of recombinant/isolated IDO2 has been developed using cytochrome b (5) as an electron donor. This, the first description of the interaction between IDO2 and cytochrome b (5), provides further evidence of the presence of a physiological electron carrier necessary for activity of enzymes in the "IDO family". Using this assay, the kinetic activity and substrate range of IDO2 were shown to be different to those of IDO1. 1-Methyl-D-tryptophan, a current lead IDO inhibitor used in clinical trials, was a poor inhibitor of both IDO1 and IDO2 activity. This suggests that its immunosuppressive effect may be independent of pharmacological inhibition of IDO enzymes, in the mouse at least. The different biochemical characteristics of the mouse IDO proteins suggest that they have evolved to have distinct biological roles.

Structural organization of lower marine nonvertebrate calmodulin genes.

The troponin C (TnC) superfamily genes generally possess five introns, and the positions where they are inserted are well conserved except for the fourth intron. Based on a structural comparison of TnC genes, we proposed that the common ancestor of TnC or TnC superfamily genes had no intron corresponding to the modern fourth intron, and therefore members of the superfamily have gained the fourth intron independently within each lineage. Here, we cloned calmodulin (CaM, one of the members of the TnC superfamily) cDNAs from two lower marine nonvertebrates, the sea anemone, Metridium senile, belonging to the Cnidaria, and the sponge, Halichondria okadai, belonging to the Porifera, and also determined their genomic organization. Chordate CaM genes generally possess five introns, but neither sea anemone nor sponge CaM has anything corresponding to the fourth intron of chordate CaMs, suggesting that the early metazoan CaM must have had only four introns. The modern fourth intron of chordate CaMs was acquired within the chordate lineage after nonvertebrate/chordate divergence. This notion concurs with our proposal explaining the evolution of the TnC superfamily genes.

Genomic structure of the sandworm, Perinereis vancaurica tetradentata, troponin C.

Troponin C (TnC) superfamily genes essentially possess five introns, the positions of all but the fourth being highly conserved. The fourth intron is frequently absent from protostomian invertebrate genes, such as calmodulin or TnC. We previously proposed that the common ancestor of TnC superfamily genes never possessed an intron corresponding to today's fourth introns, and that members of the superfamily independently gained a fourth intron in the evolutionary pathway of each lineage. In the present study, we isolated the TnC cDNA from the sandworm, Perinereis vancaurica tetradentata and determined its genomic structure. Sandworm TnC appears to exist as a single copy gene consisting of six exons and five introns. The positions of the first, second, third and fifth introns are identical to other TnCs, but that of the fourth intron is unique. This is in good agreement with the above-mentioned scheme, i.e. the gain of the fourth intron of sandworm TnC might have occurred within the annelid lineage after annelida/mollusca divergence.

The genomic structure of the scallop, Patinopecten yessoensis, troponin C gene: a hypothesis for the evolution of troponin C.

Two cDNAs encoding troponin C (TnC) isoforms are isolated from the scallop, Patinopecten yessoensis, striated adductor muscle. The sequential differences between these isoforms, named TnC(long) and TnC(short), are restricted in several residues of the C-terminal region. TnC(long) is commonly expressed in both the striated and the smooth adductor muscle; however, TnC(short) is only in the striated adductor muscle. The TnC gene is a single copy gene in the scallop, thus they are expressed through the alternative splicing from the same gene. The scallop TnC gene is constructed from five exons and four introns, and positions of introns are identical with chordate TnC genes, although the scallop TnC possesses no corresponding intron to the fourth intron of chordates. The loss of this intron is also observed in Drosophila TnC; these may be remnants of their ancestor, namely the early metazoan TnC gene might be a five exons-four introns structure. In addition, the absence of the corresponding intron is also observed among protostomian calmodulins (CaMs), a molecule closely related to TnC. This suggests that the common ancestor gene of the TnC superfamily might also be a five exons-four introns structure. Assuming this to be true, the discordance of the fourth intron positions observed among members of the family is well explained by the evolutionary independent gain of the intron on each member's lineage.

Genomic structure of the amphioxus calcium vector protein.

Calcium vector protein (CaVP) is an EF-hand Ca(2+)-binding protein, which is unique to the protochordate, amphioxus. CaVP is supposed to act as a Ca(2+) signal transductor, but its exact function remains unknown. Not only its function but also its exact evolutionary relationship to other Ca(2+)-binding proteins is unclear. To investigate the evolution of CaVP, we have determined the complete sequences of CaVP cDNAs from two amphioxus species, Branchiostoma lanceolatum and B. floridae, whose open reading frame cDNA and amino acid sequences show 96.5 and 98.2% identity, respectively. We have also elucidated the structure of the gene of B. floridae CaVP, which is made up of seven exons and six introns. The positions of four of the six introns (introns 1, 2, 3, and 5) are identical with those of calmodulin, troponin C, and the Spec protein of the sea urchin. These latter proteins belong to the so-called troponin C superfamily (TnC superfamily) and thus CaVP likely also belongs to this family. Intron 6 is positioned in the 3' noncoding region and is unique to CaVP, so it may represent a landmark of the CaVP lineage only. The position of intron 4 is not conserved in the genes of the TnC superfamily or CaVP, and seems to result from either intron sliding or the addition of an intron (randomly inserted into or close to domain III) to the genes of the TnC superfamily during their evolution.

The structural organization of the ascidian, Halocynthia roretzi, calmodulin genes. The vicissitude of introns during the evolution of calmodulin genes.

Two distinct calmodulin (CaM) genes are isolated from the ascidian, Halocynthia roretzi, (Hr-CaM A and Hr-CaM B) and those structures are determined. There are three nucleotide substitutions, producing two amino acid differences between Hr-CaM A and Hr-CaM B, and those are corresponding to two of the known eight variable residues among metazoan CaMs. Both Hr-CaM A and Hr-CaM B are constructed from six exons and five introns, and the positions of introns are identical. The positions of introns of Hr-CaMs are also identical with those of vertebrate CaMs, except third introns. The third introns of Hr-CaMs are inserted at 28bp upstream when compared with vertebrate CaMs. Thus, sliding of the third intron might have occurred in only the ascidian lineage prior to the gene duplication that also occurred only in that lineage. In addition, with the comparison of the intron positions, we attempt to investigate the vicissitude of introns during the evolution of metazoan CaMs.

Cloning and sequencing of three C-type lectins from body surface mucus of the land slug, Incilaria fruhstorferi.

Three C-type lectins of 15 kDa were isolated from the water-soluble fraction (WSF) of the body surface mucus of the land slug, Incilaria fruhstorferi. Based on their partial amino acid sequences, the nucleotide sequences of cDNAs encoding these lectins, named incilarin A, B and C, were determined. cDNAs of incilarin A, B and C consisted of 673, 663 and 715 bp, and deduced amino acids were 150, 149 and 156 residues, respectively. All three lectins had signal peptides of 17 amino acid residues at their N-termini. They showed 44-55% amino acid sequence identity with each other, and lower but significant homology with the other animal C-type lectins and antifreeze protein. Incilarin A and B seem to possess two intramolecular disulfide bonds in the carbohydrate-binding domain (CRD) conserved among the animal C-type lectins, however, one of these bonds is absent in incilarin C.

Diversity of the troponin C genes during chordate evolution.

To elucidate the diversity of troponin C (TnC) during chordate evolution, we determined the organization of TnCs from the amphioxus, the lamprey, and the frog. Like the ascidian, the amphioxus possesses a single gene of TnC, and the fundamental gene structure is identical with the ascidian TnC. However, because alternative splicing does not occur in amphioxus, the potential for generation of TnC isoforms through this event arises only in the ascidian lineage. From the frog Xenopus laevis, two distinct cDNAs encoding fTnC isoforms and a single s/cTnC cDNA were determined. The duplication of the fTnC gene may be a character of only Xenopus or closely related species. The lamprey possesses two cDNAs each encoding fTnC and s/cTnC. The lamprey is the earliest diverged species among vertebrates, and thus it is supposed that the presence of both fTnC and s/cTnC is universal among vertebrate species, and that the gene duplication might have occurred at a vertebrate ancestor after the protochordate/vertebrate divergence. The position of the 4th intron is 3.24/0 in protochordate TnC genes, but at 3. 11/2 in vertebrate fTnCs and s/cTnCs. It is suggested that the 4th intron sliding might have occurred prior to the gene duplication.

Primary structure of troponin I isoforms from the ascidian Halocynthia roretzi.

The solitary ascidian Halocynthia roretzi possesses three types of muscle: the larval tail striated muscle, the adult heart striated muscle, and the adult body wall smooth muscle. The troponin complex is observed in all types of muscle, and the isoform sequences and expression patterns of two of the three troponin components, troponins C and T, have been reported. In this study, we have determined cDNA sequences of the three TnI isoforms from H. roretzi. One of the three isoforms (adult TnI), expressed in adult body wall smooth muscle and heart muscle, was composed of 173 amino acids, being similar to vertebrate fast and slow skeletal TnIs in length. The other two isoforms (larval TnI alpha and TnIbeta) were isolated from a cDNA library of larvae. Both larval TnIs were composed of 142 amino acids, with truncation amounting to ca. 30 amino acid residues at the C-termini. These larval TnIs are the smallest known TnIs. The position of the last intron of these TnIs was also determined. When compared with vertebrate TnI genes, the last intron of the ascidian adult TnI gene is located at 6 nucleotides downstream, and the introns of the two larval TnIs are positioned at 9 nucleotides upstream. These results suggest that H. roretzi TnI is encoded by at least three genes.

Structure of the ascidian, Halocynthia roretzi, troponin C gene.

Two distinct cDNAs encoding troponin C (TnC) isoforms were isolated from the ascidian, Halocynthia roretzi. One is expressed in adult body wall smooth muscle and heart muscle, and the other in larval striated muscle. The H. rorezti gene is composed of 7 exons separated by 6 introns, and Southern blot analysis showed that TnC is a single copy gene product. The two isoforms of TnC were derived through the alternative splicing of the third exon.

Electrospray ionization mass spectrometric composition of the 400 kDa hemoglobin from the pogonophoran Oligobrachia mashikoi and the primary structures of three major globin chains.

Maximum entropy analysis of the electrospray ionization mass spectra of the native, carbamidomethylated, reduced and reduced and carbamidomethylated forms of the extracellular ca. 400 kDa hemoglobin of the pogonophoran Oligobrachia mashikoi has shown it to consist of eight globin chains: (a1-a5), 14861.1, 14937.1, 15040.7, 15070.6 and 15310.6 Da and b-dl, 15173.2, 15605.1 and 14775.4 Da, respectively. Although chains a1-a5 are monomeric, chains b + c form a disulfide-bonded dimer of 30776.8 Da and chains b + c + d1 form a disulfide-bonded trimer of 45551.9 Da. The major chains a5, b and c were separated by reverse-phase chromatography, and their cDNA's amplified by PCR using redundant oligomers based on their N-terminal amino-acid sequences. The complete amino-acid sequences of chains a5 (142 residues), b (140 residues) and c (147 residues) were derived from protein and cDNA sequencing and represent the first pogonophoran globin sequences. They have a high percent identity (35-52%) with the globin chains of the approximately 3500 kDa hexagonal bilayer hemoglobins from the annelids Lumbricus and Tylorrhynchus and the vestimentiferan Lamellibrachia, suggesting a very close relationship among the phyla Annelida, Pogonophora and Vestimentifera. Two free cysteine residues (Cys-73 and Cys-83), which we proposed to be the most probable candidates for the sulfide-binding sites in the Lamellibrachia chains (Suzuki, T., Takagi, T. and Ohta, S. (1990) Biochem. J. 266, 221-225), are also conserved in three chains (Cys-73 for chains b and c, and Cys-83 for chain a5) of Oligobrachia hemoglobin, in agreement with the probable role of the hemoglobin in the binding and transport of sulfide to the symbiotic bacteria which provide the metabolic fuel in the two phyla.

The giant extracellular hemoglobin from the polychaete Neanthes diversicolor. The cDNA-derived amino acid sequence of linker chain L2 and the exon/intron boundary conserved in linker genes.

The 4000 kDa extracellular hemoglobin from the polychaete Neanthes diversicolor consists of three types of subunits; three 15 kDa monomers (chains M1, M2 and M3), a 45 kDa disulfide-bonded trimer of chains T1, T2 and T3, and two 50-55 kDa disulfide-bonded homodimeric linkers (chains L1 and L2). The latter linker subunits are essential for the assembly of the other heme-containing subunits, monomers and a trimer. The cDNA encoding the linker chain L2 was amplified by polymerase chain reaction (PCR), and the cDNA-derived amino acid sequence of 235 residues has been determined. The sequence showed 22-75% identity with other linker chains. All of the linker sequences examined so far have a highly conserved cysteine-rich segment at positions 89-130: Xaa3-Cys-Xaa6-Cys-Xaa6-Cys-Xaa6-Cys-Asp-Gly-X aa2-Asp-Cys-Xaa4-Asp-Glu-Xaa4-Cys, and the motif corresponds exactly to the cysteine-rich repeats of the ligand-binding domains of vertebrate low-density lipoprotein (LDL) receptors (Suzuki, T. and Riggs, A.F. (1993) J. Biol. Chem. 268, 13548-13555). A 287 bp intron interrupts the coding sequence of Neanthes L2 gene just at the N-terminal boundary of this motif, and the position of the splice junction was exactly conserved in Neanthes and Lumbricus linker genes. This suggests that the intron has been conserved for at least 450 million years in annelid linker genes. The evolutionary origin of the remaining parts of linker chains is unclear, but it is noteworthy that the topology of the two intrachain disulfide bridges in the C-terminal segment of linker chains is homologous with that of the carbohydrate-recognition domain of animal C-type lectin.