Amino acid sequence of the coelomic C globin from the sea cucumber Caudina (Molpadia) arenicola.

The sequence of a globin from a marine invertebrate, the sea cucumberCaudina (Molpadia) arenicola (Echinodermata), is reported. This globin, chain C, is one of four major globins found in coelomic red cells in this organism and is the second to be sequenced. Chain C consists of 157 residues, is amino-terminally acetylated, and has an extended amino-terminal region. This globin shares a 60% sequence identity with the other sequencedC. arenicola globin, D chain (Mauriet al., Biochem. Biophys. Acta 1078, 63–67, 1991), but has a 93.6% identity with a globin from another sea cucumber,Paracaudina chilensis (Suzuki,Biochem. Biophys. Acta, 998, 292–296, 1989).     

Amino acid sequence of the coelomic C globin from the sea cucumberCaudina (Molpadia) arenicola

The sequence of a globin from a marine invertebrate, the sea cucumberCaudina (Molpadia) arenicola (Echinodermata), is reported. This globin, chain C, is one of four major globins found in coelomic red cells in this organism and is the second to be sequenced. Chain C consists of 157 residues, is amino-terminally acetylated, and has an extended amino-terminal region. This globin shares a 60% sequence identity with the other sequencedC. arenicola globin, D chain (Mauriet al., Biochem. Biophys. Acta 1078, 63–67, 1991), but has a 93.6% identity with a globin from another sea cucumber,Paracaudina chilensis (Suzuki,Biochem. Biophys. Acta, 998, 292–296, 1989).     

Amino acid sequence of the dimeric hemoglobin (Hb I) from the deep-sea cold-seep clam Calyptogena soyoae and the phylogenetic relationship with other molluscan globins

The deep-sea cold-seep clam Calyptogena soyoae has two homodimeric hemoglobins (Hbs I and II) in erythrocytes. The complete amino acid sequence of Hb I has been determined. It is composed of 144 amino acid residues, has a high content of hydrophobic residues, and a calculated molecular weight of 16,350 including a heme group. The sequence of Calyptogena Hb I showed high homology (42% identity) with that of Calyptogena Hb II (Suzuki, T., Takagi T. and Ohta, S. (1989) Biochem. J. 260, 177-182), although it has a long insertion of seven residues in the C-terminal region compared with Hb II. On the other hand, it showed low homology (12-20% identity) with other molluscan globins. As well as Hb II, Calyptogena Hb I lacked the N-terminal extension of 7-9 residues characteristic of molluscan intracellular hemoglobins, and the distal (E7) histidine was replaced by glutamine. A phylogenetic tree was constructed from 13 molluscan globins belonging to the five families Aplysiidae, Galeodidae, Potamididae, Arcidae and Vesicomyidae. The globin sequences of Calyptogena (Vesicomyidae) were found to be rather distant from other globin sequences, suggesting that they might conserve a primitive form of molluscan globins.     

Axolotl hemoglobin: cDNA-derived amino acid sequences of two alpha globins and a beta globin from an adult Ambystoma mexicanum

Erythrocytes of the adult axolotl, Ambystoma mexicanum, have multiple hemoglobins. We separated and purified two kinds of hemoglobin, termed major hemoglobin (Hb M) and minor hemoglobin (Hb m), from a five-year-old male by hydrophobic interaction column chromatography on Alkyl Superose. The hemoglobins have two distinct alpha type globin polypeptides (alphaM and alpham) and a common beta globin polypeptide, all of which were purified in FPLC on a reversed-phase column after S-pyridylethylation. The complete amino acid sequences of the three globin chains were determined separately using nucleotide sequencing with the assistance of protein sequencing. The mature globin molecules were composed of 141 amino acid residues for alphaM globin, 143 for alpham globin and 146 for beta globin. Comparing primary structures of the five kinds of axolotl globins, including two previously established alpha type globins from the same species, with other known globins of amphibians and representatives of other vertebrates, we constructed phylogenetic trees for amphibian hemoglobins and tetrapod hemoglobins. The molecular trees indicated that alphaM, alpham, beta and the previously known alpha major globin were adult types of globins and the other known alpha globin was a larval type. The existence of two to four more globins in the axolotl erythrocyte is predicted.     

Abalone myoglobins evolved from indoleamine dioxygenase: The cDNA-derived amino acid sequence of myoglobin fromNordotis madaka

The cDNA for the unusual 41 kD myoglobin of the abaloneNordotis madaka was amplified by polymerase chain reaction (PCR), and the cDNA-derived amino acid sequence of 378 residues was determined. As with the myoglobin of the related abaloneSulculus diversicolor (Suzuki and Takagi,J. Mol. Biol. 228, 698–700, 1992), the sequence ofNordotis myoglobin showed no significant homology with any other globins, but showed high homology (35% identity) with vertebrate indoleamine 2,3-dioxygenase, a tryptophan degrading enzyme containing heme. The amino acid sequence homology betweenNordotis andSulculus myoglobins was 87%. These results support our previous idea that the abalone myoglobins evolved from a gene for indoleamine dioxygenase, but not from a globin gene, and therefore all of the hemoglobins and myoglobins are not homologous. Thus, abalone myoglobins appear to be a typical case of convergent evolution.     

Abalone myoglobins evolved from indoleamine dioxygenase: The cDNA-derived amino acid sequence of myoglobin fromNordotis madaka

The cDNA for the unusual 41 kD myoglobin of the abaloneNordotis madaka was amplified by polymerase chain reaction (PCR), and the cDNA-derived amino acid sequence of 378 residues was determined. As with the myoglobin of the related abaloneSulculus diversicolor (Suzuki and Takagi,J. Mol. Biol. 228, 698–700, 1992), the sequence ofNordotis myoglobin showed no significant homology with any other globins, but showed high homology (35% identity) with vertebrate indoleamine 2,3-dioxygenase, a tryptophan degrading enzyme containing heme. The amino acid sequence homology betweenNordotis andSulculus myoglobins was 87%. These results support our previous idea that the abalone myoglobins evolved from a gene for indoleamine dioxygenase, but not from a globin gene, and therefore all of the hemoglobins and myoglobins are not homologous. Thus, abalone myoglobins appear to be a typical case of convergent evolution.     

Amino acid sequence of the monomer subunit of the giant extracellular hemoglobin of the aquatic oligochaete, Tubifex tubifex

The extracellular hemoglobin of the aquatic oligochaete Tubifex tubifex consists of four subunits: a monomer of 16.5 kDa, a disulfide-bonded trimer of about 50 kDa and at least two subunits of about 30 kDa. The complete amino acid sequence of the monomeric subunit was determined: it consists of 141 amino acid residues and has a molecular mass of 16,286 Da including a heme group. 39 residues (28%) were found to be identical with those in the corresponding positions in the monomeric globin chains from Lumbricus terrestris, Pheretima sieboldi, and Tylorrhynchus heterochaetus. Tubifex and Lumbricus are most similar, with 75 amino acid identities (53%). There are eight invariant residues amongst these monomeric globins and the intracellular monomeric globin of Glycera and the human beta-globin. The monomeric globin from Tubifex aligns best with those of group A, globins which have a Cys in their second position and an invariant Lys-Val-Lys at positions 9-11 [Gotoh et al. (1987) Biochem. J. 241, 441-445]. The two cysteine residues, at positions 2 and 131, appear to be disulfide-bonded.     

A structural domain of the covalent polymer globin chains of artemia. Interpretation of amino acid sequence data

Artemia is unusual in having extracellular hemoglobins of Mr 260,000 comprising two globin chains (Mr 130,000), each of which is a polymer of eight covalently linked domains of about Mr 16,000. The amino acid sequence of one of these domains (E1) has been determined. It has 147 residues and Mr of 17,574 including heme. Sequence alignment revealed 19.0% identity with sperm whale myoglobin, whereas other vertebrate and invertebrate globins had between 13 and 24% identity. However, a much higher percentage of residues has a similar side chain character, suggesting that the domain E1 is very similar to other globins in showing the myoglobin fold. Template model building based on the known three-dimensional structure of myoglobin further supports this conclusion. Conversely, the differences between E1 and other globins are believed to reflect differences in the packing of the domains, first in a covalent polymeric subunit containing eight hemes and subsequently by association of two of these subunits as dimers. These findings provide further evidence for the versatility of the myoglobin fold.     

Prosimian hemoglobins. III. The primary structures of the duplicated alpha-globin chains of Lemur variegatus

The amino acid sequences of the alpha chains of hemoglobins purified from Lemur variegatus erythrocytes have been determined. The sequences were determined primarily from peptides generated from treatment of the isolated alpha chains with cyanogen bromide or warm formic acid. The ordering of the peptides from both alpha globins was based on the homology between lemur hemoglobins and those of other primates. The genetic difference at position 15 (Asn vs. Lys) explains the phenotypic characteristic of two hemoglobin species during alkaline electrophoresis. The function of certain residues is discussed in the context of other known sequences. The dispersion of the amino acid changes noted in lemur species falls mostly within the first 75 residues of the alpha chain (exons 1 and 2). The extent of divergence of the L. variegatus alpha-globin chains from the Lemur fulvus alpha globin is similar to that seen for the beta-globin chains of these species. This degree of separation (11-16 residues) is consistent with an extended period of independent evolution by these congeneric species after their divergence.     

The amino acid sequences of two alpha chains of hemoglobins from Komodo dragon Varanus komodoensis and phylogenetic relationships of amniotes

To elucidate phylogenetic relationships among amniotes and the evolution of alpha globins, hemoglobins were analyzed from the Komodo dragon (Komodo monitor lizard) Varanus komodoensis, the world's largest extant lizard, inhabiting Komodo Islands, Indonesia. Four unique globin chains (alpha A, alpha D, beta B, and beta C) were isolated in an equal molar ratio by high performance liquid chromatography from the hemolysate. The amino acid sequences of two alpha chains were determined. The alpha D chain has a glutamine at E7 as does an alpha chain of a snake, Liophis miliaris, but the alpha A chain has a histidine at E7 like the majority of hemoglobins. Phylogenetic analyses of 19 globins including two alpha chains of Komodo dragon and ones from representative amniotes showed the following results: (1) The a chains of squamates (snakes and lizards), which have a glutamine at E7, are clustered with the embryonic alpha globin family, which typically includes the alpha D chain from birds; (2) birds form a sister group with other reptiles but not with mammals; (3) the genes for embryonic and adult types of alpha globins were possibly produced by duplication of the ancestral alpha gene before ancestral amniotes diverged, indicating that each of the present amniotes might carry descendants of the two types of alpha globin genes; (4) squamates first split off from the ancestor of other reptiles and birds.      

The cDNA sequences encoding two components of the polymeric fraction of the intracellular hemoglobin of Glycera dibranchiata

The intracellular hemoglobin of the polychaete Glycera dibranchiata consists of several components, some of which self-associate into a "polymeric" fraction. The cDNA library constructed from the poly(A+) mRNA of Glycera erythrocytes (Simons, P. C., and Satterlee, J. D. (1989) Biochemistry 28, 8525-8530) was screened with two oligodeoxynucleotide probes corresponding to the amino acid sequences MEEKVP and AMNSKV. Each of the two probes identified a full-length positive insert; these were sequenced using the dideoxynucleotide chain termination method. One clone was 630 bases long and contained 36 bases of 5'-untranslated RNA, a reading frame of 441 bases coding for the 147 amino acids of globin P2 including the residues MEEKVP, and a 3'-untranslated region of 153 bases. The other clone was 540 bases long and contained 24 bases of 5'-untranslated RNA, an open reading frame of 441 bases coding for globin P3 including the residues AMNSKV, and a 3'-untranslated region of 75 bases. The inferred amino acid sequences of the two globins were in agreement with the partial amino acid sequences obtained by chemical methods. The P2 and P3 globin sequences, together with the previously determined P1 sequence of a complete insert and partial sequences P4, P5, and P6 obtained from partial inserts (Zafar, R. S., Chow, L. H., Stern, M. S., Vinogradov, S. N., and Walz, D. A. (1990) Biochim. Biophys. Acta, in press) suggest that there are at least six components in the polymeric fraction of Glycera hemoglobin, which is in agreement with the results of polyacrylamide gel electrophoresis in Tris/glycine buffer, pH 8.3, 6 M urea. Nothern and dot blot analyses of Glycera erythrocyte poly(A+) mRNA using the foregoing two cDNA probes clearly demonstrated the presence of mature messages encoding both types of globins. Comparison of the polymeric sequences P1, P2, and P3 with the "monomeric" globins M-II and M-IV using the alignment and templates of Bashford et al. (Bashford, D., Chothia, C., and Lesk, A. M. (1987) J. Mol. Biol. 196, 199-216) showed that all five globins have identical residues at 39 positions. At 44 positions, the three polymeric globins share identical residues that differ from the identical residues at the corresponding locations in the monomeric sequences M-II and M-IV including position E7, where the latter have leucine instead of the distal histidine. At 15 positions, there occurs an alteration from polar to nonpolar or from a small nonpolar to a larger nonpolar residue in going from the monomeric to the polymeric globins.(ABSTRACT TRUNCATED AT 400 WORDS)     

Recombinant protein sequences can trigger methylation of N-terminal amino acids in Escherichia coli.

Recombinant human hemoglobin rHb1.1 has been genetically engineered with the replacement of the wild-type valine residues at all N-termini with methionine, an Asn 108 Lys substitution on the beta globins, and a fusion of the two alpha globins with a glycine linker. When rHb1.1 was expressed in Escherichia coli, methylation of the N-terminal methionine of the alpha globin was discovered. Another mutant has been engineered with the alpha globin gene coding for N-terminal methionine followed by an insertion of alanine. Characterization of expressed hemoglobin from this variant revealed a methylated N-terminal alanine that occurred through two posttranslational events: initial excision of the N-terminal methionine, followed by methylation of alanine as the newly generated N-terminus. No methylation was observed for variants expressed with wild-type valine at the N-terminus of the alpha globin. The methylation of N-terminal amino acids was attributed to a specific protein sequence that can trigger methylation of proteins expressed in E. coli. Here we demonstrate that proline at position 4 in the protein sequence of alpha globin seems an essential part of that signaling. Although N-terminal methylation has been observed previously for native E. coli proteins with similar N-terminal sequences, methylation of the recombinant globins has allowed further delineation of the recognition sequence, and indicates that methylation of heterologous proteins can occur in E. coli.     

Characterization and expression of embryonic and adult globins of the teleost Oryzias latipes (medaka)

Using the teleost Oryzias latipes (medaka), we isolated three embryonic globin cDNAs (em.alpha-0, em.alpha-1, and em.beta-1) from the embryos 5 days after fertilization (at 30 degrees C) and two adult globin cDNAs (ad.alpha-1 and ad.beta-1) from the kidney of the fully-grown adult fish, and predicted their amino acid sequences. Molecular phylogenetic analysis showed that the embryonic globins were highly homologous in amino acid sequence to the embryonic globins previously identified in rainbow trout and zebrafish, and that they formed a monophyletic group among the teleostean globin molecules. They were clearly discriminated from the adult globin of the medaka. RT-PCR analysis showed that the embryonic globin mRNAs were intensely expressed in stage 30 and 38 embryos and in young fish 30 days after hatching. The level of expression decreased drastically after the young fish stage, and was low in fully-grown adult fish. The adult alpha globin mRNA ad.alpha-1 was scarcely expressed in the embryos, and the level of expression gradually increased in young to fully-grown adult fish. Unexpectedly, the adult beta globin mRNA ad.beta-1 was expressed throughout life, from the early embryonic stage to the fully-grown adult stage. This expression profile was quite different from that of the rainbow trout previously investigated. Some globins of the medaka were expressed both in primitive hematopoiesis and in definitive hematopoiesis.     

The amino acid sequence of hemoglobin III from the symbiont-harboring clamLucina pectinata

The cytoplasmic hemoglobin III from the gill of the symbiont-harboring clamLucina pectinata consists of 152 amino acid residues, has a calculated M_m of 18,068, including heme, and has N-acetyl-serine as the N-terminal residue. Based on the alignment of its sequence with other vertebrate and nonvertebrate globins, it retains the invariant residues Phe45 at position CD1 and His98 at the proximal position F8, as well as the highly conserved Trp16 and Pro39 at positions A12 and C2, respectively. The most likely candidate for the distal residue at position E7 is Gln66.Lucina hemoglobin III shares 95 identical residues with hemoglobin II (J. D. Hockenhull-Johnsonet al., J. Prot. Chem. 10, 609–622, 1991), including Tyr at position B10, which has been shown to be capable of entering the distal heme cavity and placing its hydroxyl group within a 2.8 Å of the water molecule occupying the distal ligand position, by modeling the hemoglobin II sequence using the crystal structure of sperm whale metmyoglobin. The amino acid sequences of the twoLucina globins are compared in detail with the known sequences of mollusc globins, including seven cytoplasmic and 11 intracellular globins. Relative to 75% homology between the twoLucina globins (counting identical and conserved residues), both sequences have percent homology scores ranging from 36–49% when compared to the two groups of mollusc globins. The highest homology appears to exist between theLucina globins and the cytoplasmic hemoglobin ofBusycon canaliculatum.     

Amino acid sequence of the beta-chain of the tetrameric haemoglobin of the bivalve mollusc, Anadara trapezia

The amino acid sequence of the beta-chain of the principal haemoglobin from A. trapezia has been determined. The sequence was deduced from the sequences of tryptic peptides, which were fractionated using highperformance liquid chromatography and peptide mapping. Additional sequence data, particularly for the large tryptic peptides, was obtained from enzyme digests of both cyanogen bromide fragments and large citraconyltryptic peptides. The beta-chain has 151 residues which is longer than all the other sequenced haemoglobin chains except the alpha-chain of A. trapezia, which is 153 residues in length. The residues corresponding to those normally in the D helix are absent in this beta-chain. The additional residues are contributed by an extension of the N-terminal region, which was also found to be acetylated. Comparison of the beta-chain amino acid sequence with that of the alpha-chain of A. trapezia, the dimeric chain of A. trapezia, and the dimeric chain of A. broughtonii showed 53% identity in each case. In the E and F helices, the homology is particularly noticeable. There is 100% homology in the F helix of all four chains. The dimeric globin of A. trapezia also shows 100% homology with the beta-chain in the E helix, while the alpha-chain shows 75%. If the tertiary structure of the alpha- and beta-chains of A. trapezia haemoglobin is the same as that of horse haemoglobin, then there are many changes in the alpha 1 and beta 2 contact site residues.     

The amino acid sequence of hemoglobin III from the symbiont-harboring clamLucina pectinata

The cytoplasmic hemoglobin III from the gill of the symbiont-harboring clamLucina pectinata consists of 152 amino acid residues, has a calculated M_m of 18,068, including heme, and has N-acetyl-serine as the N-terminal residue. Based on the alignment of its sequence with other vertebrate and nonvertebrate globins, it retains the invariant residues Phe45 at position CD1 and His98 at the proximal position F8, as well as the highly conserved Trp16 and Pro39 at positions A12 and C2, respectively. The most likely candidate for the distal residue at position E7 is Gln66.Lucina hemoglobin III shares 95 identical residues with hemoglobin II (J. D. Hockenhull-Johnsonet al., J. Prot. Chem. 10, 609–622, 1991), including Tyr at position B10, which has been shown to be capable of entering the distal heme cavity and placing its hydroxyl group within a 2.8 Å of the water molecule occupying the distal ligand position, by modeling the hemoglobin II sequence using the crystal structure of sperm whale metmyoglobin. The amino acid sequences of the twoLucina globins are compared in detail with the known sequences of mollusc globins, including seven cytoplasmic and 11 intracellular globins. Relative to 75% homology between the twoLucina globins (counting identical and conserved residues), both sequences have percent homology scores ranging from 36–49% when compared to the two groups of mollusc globins. The highest homology appears to exist between theLucina globins and the cytoplasmic hemoglobin ofBusycon canaliculatum.     

Sequence analysis of a full-length cDNA for the murine pro alpha 2(I) collagen chain: comparison of the derived primary structure with human pro alpha 2(I) collagen.

Comparison of the nucleotide sequence and primary structure of murine and human pro alpha 2(I) collagen indicates a high degree of homology: 87% at the nucleotide level and 87% at the amino acid level, with the greatest degree of variability in the amino- and carboxy-pro-peptide domains. The homology is greatest in the triple helical domain, repeating [Gly-X-Y]338, exhibiting 90% homology at the amino acid level, with only X and Y position residue substitutions. The X and Y residues show 86% homology between murine and human pro alpha 2(I) collagen triple helices, with no truly nonconservative substitutions.     

The complete amino acid sequences of four globins from the land leech Haemadipsa zeylanica var. japonica

The amino acid sequences of four globins from the land leech, Haemadipsa zeylanica var. japonica, were determined using nucleotide sequencing and protein sequencing. The mature globin-molecules were composed of 146 amino acid residues for M-1 globin, 156 for M-2 globin, 143 for D-1 globin, and 149 for D-2 globin. Alignment of the four kinds of globins by Clustal X revealed 22 invariant amino acids. The four globins were 26–33% identical. A striking feature of amino acid alteration was: the replacement of the E7 distal-His of D-1 globin by phenylalanine because histidine is conserved among the rest of the globins of H. zeylanica, those of other representative species (Lumbricus and Tylorrhynchus) of Annelida and most other hemoglobins. A phylogenetic tree constructed of 18 globin structures including two species of leeches, H. zeylanica (a land leech) and Macrobdella decora (a freshwater leech), T. heterochaetus (a representative species of polychaetes), L. terrestris (a representative species of oligochaetes), and human α and β globins strongly indicated that the leech globins first separated from globin lineage of annelids.     

海参i型溶菌酶基因及其编码产物的结构特点

通过RT-PCR 和 RACE PCR技术,从海参(Stichopus japonicus)体壁中克隆得到一种溶菌酶基因(GenBank：EF036468).生物信息软件分析表明,其中全长cDNA为 713 bp,5′非编码区（UTR）246 bp,3′UTR 29 bp,开放阅读框438 bp,编码145个氨基酸,包括溶菌酶成熟肽124个氨基酸和信号肽21个氨基酸.对海参溶菌酶与多种无脊椎动物的c、g和i型溶菌酶进行分析比较,发现它与i型溶菌酶有较高的同源性,并具有i型溶菌酶高度保守的2个活性位点,即Glu34和Ser50.活性位点附近具有i型溶菌酶的一段特有的氨基酸保守序列MDVGSLSCG(P/Y)(Y/F)QIK,所以推断克隆的海参溶菌酶为i型.另外,通过搜索蛋白保守结构域数据库,发现海参溶菌酶与医用水蛭失稳酶相似性最高,并且这2个酶的三级结构模型也极其相似.因此推测,海参i型溶菌酶具有双功能特性,既能作用于细菌细胞壁的糖苷键使细胞裂解,又具有失稳酶的一些生化功能,能够水解纤维蛋白,这些特点在海参自溶过程中发挥重要的作用.