Multimeric hemoglobin of the Australian brine shrimp Parartemia

The hemoglobin molecule of the commercially important brine shrimp Artemia sp. has been used extensively as a model for the study of molecular evolution. It consists of nine globin domains joined by short linker sequences, and these domains are believed to have originated through a series of duplications from an original globin gene. In addition, in Artemia, two different polymers of hemoglobin, called C and T, are found which differ by 11.7% at the amino acid level and are believed to have diverged about 60 MYA. This provides a set of data of 18 globin domain sequences that have evolved in the same organism. The pattern of amino acid substitution between these two polymers is unusual, with pairs of equivalent domains displaying differences of up to 2.7-fold in total amino acid substitution. Such differences would reflect a similar range of molecular-clock rates in what appear to be duplicate, structurally equivalent domains. In order to provide a reference outgroup, we sequenced the cDNA for a nine-domain hemoglobin (P) from another genus of brine shrimp, Parartemia zietziana, which differs morphologically and ecologically from Artemia and is endemic to Australia. Parartemia produces only one hundredth the amount of hemoglobin that Artemia produces and does not upregulate production in response to low oxygen partial pressure. Comparison of the globin domains at the amino acid and DNA levels suggests that the Artemia globin T gene has accumulated substitutions differently from the Parartemia P and Artemia C globin genes. We discuss the questions of accelerated evolution after duplication and possible functions for the Parartemia globin.     

Variant subunit specificity in the quaternary structure of Artemia hemoglobin

The brine shrimp Artemia has three extracellular hemoglobins (Hbs) that are developmentally expressed and exhibit distinct oxygen-binding characteristics (Heip, Moens, and Kondo 1978; Heip et al. 1978 ). These Hbs are composed of two polymers, each of which comprises nine covalently linked globin domains. Although the cDNA sequences of two nine-domain globins from Artemia have been published, there is evidence for the existence of further expressed globin genes (Manning, Trotman, and Tate 1990 ). In the present study extensive analysis at the cDNA and genomic levels was performed in order to determine the globin gene copy number in Artemia. Sequence and Southern analysis suggest that four Hb polymers (T1, T2, C1, and C2) are expressed in Artemia. In addition, there is also at least one globin pseudogene. Protein sequencing of the native Hbs revealed that there are limitations on which two polymers can associate. The composition of the Hbs has been determined to be: Hb I, C1C2; Hb II, C1T2; and Hb III, T1T2. These pairings allow the levels of the three Artemia Hbs to be regulated independently by polymer expression alone, therefore explaining the previously inconsistent developmental and hypoxia-induced expression patterns.     

The polymeric hemoglobin molecule of Artemia. Interpretation of translated cDNA sequence of nine domains.

Translated cDNA for Artemia hemoglobin provided sequence data for almost nine domains, from the fourth residue of the A helix of one domain through 1405 residues to a stop codon after the ninth domain. The domain sequences were all different (homology between pairs 17-38%) but aligned well with each other and with conventional globins, satisfying the requirements for Phe at CD1, His at F8 and most other highly conserved features of globins including His at E7. Features found to be characteristic of Artemia globin and present in all nine domains were Phe at B10, Tyr at C4, Gly at F5, Phe at G5 and Gly at H22. Approximately 14 residues including a consensus -Val-Asp-Pro-Val-Thr-Gly-Leu- were available to form the linker between each pair of domains. The Artemia sequence data were compared with the crystal structures of Chironomus thummi thummi erythrocruorin III and sperm whale myoglobin in order to identify features of structural similarity and to examine the consequences of the differences. The Artemia sequences were compatible with the main helices and critical features of the globin fold. Possible modifications to the C helix, FG turn, and GH turn were studied in terms of molecular coordinates.     

Androglobin: a chimeric globin in metazoans that is preferentially expressed in Mammalian testes

Comparative genomic studies have led to the recent identification of several novel globin types in the Metazoa. They have revealed a surprising evolutionary diversity of functions beyond the familiar O(2) supply roles of hemoglobin and myoglobin. Here we report the discovery of a hitherto unrecognized family of proteins with a unique modular architecture, possessing an N-terminal calpain-like domain, an internal, circular permuted globin domain, and an IQ calmodulin-binding motif. Putative orthologs are present in the genomes of many metazoan taxa, including vertebrates. The calpain-like region is homologous to the catalytic domain II of the large subunit of human calpain-7. The globin domain satisfies the criteria of a myoglobin-like fold but is rearranged and split into two parts. The recombinantly expressed human globin domain exhibits an absorption spectrum characteristic of hexacoordination of the heme iron atom. Molecular evolutionary analyses indicate that this chimeric globin family is phylogenetically ancient and originated in the common ancestor to animals and choanoflagellates. In humans and mice, the gene is predominantly expressed in testis tissue, and we propose the name "androglobin" (Adgb). Expression is associated with postmeiotic stages of spermatogenesis and is insensitive to experimental hypoxia. Evidence exists for increased gene expression in fertile compared with infertile males.     

Structural interpretation of the amino acid sequence of a second domain from the Artemia covalent polymer globin

Artemia has a complex extracellular hemoglobin of Mr 260,000 comprising two globin chains (Mr 130,000) each of which is a polymer of eight covalently linked domains of Mr 16,000. The primary structure of this polymeric globin was studied to understand how globin folded domains are ordered within a globin chain and, in turn, how the latter associate into a functional hemoglobin molecule. Here we report the amino acid sequence of a second domain, E7 (Mr 16,081, excluding the heme), and interpretations of sequence data by computer-assisted alignment and modeling. This clearly shows that, as with domain E1 (Moens, L., Van Hauwaert, M.-L., De Smet, K., Geelen, D., Verpooten, G., Van Beeumen, J., Wodak, S., Alard, P., & Trotman, C. (1988) J. Biol. Chem. 263, 4679-4685), domain E7 is compatible with a globin folded structure of the beta-type chain. Several specific differences of domains E7 and E1 from the classic globins are identified. They possibly can be interpreted in terms of specific requirements for a double octameric functional molecule.     

Daphnia pulex didomain hemoglobin: structure and evolution of polymeric hemoglobins and their coding genes.

The high-molecular-weight extracellular hemoglobin of Daphnia pulex is composed of at least three different didomain globin chains. The primary structure of one of these chains was determined at the protein and cDNA levels. Each globin domain of the polypeptide chain displays the standard structural characteristics. The first domain is preceded by a 30-residue extension containing an 18-residue unprecedented threonine-rich segment and a 12-residue preA segment which is homologous to the preA segments of other nonvertebrate globin chains. Both domains are linked together by a preA' segment, which is homologous to other preA segments and lacks the threonine-rich segment. Dimerization of the globin chains by the formation of a disulphide bridge linking the unique cysteines near the amino-termini results in a covalent, vertebrate-like tetradomain structure. The flexible amino-terminal extension most likely facilitates dimerization. The gene coding for this globin chain is interrupted by six small introns. Each domain displays two intradomain introns at the conserved positions B12.2 and G7.0. A precoding intron occurs at position preA(-27.0) and a bridge intron at occurs preA'(-13.2). We propose a crossover event as the most likely mechanism for duplication. Arthropod globin trees reflect the added effects of gene diversification, gene duplication, and species evolution. The position of monodomain intracellular globins in the tree suggests that they resemble the ancestral globin more than the derived didomain extracellular globins do.     

Aspects of the Origin and Evolution of Non-Vertebrate Hemoglobins

Hemoglobins, found in members of almost all invertebrate phyla,display an extraordinary diversity of form and function. Althoughsome are intracellular with chains and assemblies similar insize to those of vertebrates, others are giant extracellularproteins with masses as large as 8,000 kilodaltons. Two verydifferent strategies have evolved for the stabilization of theselarge molecules. The first is the formation of both intra- andinterchain disulfide bonds that effectively immobilize segmentsof the protein, and the second is the evolution by gene duplicationof multi-domain chains with from two to eighteen myoglobin-like,heme-containing domains in a single polypeptide that may beas large as {small tilde}260 kilodaltons. The genes for vertebrateglobins have a characteristic two-intron, threeexon structure.The gene encoding chain c of the hemoglobin of the earthwormLumbricus terrestris has precisely the same organization andsplice junction positions. This shows that these positions havebeen conserved for at least 600 million years, the estimatedtime of divergence of annelids and the ancestor to chordates.The gene encoding a hemoglobin (leghemoglobin) of higher plantsshows exactly the same splice junctions as in the globin genesof vertebrates except that the middle exon is split by an additionalintron which is believed to have been lost early in animal evolution.The occurrence of hemoglobins in diverse higher plants suggeststhat they might be present in all plants albeit at very lowconcentrations and perhaps serving an enzymatic function. Hemoglobin,broadly defined as a heme-containing protein capable of reversiblecombination with oxygen, also occurs in bacteria and fungi.The discovery of a bacterial hemoglobin that is 26% identicalwith lupin leghemoglobin indicates a procaryotic origin. Thepossibility that hemoglobin may have evolved from a cytochromeis suggested by the presence of hemoglobins in the yeast, Candida,and the bacterium, Alcaligenes, that contain both heme and flavin.They must therefore have evolved by the fusion of the genesfor two different proteins. However, the possible homology ofthe heme domains with plant or animal hemoglobins remains tobe determined. The lactic dehydrogenase of yeast, cytochromeb2, is also a soluble flavoheme protein with a heme domain thatis homologous with mammalian cytochrome b5. Globin may haveevolved in part from a member of the cytochrome b5 family, butif so, the event must have occurred so early that only a borderlineperhaps random correspondence of amino acid sequences remains     

Two-domain hemoglobin from the blood clam,Barbatia lima. The cDNA-derived amino acid sequence

The blood clam,Barbatia lima, from Kochi, Japan, expresses a tetrameric (α ₂ β ₂) and a polymeric hemoglobin in erythrocytes. The latter hemoglobin is composed of unusual 34-kDa hemoglobin with a two-domain structure, and its molecular mass (about 430 kDa) is exceptionally large for an intracellular hemoglobin. The 3′ and 5′ parts of the cDNA ofB. lima two-domain globin have been amplified separately by polymerase chain reaction and the complete nucleotide sequence of 1147 bp was determined. The open reading frame is 930 nucleotides in length and encodes a protein with 309 amino acid residues, of which 73 amino acids were identified directly by protein sequencing. The mature protein begins with the acetylated Ser, and thus the N-terminus Met is cleaved. The molecular mass for the protein was calculated to be 35,244 Da. The cDNA-derived amino acid sequence ofB. lima two-domain globin shows 89% homology with that of two-domain globin fromB. reeveana, a North American species. The sequence homology between the two domains is 75%, suggesting that the two-domain globin resulted from the gene duplication of an ancestral 17-kDa globin.     

Deoxynucleotide sequence of an insect cDNA codes for an unreported member of the Chironomus thummi globin family

Synthetic oligonucleotides served as probes to isolate insect globin clones from a Chironomus thummi cDNA bank. The cDNA insert of one clone (pC-S9) was completely sequenced by the dideoxy termination procedure. Beginning at the 5' end of the coding region, the 584 base pair sequence encodes most of an N-terminal hydrophobic signal sequence and the complete sequence for a mature secreted globin, and contains a polyadenylation recognition site 3' to an appropriate stop codon. The inferred amino acid sequence is that of an unreported variant of hemoglobin VIIB. Based on the number of differences between Hb VIIB chains, the pC-S9 gene has been evolutionarily independent longer than the other (two) members of the globin VIIB subfamily.     

Organization of sequences of avian globin mRNA.

Formamide polyacrylamide gel electrophoresis shows that chicken globin mRNA contains about 6.50 nucleotides, and since only 435 of these code for globin, a further 215 are not translated, and their function and position are not known. This work has produced the following conclusions. 1. 45-50 of these untranslated nucleotides are present as poly (A) at the 3' terminus. 2. The 3' untranslated region of chicken globin mRNA is at least 90 nucleotides in length. This minimal estimate is based on data derived from hybridization of defined lenghts of chicken globin cDNA to rabbit globin mRNA. The percentage of avian globin cDNA sequences which hybridize to rabbit globin mRNA is directly proportional to the length of the cDNA in each case. This relationship holds for lengths of cDNA from 115 up to 620 nucleotides. The low percentage homology for short cDNA molecules is not due to their being short per se. In homologous mRNA excess hybridizations (chicken cDNA/chicken mRNA), all cDNA preparations were completely protected from S1 nuclease digestion. 3. It is probable that there is greater evolutionary divergence in the 3' untranslated region of chicken and rabbit globin mRNA when compared with the coding regions of these molecules; The combined data is sued to formulate a regional map of chicken globin mRNA,     

A primitive myoglobin from Tetrahymena pyriformis: its heme environment, autoxidizability, and genomic DNA structure

A myoglobin-like protein isolated from Tetrahymena pyriformis is composed of 121 amino acid residues. This is much smaller than sperm whale myoglobin by 32 residues, suggesting a distinct origin from the common globin gene. We have therefore examined this unique protein for its structural, spectral and stability properties. As a result, the rate of autoxidation of Tetrahymena oxymyoglobin (MbO(2)) was found to be almost comparable to that of sperm whale MbO(2) over a wide range of pH 4-12 in 0.1 M buffer at 25 degrees C. Moreover, both pH profiles exhibited the remarkable proton-assisted process, which can be performed in sperm whale myoglobin by the distal (E7) histidine as its catalytic residue. These kinetic observations are also in full accord with spectral examinations for the presence of a distal histidine in ciliated protozoa myoglobin. At the same time, we have isolated the globin genes both from T. pyriformis and Tetrahymena thermophila, and found that there is no intron in their genomic structures. This is in sharp contrast to previous reports on the homologous globin genes from Paramecium caudatum and Chlamydomonas eugametos. Rather, the Tetrahymena genes seemed to be related to the cyanobacterial globin gene from Nostoc commune. These contracted or truncated globins thus have a marked diversity in the cDNA, protein, and genomic structures.     

IN SITU LOCALIZATION OF GLOBIN MESSENGER RNA FORMATION : II. After Treatment of Friend Virus-Transformed Mouse Cells with Dimethyl Sulfoxide

Globin messenger RNA (mRNA) levels in Friend virus-transformed mouse cells have been estimated by in situ hybridization of DNA copy (cDNA) to fixed preparations of cells and by hybridization of cDNA to extracted cytoplasmic RNA in true solution. The results obtained by both methods agree in showing that a low level of globin mRNA can be detected in untreated Friend cells. The levels of hemoglobin and globin mRNA have also been correlated after treatment of Friend cells with dimethyl sulfoxide (DMSO). The results obtained by both experimental approaches show that there is a minimum period of treatment with DMSO required in order that Friend cells may become hemoglobinized, and that this period coincides with the time when globin mRNA accumulates. Moreover, bromodeoxyuridine prevents both hemoglobin and globin mRNA accumulation.     

Characterization of a cathepsin L-associated protein in Artemia and its relationship to the FAS-I family of cell adhesion proteins.

We reported previously that the major cysteine protease in embryos and larvae of the brine shrimp, Artemia franciscana, is a heterodimeric protein consisting of a catalytic subunit (28.5 kDa) with a high degree of homology with cathepsin L, and a noncatalytic subunit (31.5 kDa) of unknown function. In the study reported here the noncatalytic subunit, or cathepsin L-associated protein (CLAP), was separated from cathepsin L by chromatography on Mono S and found to contain multiple isoforms with pIs ranging from 5.9 to 6.1. Heterodimeric and monomeric cathepsin L showed similar activity between pH 5 and 6.5, while the heterodimer was about twice as active as monomeric cathepsin L below pH 5. The heterodimer was more stable than the monomer between pH 6 and 7.4 and at 30-50 degrees C. Artemia CLAP and cathepsin L are present in nearly equimolar amounts at all stages in the life cycle and most abundant in encysted eggs and embyros. Moreover, CLAP, either free or as a complex with cathepsin L, was resistant to hydrolysis by cathepsin L. Two clones coding for CLAP were isolated from an Artemia embryo cDNA library and sequenced. Both clones have nearly identical open reading frames, but show differences at the 5'- and 3'-termini. Each cDNA clone has an extensive 3'-untranslated region containing 70-72% A+T. The deduced amino acid sequence of CLAP cDNA revealed two domains which were very similar to domains in fasciclin I and other cell adhesion proteins. The nucleotide sequences of clones 1 and 2 have been entered into the NCBI database (AY307377 and AY462276). This study supports the view that the noncatalytic subunit of the heterodimeric cysteine protease in Artemia stabilizes cathepsin L at various pH and temperatures normally inconsistent with cathepsin L from other organisms, and that CLAP serves as a docking mechanism for cathepsin L at nonlysosomal sites in Artemia embryos.     

Protein structure and cDNA nucleotide sequence of the Japanese quail alpha A globin.

The primary structure of the alpha polypeptide chain (alpha A) of the major component (QII) of Japanese quail hemoglobin was determined by protein and cDNA sequence analysis. The amino-acid sequences of all the soluble tryptic peptides were determined by the conventional protein sequencing technology. The sequence of the remaining portion, which contained an insoluble "core region", was determined through determination of the cDNA nucleotide sequence. The cDNA clones coding for the alpha A globin were isolated from the quail reticulocyte cDNA library, mapped by restriction enzyme digestion, and the nucleotide sequence was determined completely. The primary structure of quail alpha A globin shows a close similarity to that of chicken alpha A globin.     

Cloning of a new mouse foetal beta-globin mRNA sequence.

A novel globin cDNA recombinant (pFG5) has been isolated from a 14-15 day Porton mouse foetal liver cDNA library. It codes for a beta-like globin mRNA expressed in foetal liver-derived erythroblasts and erythrocytes but not in adult reticulocytes nor in yolk sac derived nucleated erythrocytes. It is also found in Friend cells induced to differentiate by DMSO. The nucleotide sequence of pFG5 confirms that it does not code for the beta major or beta minor globin chains nor the embryonic epsilon Y2 globin chain; but it is identical to the published partial sequence of the epsilon Y3 globin gene over the region of overlap (78 nucleotides).     

The heterogeneity of the polymeric intracellular hemoglobin of Glycera dibranchiata and the cDNA-derived amino acid sequence of one component

The erythrocytes of the marine polychaete Glycera dibranchiata contain a number of different, single-chain hemoglobins, some of which self-associate into a 'polymeric' fraction. An oligodeoxynucleotide probe was synthesized based on partial amino acid sequences determined by chemical methods, and used to screen a cDNA library constructed from the poly(A+)mRNA of Glycera erythrocytes (Simons, P.C. and Satterlee, J.D. (1989) Biochemistry 28, 8525-8530). The longest positive inserts found were sequenced using the dideoxy nucleotide chain termination method. One complete clone was obtained: clone 5A, 816 bases long, contained 59 bases of 5'-untranslated RNA, an open reading frame of 441 bases coding for 147 amino acids and a 3'-untranslated region of 316 bases. The derived amino acid sequence of Glycera globin P1 was in agreement with the partial amino acid sequences obtained by chemical methods. Three additional inserts obtained in the screening were also sequenced: the inferred amino acid sequences proved to be partial globin sequences which were different from each other and from the sequence of P1. Thus, the 'polymeric' fraction of the intracellular hemoglobin of Glycera probably consists of at least four different globin chains much like the 'monomeric' fraction. Comparison of the 'polymeric' sequence with the two known 'monomeric' sequences, M-II and M-IV, shows that they share 54 identical residues. At 74 positions, the identical residues in M-II and M-IV differ from the corresponding residue in P1, including at E-7, where P1 has a distal His, in contrast to Leu in M-II and M-IV. The alignment of Bashford et al. ((1987) J. Mol. Biol. 196, 199-216) and their templates were used to examine the principal differences between the two types of Glycera globin sequences. They appear to consist of uncommon surface amino acid residues at positions C6 (Phe vs. Ala), E10 (Val vs. Lys), E17 (Lys vs. Val), G1 (Arg vs. Lys), G10 (Met vs. Ala) and H5 (Arg vs. Lys). One or more of these residues could be responsible for the self-association exhibited by the 'polymeric' Glycera globins.     

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)     

Two-domain hemoglobin from the blood clam,Barbatia lima. The cDNA-derived amino acid sequence

The blood clam,Barbatia lima, from Kochi, Japan, expresses a tetrameric ( _{2 2}) and a polymeric hemoglobin in erythrocytes. The latter hemoglobin is composed of unusual 34-kDa hemoglobin with a two-domain structure, and its molecular mass (about 430 kDa) is exceptionally large for an intracellular hemoglobin. The 3 and 5 parts of the cDNA ofB. lima two-domain globin have been amplified separately by polymerase chain reaction and the complete nucleotide sequence of 1147 bp was determined. The open reading frame is 930 nucleotides in length and encodes a protein with 309 amino acid residues, of which 73 amino acids were identified directly by protein sequencing. The mature protein begins with the acetylated Ser, and thus the N-terminus Met is cleaved. The molecular mass for the protein was calculated to be 35,244 Da. The cDNA-derived amino acid sequence ofB. lima two-domain globin shows 89% homology with that of two-domain globin fromB. reeveana, a North American species. The sequence homology between the two domains is 75%, suggesting that the two-domain globin resulted from the gene duplication of an ancestral 17-kDa globin.