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)     

Complete amino acid sequence of theGlycera dibranchiata monomer hemoglobin Component IV: Structural implications

The globin derived from the monomer Component IV hemoglobin of the marine annelid,Glycera dibranchiata, has been completely sequenced, and the resulting information has been used to create a structural model of the protein. The most important result is that the consensus sequence of Component IV differs by 3 amino acids from a cDNA-predicted amino acid sequence thought earlier to encode the Component IV hemoglobin. This work reveals that the histidine (E7), typical of most heme-containing globins, is replaced by leucine in Component IV. Also significant is that this sequence is not identical to any of the previously reportedGlycera dibranchiata monomer hemoglobin sequences, including the sequence from a previously reported crystal structure, but has high identity to all. A three-dimensional structual model for monomer Component IV hemoglobin was constructed using the published 1.5 å crystal structure of a monomer hemoglobin fromGlycera dibranchiata as a template. The model shows several interesting features: (1) a Phe31 (B10) that is positioned in the active site; (2) a His39 occurs in an interhelical region occupied by Pro in 98.2% of reported globin sequences; and (3) a Met41 is found at a position that emerges from this work as a previously unrecognized heme contact.Abbreviations used GMHX the holo-protein (including b-type heme, Glycera dibranchiata monomer hemoglobin Component X (X=2, 3, or 4) - GMGX the apo-protein, or globin, Glycera dibranchiata monomer globin derived from Component X (X=2, 3, or 4) - rec-gmg the globin derived from a recombinant holoprotein of a Glycera dibranchiata monomer hemoglobin, rec-gmh, whose sequence has been inferred from an isolated cDNA insert - CB label refers to peptides generated from cyanogen bromide cleavage of GMG4 - HPLC high-performance liquid chromatography - T label refers to peptides generated from trypsin digests of GMG4 - Mb myoglobin - MCS monomer hemoglobin crystal structure from Glycera dibranchiata. H, N-terminal sequence of GMG4 - SWMb sperm whale myoglobin     

Isoelectric focusing purity criteria and 1H NMR detectable spectroscopic heterogeneity in the major isolated monomer hemoglobins from Glycera dibranchiata

Three major monomeric hemoglobins have been isolated from the erythrocytes of Glycera dibranchiata. Their importance to structure-function studies of heme proteins lies in the fact that they have been shown to possess an exceptional amino acid substitution. In these proteins, the E-7 position is occupied by leucine rather than the more common distal histidine. This substitution alters the polarity of the heme ligand binding environment compared to myoglobin. Due to this, the G. dibranchiata monomer hemoglobins are attracting much attention. However, until now no purity criterion has been developed. Here we demonstrate that, for all of the Glycera monomer hemoglobins, multiple line patterns are shown on high-voltage isoelectric focusing (IEF) gels. Most of these lines are shown to be a consequence of heme-related phenomena and can be understood on the basis of changes in oxidation and ligation state of the heme iron. The multiple line pattern does not indicate significant impurities in the monomer hemoglobin preparations. Similar behavior is also demonstrated for horse heart myoglobin. The multiple line patterns on IEF gels disappear when gels of the apoproteins alone are focused. Single bands occur in this case for all of the monomer hemoglobins except component II, which displays two bands, one major and one minor. The minor band is found to be a modified apoprotein form. It is sensitive to apoprotein handling prior to focusing and depends upon whether the IEF gel is prefocused or not. From this analysis, IEF is shown to be a valuable purity criterion, and the purity of our monomer hemoglobin component II preparation is 97% one globin.(ABSTRACT TRUNCATED AT 250 WORDS)     

Molecular cloning and nucleotide sequence of cDNA coding for rat brain cholecystokinin precursor

A mixture of 14-mer oligodeoxynucleotides was used for the screening of a cDNA clone coding for a cholecystokinin (CCK) precursor from a cDNA library for rat brain microsomal poly(A)RNA. The longest insert is 718 bp long which was verified to contain a nearly full-length cDNA sequence coding for rat CCK precursor, because the size of CCK mRNA was estimated to be about 850 bases long by Northern blotting analysis. Sequence analysis revealed 110 bp in the 5'-untranslated region, 345 bp in the amino acid coding region corresponding to the CCK precursor and 263 bp in the 3'-noncoding region which contains polyadenylation signal AUUAAA and the poly(A) sequence. The precursor may contain a 28 amino acid signal peptide and 12 additional amino acids at the carboxyl terminus.     

Complete murine cDNA sequence, genomic structure, and tissue expression of the high mobility group protein HMG-I(Y)

A cDNA coding for the non-histone chromosomal protein HMG-I, or its isoform HMG-Y, was isolated from a murine Friend cell library using synthetic oligonucleotide hybridization probes. Sequence analysis showed that the 1670-base pair full length cDNA insert consists of a 201-base pair, G/C-rich (74%), 5'-untranslated region, a 288-base pair amino acid coding sequence, and an unusually long 1182-base pair 3'-untranslated region. The deduced 96-residue amino acid coding sequence of the murine HMG-I(Y) cDNA is very similar to the reported amino acid sequence of human HMG-I, except that it lacks 11 internal amino acids reported in the human protein. Based on Southern blot hybridization analysis of genomic DNA, there appear to be fewer than five copies of HMG-I(Y) genes in the haploid murine genome. These murine HMG-I(Y) genes contain a large (at least 890 base pairs) exon that includes most, or all, of the 3'-untranslated region; whereas the much shorter 5'-untranslated region and amino acid coding sequences are interrupted by at least one intron. A single size class (approximately 1700 nucleotides in murine cells and 2000 nucleotides in human cells) of HMG-I(Y) mRNAs was detected at high levels in total RNA extracts from rapidly dividing, transformed cells, but to a lesser extent, or not at all, in extracts from slowly or non-dividing cells.     

Anomalous pH dependence of the heme-bound carbon monoxide spectroscopic properties in the Glycera dibranchiata monomer hemoglobin fraction compared to vertebrate hemoglobins

The pH dependence of infrared and NMR spectroscopic parameters for carbon monoxide bound to human, equine, rabbit and Glycera dibranchiata monomer fraction hemoglobins has been examined. In all cases, the vertebrate hemoglobins exhibit CO vibrations and 13CO chemical shifts which are pH dependent, whereas the invertebrate hemoglobin does not. The Glycera dibranchiata monomer fraction exhibits the highest wavenumber CO vibration (1970 cm-1) and the most shielded chemical shift (206.2 ppm). The pH behavior of the vertebrate CO-hemoglobins is that the heme-coordinated carbon monoxide chemical shifts and principal infrared vibrations tend toward the values observed for the G. dibranchiata CO-hemoglobin fraction. These results are interpreted as originating in protonation of the distal histidine (E-7) in the vertebrate hemoglobins. The anomalous values for Glycera dibranchiata are concluded to be due to the absence of a distal histidine (E-7 His----Leu) in the heme pocket and not to gross structural dissimilarities between the proteins of the different species examined. Primary sequence similarity matrices have been constructed to compare the functional classes of amino acids at homologous positions for the CD and E helices and for the primary heme contacts in human, equine, sperm whale myoglobin, and the Glycera dibranchiata monomer hemoglobin to illustrate this point. They reveal a high correspondence for all globins and do not correlate with the spectroscopic parameters of heme-coordinated CO.     

Cloning of the gene and amino acid sequence for glucose 6-phosphate dehydrogenase from Leuconostoc mesenteroides

Amino acid sequencing of glucose 6-phosphate dehydrogenase (Glc6PD) from Leuconostoc mesenteroides yielded sequence for over 75% of the protein. Two oligonucleotides based on the amino acid sequence were used to isolate a partial Glc6PD gene clone (pLmz delta N65), from a pUC9 library, containing 85% of the coding sequence and the 3'-untranslated DNA, but lacking the 5'-noncoding DNA sequence and the portion of the gene encoding the 65 N-terminal amino acids. Attempts to obtain a full-length clone from lambda libraries were unsuccessful, possibly due to restriction of L. mesenteroides DNA by Escherichia coli host cells. The 5'-untranslated DNA was amplified by the polymerase chain reaction and partially sequenced. To obtain unmodified DNA for the gene, oligonucleotides corresponding to the 5'- and 3'-noncoding sequences were used to amplify the gene by the polymerase chain reaction, and a 1.8-kilobase pair fragment was isolated and cloned into pUC19. The recombinant plasmid, pLmz, contains the entire Glc6PD gene and expresses the gene in E. coli. pLmz was sequenced showing that the enzyme consists of 485 amino acids. L. mesenteroides Glc6PD is 31% identical to the human enzyme.     

Isolation and characterization of cDNA for chicken muscle adenylate kinase

A cDNA clone for muscle adenylate kinase was isolated from a cDNA library of chick skeletal muscle poly(A)+ RNA, and the DNA sequence was determined. The cDNA insert had 854 nucleotides, which consisted of the 5'-untranslated sequence of 57 nucleotides, the sequence of 582 nucleotides coding for 194 amino acids, and the 3'-untranslated sequence of 163 nucleotides and the poly(A) tail of 52 nucleotides. The amino acid sequence predicted from the nucleotide sequence was highly homologous with the reported sequences of human, calf, porcine, and rabbit muscle adenylate kinases. RNA blot analysis of poly(A)+ RNA from various chicken tissues revealed a single species of mRNA of approximately 850 nucleotides and its tissue-specific distribution. The induction of muscle adenylate kinase mRNA synthesis during the chick embryogenesis was also demonstrated by the blot analysis. Southern blot analysis indicated a single gene for muscle adenylate kinase in the chicken genome.     

Molecular cloning and expression in Escherichia coli of a cDNA encoding human pancreatic elastase 2

We have cloned a DNA that is complementary to the messenger RNA that encodes human pancreatic elastase 2 from a human pancreatic cDNA library using a cloned cDNA for rat pancreatic elastase 2 messenger RNA. This complementary DNA contains the entire protein coding region of 807 nucleotides which encodes preproelastase of 269 amino acids, and 4 and 82 nucleotides of the 5'- and 3'-untranslated sequences, respectively. When this deduced amino acid sequence was compared with known amino acid sequences it showed 82% homology with rat pancreatic elastase 2. This deduced sequence also contains a 16-amino-acid peptide identical with the N-terminal sequence determined for native human pancreatic proelastase 2. Taking the above findings together, we conclude that the cloned cDNA encodes a mature enzyme of 241 amino acids including 16 and 12 amino acids for a signal peptide and an activation peptide, respectively. Moreover, the predicted key amino acid residues involved in determining the substrate specificity of mammalian pancreatic elastase 2 are retained in the human enzyme. Cloned human pancreatic elastase 2 cDNA was expressed in E. coli as a mature and pro-form protein. Both resulting proteins showed immunoreactivity toward anti-elastase serum and enzymatic activity. We have also cloned and sequenced a porcine pancreatic elastase 2 cDNA.     

Characterization of an associated microfibril protein through recombinant DNA techniques.

The complete primary structure of a new extracellular protein associated with elastic fiber microfibrils was determined by recombinant DNA techniques. Antiserum to insoluble bovine ocular zonule protein was used to screen a lambda gt11 cDNA expression library constructed from whole chick embryo poly(A)+ RNA. The cDNAs encoding immunoreactive fusion polypeptides were then used to rescreen the library by plaque hybridization. Nucleotide sequencing of overlapping cDNA clones revealed an open translation reading frame of 1326 bases beginning at an initiation start sequence and ending at a stop codon. The contiguous cDNA sequence contains a 3'-untranslated region of 563 bases with a possible polyadenylation site 16 bases upstream from the poly(A) tail. Primer extension of chick aortic mRNA taken together with the sequence data, reveals a 5'-untranslated region of 95 bases extending upstream from the translation start site. Northern blot analyses indicated that the isolated cDNA hybridized with a 2.1-kilobase mRNA in preparations of whole chick embryo and chick embryonic aortic, heart, and muscle RNAs. The initial translation protein encoded by the cDNA is 53,932 kDa and possesses a hydrophilic amino acid composition with glutamic acid comprising 22% of the total amino acid residues. Antiserum was elicited to a synthetic peptide sequence (14 amino acids) encoded within the deduced protein primary structure. Western blots of extracted proteins from chick embryonic aortae cultured in the presence of beta-aminopropionitrile showed that the medium and a mild salt extract contained an immunoreactive protein possessing an apparent molecular mass of 58,000 whereas harsh denaturants extracted a 32,000-kDa protein. Pulse-chase experiments using radiolabeled lysine showed that the newly synthesized 58,000-kDa protein was chased into a 32,000-kDa protein within a 2-24-h period. Immunoelectron microscopy of tissue sections from chick aortae, bovine nuchal ligament, and human ocular zonules showed that the peptide-elicited antibody localized specifically to ultrastructurally definable microfibril structures.     

The isolation of cDNA clones for human apolipoprotein E and the detection of apoE RNA in hepatic and extra-hepatic tissues.

We have isolated cDNA clones coding for apolipoprotein E (apoE) from a cDNA library prepared from adult human liver mRNA. Mixtures of 128 different oligonucleotides, 17 residues long were synthesised to be complementary to regions of the mRNA corresponding to amino acids 1-6 and 151-156. Five independent apoE clones were selected by direct screening of 5000 recombinants with the two oligonucleotide mixtures. Two overlapping clones contain the 3'-untranslated sequence, the entire coding sequence and an additional 30 bases 5' to the amino terminus of the mature protein. The DNA sequence has been determined spanning the known sites of amino acid substitutions which account for the observed protein polymorphism of apoE. Using the clones as probes in Northern blot analysis of total human liver and kidney RNAs and leucocyte poly(A)+ RNA we have detected a single species of mRNA in liver and kidney of 1.2 kb and two larger species in leucocyte RNA. The level of expression of the mRNA in kidney is approximately 10% of that in liver while the level of apoE RNA sequences in the leucocytes is less than 1% of that in the liver.     

Globin gene family evolution and functional diversification in annelids

Globins are the most common type of oxygen-binding protein in annelids. In this paper, we show that circulating intracellular globin (Alvinella pompejana and Glycera dibranchiata), noncirculating intracellular globin (Arenicola marina myoglobin) and extracellular globin from various annelids share a similar gene structure, with two conserved introns at canonical positions B12.2 and G7.0. Despite sequence divergence between intracellular and extracellular globins, these data strongly suggest that these three globin types are derived from a common ancestral globin-like gene and evolved by duplication events leading to diversification of globin types and derived functions. A phylogenetic analysis shows a distinct evolutionary history of annelid extracellular hemoglobins with respect to intracellular annelid hemoglobins and mollusc and arthropod extracellular hemoglobins. In addition, dehaloperoxidase (DHP) from the annelid, Amphitrite ornata, surprisingly exhibits close phylogenetic relationships to some annelid intracellular globins. We have characterized the gene structure of A. ornata DHP to confirm assumptions about its homology with globins. It appears that it has the same intron position as in globin genes, suggesting a common ancestry with globins. In A. ornata, DHP may be a derived globin with an unusual enzymatic function.     

cDNA structure of murine C4b-binding protein, a regulatory component of the serum complement system

A cDNA library representing total poly(A+) RNA from the livers of male B10.WR mice was screened with a 1097 base pair (bp) probe obtained from a partial human C4b-binding protein (C4BP) cDNA clone. Two cDNA clones were isolated, the largest of which was sequenced and found to be 1889 bp in length exclusive of the poly(A) tail. The predicted mouse C4BP polypeptide chain encoded by 1239 bp is 413 amino acid residues in length and has a calculated molecular weight of 45,281. The 370-nucleotide sequence upstream from the codon for the predicted amino terminus contains two possible in-phase translational start signals which yield leader sequences of 56 and 13 amino acid residues, respectively. The 3'-untranslated region is 277 bp long, and there are two potential overlapping poly(A) recognition signals, AATTAA and ATTAAAA, located 26 and 25 bp, respectively, upstream from the poly(A) tail; these are preceded by five other potential polyadenylation signals. Beginning at the amino terminus and continuing through to residue 358, there are six contiguous regions of internal homology, each about 60 amino acids in length. The carboxy-terminal 55 amino acid sequence shares no homology with the repeating units. Extensive homology was found with human C4BP at the amino acid level (61%) as well as at the nucleotide level for both the coding and 3'-untranslated regions. Significant differences, however, were observed between mouse and human C4BP.(ABSTRACT TRUNCATED AT 250 WORDS)     

Molecular cloning and sequencing of the beta-isopropylmalate dehydrogenase gene from the cyanobacterium Spirulina platensis.

The gene for beta-isopropylmalate dehydrogenase (EC 1.1.1.85) of Spirulina platensis (leuB) was cloned from a lambda EMBL3 genomic library by heterologous hybridization using the Nostoc UCD 7801 leuB gene as a probe. The sequence of the entire leuB coding region was determined as well as 645 bp of 5' flanking region and 956 bp of 3' flanking region. DNA sequencing revealed an open reading frame of 1065 nucleotides capable of encoding a polypeptide of 355 amino acids. Homologies between the amino acid sequence deduced from the nucleotide sequence of the S. platensis leuB gene and the amino acid sequences published for corresponding proteins either from bacteria or yeasts are 45% or more. Northern hybridization analysis indicated that the S. platensis leuB gene is transcribed as a single monocistronic RNA of approximately 1200 bases.     

cDNA cloning of the Octopus dofleini hemocyanin: sequence of the carboxyl-terminal domain

A cDNA library was constructed in pUC 19, using poly(A+) RNA purified from Octopus dofleini branchial gland, which is the site of hemocyanin biosynthesis in cephalopods. The library was screened with an oligonucleotide probe derived from a portion of the partially known sequence of the C-terminal domain of Paroctopus dofleini dofleini. The clone with the longest insert--called pHC1--was sequenced and used as a probe for Northern blotting. It hybridized to a 9.5-kb RNA species, which was also visible as a band after ethidium bromide staining. The cDNA insert (approximately 1200 bp) of pHC1 contained an open reading frame of 1071 bp coding for 357 amino acids. In this insert, a region coding for 42 amino acids from the N-terminal end of the C-terminal domain is missing. These were obtained by sequencing a cloned primer extension product. By comparing our sequence with Helix pomatia beta c-hemocyanin unit D, we found 42.9% identical and 11.5% similar residues. One putative copper binding site (site B) was identified by homology to Helix hemocyanin and arthropodan hemocyanin. The location of a second possible site was identified.     

Bovine lactoferrin mRNA: sequence, analysis, and expression in the mammary gland.

The mRNA sequence for bovine lactoferrin expressed in the mammary gland was determined by sequencing three over lapping cDNA clones and by direct sequencing of the mRNA. The mRNA (2351 bases) codes for a 708 amino acid protein with a 19 amino acid signal peptide immediately preceding a sequence identical to the N-terminal 40 amino acids reported for bovine lactoferrin. A putative destabilizing sequence (AUUUA) was identified in the 3'-untranslated region. The nucleic acid sequence and deduced amino acid sequence are highly homologous with other transferrin family members. Lactoferrin mRNA concentrations in bovine mammary tissue were quite low two days before parturition and during lactation but were high three days after the cessation of milking, a sharp contrast from the pattern of regulation of the other milk proteins.     

The laminin B2 chain has a multidomain structure homologous to the B1 chain

Laminin (Mr = 850,000) is a large basement membrane-specific glycoprotein composed of three chains: A, B1, and B2. Previously, we have reported the primary structure of the B1 chain of mouse laminin deduced from sequencing cDNA clones (Sasaki M., Kato, S., Kohno, K., Martin, G. R., and Yamada, Y. (1987) Proc. Natl. Acad. Sci. U.S.A. 84, 935-939). Here we report the isolation of overlapping cDNA clones spanning 7642 bases which encode the entire B2 chain. The nucleotide sequence of the clones contains an open reading frame of 4821 bases coding for a protein of 1607 amino acids including 33 amino acids of a presumptive signal peptide. The mRNA for the B2 chain contains 2.5 kilobases of 3'-untranslated region. The deduced amino acid sequence indicates that the B2 chain consists of six distinct domains, including two domains with alpha-helical, coiled-coil structures, two domains with cysteine-rich homologous repeats, and two globular domains. These structural features of the B2 chain are similar to those of the B1 chain. In addition, the amino acid sequences of the B2 and B1 chains demonstrate considerable homology, suggesting that the genes for these two chains arose from a common ancestor.