Both a short hydrophobic domain and a carboxyl-terminal hydrophilic region are important for signal function in the Escherichia coli leader peptidase

Leader peptidase, typical of inner membrane proteins of Escherichia coli, does not have an amino-terminal leader sequence. This protein contains an internal signal peptide, residues 51-83, which is essential for assembly and remains as a membrane anchor domain. We have employed site-directed mutagenesis techniques to either delete residues within this domain or substitute a charged amino acid for one of these residues to determine the important properties of the internal signal. The deletion analysis showed that a very small apolar domain, residues 70-76, is essential for assembly, whereas residues that flank it are dispensable for its function. However, point mutations with charged amino acid residues within the polar sequence (residues 77-82) slow or abolish leader peptidase membrane assembly. Thus, a polar region, Arg-Ser-Phe-Ile-Tyr-Glu, is important for the signal peptide function of leader peptidase, unlike other signals identified thus far.     

Amino acid sequence of the a subunit of human factor XIII

Factor XIII is a plasma protein that plays an important role in the final stages of blood coagulation and fibrinolysis. The complete amino acid sequence of the a subunit of human factor XIII was determined by a combination of cDNA cloning and amino acid sequence analysis. A lambda gtll cDNA library prepared from human placenta mRNA was screened with an affinity-purified antibody against the a subunit of human factor XIII and then with a synthetic oligonucleotide probe that coded for a portion of the amino acid sequence present in the activation peptide of the a subunit. Six positive clones were identified and shown to code for the a subunit of factor XIII by DNA sequence analysis. A total of 3831 base pairs was determined by sequencing six overlapping cDNA clones. This DNA sequence contains a 5' noncoding region or a region coding for a portion of a pro-piece or leader sequence, the mature protein (731 amino acids), a stop codon (TGA), a 3' noncoding region (1535 nucleotides), and a poly(A) tail (10 nucleotides). When the a subunit of human factor XIII was digested with cyanogen bromide, 11 peptides were isolated by gel filtration and reverse-phase HPLC. Amino acid sequence analyses of these peptides were performed with an automated sequenator, and 363 amino acid residues were identified. These amino acid sequences were in complete agreement with those predicted from the cDNA. The a subunit of factor XIII contained the active site sequence of Tyr-Gly-Gln-Cys-Trp, which is identical with that of tissue transglutaminase.(ABSTRACT TRUNCATED AT 250 WORDS)     

Complete primary structure of the transacylase (E2b) subunit of the human branched chain alpha-keto acid dehydrogenase complex

We isolated from a placental cDNA library by immunoscreening a cDNA clone encoding the transacylase (E2b) precursor of the human branched chain alpha-keto acid dehydrogenase (BCKDH) complex. The cDNA insert consists of 2,649 base pairs with an open reading frame of 1,431 base pairs which can be translated into 477 amino acids and a 3'-untranslated region of 1,205 base pairs. The deduced amino acid sequence includes a leader peptide of 56 amino acid residues, a lipoyl-bearing domain, a E3-binding domain and an inner core domain. A mature human E2b subunit is likely to contain 421 amino acid residues with a calculated Mr 46,322. The nucleotide sequence of the open reading frame and the deduced amino acid sequence of the human E2b shows 91.6% and 92.0% homology with those of the bovine E2b subunit, respectively.     

Molecular cloning and nucleotide sequence of cDNA encoding the entire precursor of rat liver medium chain acyl coenzyme A dehydrogenase

cDNA encoding the precursor of rat liver medium chain acyl-CoA dehydrogenase (EC 1.3.99.3) was cloned and sequenced. The longest cDNA insert isolated was 1866 bases in length. This cDNA encodes the entire protein of 421-amino acids including a 25-amino acid leader peptide and a 396-amino acid mature polypeptide. The identity of the medium chain acyl-CoA dehydrogenase clone was confirmed by matching the amino acid sequence predicted from the cDNA to the NH2-terminal and nine internal tryptic peptide sequences derived from pure rat liver medium chain acyl-CoA dehydrogenase. The calculated molecular masses of the precursor medium chain acyl-CoA dehydrogenase, the mature medium chain acyl-CoA dehydrogenase, and the leader peptide are 46,600, 43,700, and 2,900 daltons, respectively. The leader peptide contains five basic amino acids and only one acidic amino acid; thus, it is positively charged, overall. Cysteine residues are unevenly distributed in the mature portion of the protein; five of six are found within the NH2-terminal half of the polypeptide. Comparison of medium chain acyl-CoA dehydrogenase sequence to other flavoproteins and enzymes which act on coenzyme A ester substrates did not lead to unambiguous identification of a possible FAD-binding site nor a coenzyme A-binding domain. The sequencing of other homologous acyl-CoA dehydrogenases will be informative in this regard.     

Isolation of a full-length complementary DNA coding for human E1 alpha subunit of the pyruvate dehydrogenase complex

A 1.5-kilobase cDNA clone for human pyruvate dehydrogenase E1 was isolated from a lambda gt11 expression library by screening with polyclonal antiserum to the E1 alpha subunit of the porcine pyruvate dehydrogenase complex, a polyclonal antibody against bovine pyruvate dehydrogenase complex and a synthetic oligonucleotide based on the known amino acid sequence of the amino-terminal of the bovine pyruvate dehydrogenase-E1 alpha subunit. Nucleotide sequence analysis of the cDNA revealed a 5'-untranslated sequence of 72 nucleotides, a translated sequence of 1170 nucleotides, and a 3'-untranslated sequence of 223 nucleotides with a poly(A) tail. The cDNA structure predicts a leader sequence of 29 amino acids and a mature protein of 362 amino acids comprising an amino-terminal peptide identical to that of the bovine E1 alpha subunit and three serine phosphorylation sites whose sequence was also identical to those in the bovine E1 alpha subunit. The translated sequence for the mature protein differs substantially from that described by Dahl et al. (Dahl, H. H., Hunt, S. M., Hutchison, W. M., and Brown, G. K. (1987) J. Biol. Chem. 262, 7398-7403) by virtue of a frameslip between bases 390 and 594. This amended sequence is confirmed by the presence of additional restriction sites for the enzymes NaeI and HaeII at the beginning and end, respectively, of this section. The leader sequence is typical for mitochondrial enzymes being composed of a combination of neutral and basic residues. The amino acid composition is strikingly similar to that of the bovine protein. This cDNA clone hybridizes with a 1.8-kilobase mRNA on a Northern blot analysis of human fibroblasts, and a second minor band of 4.4 kilobases is also detected.     

Isolation of mouse and human cDNA clones encoding a protein expressed specifically in osteoblasts and brain tissues.

Using the differential hybridization screening method between osteoblastic and fibroblastic cells, a cDNA clone coding for an osteoblast specific protein, named OSF-1, consisting of 168 amino acid residues including a possible 32 amino acid long leader sequence, was isolated from murine osteoblastic cell line MC3T3-E1. The OSF-1 gene was shown by Northern blotting analysis to be expressed in mouse calvarial osteoblast-enriched cells and in mouse brain tissues, but not in thymus, spleen, kidney, liver, lung, testis or heart. The human counterpart was also found in cDNA libraries from human osteosarcoma cell line MG63 and normal brain tissues. DNA sequence analysis revealed four amino acid sequence differences between the mouse and human, of which only one is located in the mature protein. This extremely high sequence conservation suggests that OSF-1 plays a fundamental role in bone and brain functions.     

Cloning and sequence analysis of cDNAs encoding mammalian mitochondrial malate dehydrogenase

A cDNA clone, named ppmMDH-1 and covering a part of the porcine mitochondrial malate dehydrogenase (mMDH; L-malate:NAD+ oxidoreductase, EC 1.1.1.37) mRNA, was isolated from a porcine liver cDNA library with a mixture of 24 oligodeoxyribonucleotides as a probe. The sequences of the probe were deduced from the known sequence of porcine mMDH amino acid residues 288-293. ppmMDH-1 covered the coding region for porcine mMDH amino acid residues 17-314 and the 3' untranslated region. Subsequently, mouse mMDH cDNA clones were isolated from a mouse liver cDNA library with the ppmMDH-1 cDNA as a probe. One of the clones, named pmmMDH-1 and containing a cDNA insert of about 1350 base pairs, was selected for sequence analysis, and the primary structure of the mouse precursor form of mMDH (pre-mMDH) was deduced from its cDNA sequence. The sequenced coding regions for the porcine and mouse mMDH mRNAs showed about 85% homology. When the deduced amino acid sequence of the mouse pre-mMDH was compared with that of the porcine mMDH, they shared a 95% homology, and the mouse pre-mMDH yielded a leader sequence consisting of 24 amino acid residues and a mature mMDH, consisting of 314 amino acid residues. The leader sequence contained three basic amino acid residues, no acidic residues, and no hydrophobic amino acid stretch. The mouse mMDH leader sequence was compared with those of three other rodent mitochondrial matrix proteins.     

Molecular cloning and structural analysis of a gene from Zea mays (L.) coding for a putative receptor for the plant hormone auxin.

The major auxin-binding protein from maize coleoptiles was purified to homogeneity. The protein has an apparent mol. wt of 22 kd and binds 1-naphthylacetic acid with a KD of 2.40 x 10(-7) M. Additional antigenically related proteins, present in very low amounts, could be demonstrated in maize coleoptiles using immunodetection. Extensive protein sequence analysis of the major auxin-binding protein allowed the construction of several synthetic oligonucleotide probes which were used to isolate a cDNA coding for this protein. The cDNA corresponds to a mRNA with a 3'-poly(A)+ sequence and a single, long open reading frame of 603 bases. The open reading frame, starting 34 residues from the 5' end of the cDNA, predicts a 21,990 Dalton protein of 201 amino acids. Comparison of this deduced amino acid sequence with the partial amino acid sequences of purified auxin-binding protein, revealed a perfect match, involving a total of 53 amino acid residues. The primary amino acid sequence includes a 38-amino-acid-long N-terminal hydrophobic leader sequence which could represent a signal for translocation of this protein to the endoplasmic reticulum. An additional signal is located at the C-terminal end, consisting of the amino acids KDEL known to be responsible for preventing secretion of proteins from the lumen of the endoplasmic reticulum in eucaryotic cells. The primary sequence contains a N-glycosylation site (-asp133-thr-thr-). This site was found to be glycosylated by a high-mannose-type oligosaccharide.(ABSTRACT TRUNCATED AT 250 WORDS)     

Amino acid sequence of the b subunit of human factor XIII, a protein composed of ten repetitive segments

Factor XIII is a plasma protein that participates in the final stages of blood coagulation. The complete amino acid sequence of the b subunit of human factor XIII was determined by a combination of cDNA cloning and amino acid sequence analysis. A lambda gt11 cDNA library prepared from human liver mRNA was screened with an affinity-purified antibody against the b subunit of human factor XIII. Nine positive clones were isolated from 2 X 10(6) phage and plaque-purified. The largest cDNA insert was sequenced and shown to contain 2180 base pairs coding for a portion of the leader sequence (19 amino acids), the mature protein (641 amino acids), a stop codon (TGA), a 3' noncoding region (187 nucleotides), and a poly(A) tail. When the b subunit of human factor XIII was digested with cyanogen bromide, nine peptides were isolated by gel filtration and reverse-phase high-performance liquid chromatography. Amino acid sequence analyses of these peptides were performed with an automated sequenator, and 299 amino acid residues were identified. These amino acid sequences were in complete agreement with the amino acid sequence predicted from the cDNA. The b subunit of factor XIII contained 10 repetitive homologous segments, each composed of about 60 amino acids and 4 half-cystine residues. Each of these repeated segments is a member of a family of repeats present in human beta 2-glycoprotein I, complement factor B, and haptoglobin alpha 1 chain. Three potential Asn-linked carbohydrate attachment sites were also identified in the b subunit of factor XIII.     

The deduced protein sequence of the human carboxypeptidase N high molecular weight subunit reveals the presence of leucine-rich tandem repeats

Human plasma carboxypeptidase N is a 280-kDa tetramer with two high molecular mass (83-kDa) glycosylated subunits which protect the two 50-kDa catalytic subunits and keep them in the circulation. An initial clone for the 83-kDa subunit was obtained by screening two lambda gt11 human liver cDNA expression libraries with antiserum specific for carboxypeptidase N or the 83-kDa subunit. The libraries were rescreened with the labeled cloned cDNA, and the largest clone obtained (2536-base pair insert) was completely sequenced. The deduced protein sequence matched the sequence of several tryptic peptides from the 83-kDa subunit but did not contain the NH2-terminal sequence. The remaining portion of the protein coding sequence was synthesized by the polymerase chain reaction, cloned, and sequenced. The composite cDNA sequence is 2870 base pairs long with an open reading frame of 1608 base pair coding for a protein of 536 amino acids (Mr = 58,762). The protein sequence contains seven potential N-linked glycosylation sites and a threonine/serine-rich region which is a potential site for attachment of O-linked carbohydrate. The most striking feature is a region (residues 68-355) containing 12 leucine-rich tandem repeats of 24 residues with the following consensus sequence: P-X-X-alpha-F-X-X-L-X-X-L-X-X-L-X-L-X-X-N-X-L-X-X-L (X = any amino acid and alpha = aliphatic amino acids, I, L, or V). This repeating pattern is found in the leucine-rich alpha 2-glycoprotein and in other proteins where it might mediate interactions with macromolecules. This region also contains five sequences with heptad repeating leucine residues comprising a leucine zipper motif. The leucine-rich domain likely constitutes an important structural or functional element in the interactions of the 83- and 50-kDa subunits to form the active tetramer of carboxypeptidase N.     

Cloning and sequence analysis of beta-4 cDNA: an integrin subunit that contains a unique 118 kd cytoplasmic domain

The alpha 6 beta 4 complex is a member of the integrin superfamily of adhesion receptors. A human keratinocyte lambda gt11 cDNA library was screened using a monoclonal antibody directed against the beta 4 subunit. Two cDNAs were selected and subsequently used to isolate a complete set of overlapping cDNA clones. The beta 4 subunit consists of 1778 amino acids with a 683 amino acid extracellular domain, a 23 amino acid transmembrane domain and an exceptionally long cytoplasmic domain of 1072 residues. The deduced amino-terminal sequence is in good agreement with the published amino-terminal sequence of purified beta 4. The extracellular domain contains five potential N-linked glycosylation sites and four cysteine-rich homologous repeat sequences. The extracellular part of the beta 4 subunit sequence shows 35% identify with other integrin beta subunits, but is the most different among this class of molecules. The transmembrane region is poorly conserved, whereas the cytoplasmic domain shows no substantial identity in any region to the cytoplasmic tails of the known beta sequences or to other protein sequences. The exceptionally long cytoplasmic domain suggests distinct interactions of the beta 4 subunit with cytoplasmic proteins.     

Tissue-specific differences between heart and liver cytochrome c oxidase

Bovine liver cytochrome c oxidase has been isolated and the subunit structure of this preparation compared with that of the bovine heart enzyme. Of the 10 nuclear-coded subunits, 3 were different in the 2 tissue forms, having different migrations in sodium dodecyl sulfate-polyacrylamide gel electrophoresis, different antigenicities to antibodies made against the heart subunits, and different N-terminal amino acid sequences. Subunit ASA of heart begins with the N-terminal sequence of SSG in liver and is different in 17 of the first 33 residues including a deletion of 2 residues in the liver isoform of this subunit. Subunit CVII of liver differs from its heart counterpart in 6 of the first 37 residues while subunit CIX from liver differs from the heart isoform in 15 of the first 25 residues. No differences between tissue types were observed in partial sequencing of the remaining nuclear-coded subunits. Recently, the major portion of the sequence of subunit CIX from rat liver has been obtained by cloning and sequencing of the cDNA for this polypeptide [Suske, G., Mengel, T., Cordingley, M., & Kadenbach, B. (1987) Eur. J. Biochem. 168, 233-237]. There is a greater sequence homology of the rat and bovine liver forms of CIX than there is between the bovine heart and liver isoforms.     

Cloning, primary structure and properties of a novel human integrin beta subunit.

The originally described integrin beta subunits that define the three subfamilies of integrin heterodimers are beta 1, beta 2 and beta 3. In this paper, we describe the isolation of a cDNA coding for a novel human integrin beta subunit, designated as beta 5. The beta 5 cDNA was isolated from a human thymic epithelial cell library, using oligonucleotide probes that were designed from a region highly conserved among the known beta 1, beta 2 and beta 3 sequences. The beta 5 cDNA codes for 799 (or 796) amino acids, including a 23 amino acid leader sequence. There are 776 (or 773) amino acids in the mature protein, which includes a long extracellular domain of 696 amino acids, a transmembrane domain and an intracellular C-terminal domain of 57 amino acids. The beta 5 sequence resembled the known beta 3, beta 1 and beta 2 sequences by 55, 43 and 38%, respectively, including conservation of 56/56 cysteines. Rabbit antiserum was prepared against a 20 amino acid synthetic peptide predicted from the beta 5 C-terminal sequence. This serum immunoprecipitated a beta 5 protein that was 100,000 Mr (reduced) and 95,000 Mr (nonreduced). Only a single alpha subunit was detected in association with beta 5, and that alpha subunit was immunochemically indistinguishable from the alpha v subunit previously found as part of the vitronectin receptor complex. By immunoprecipitation, beta 5 was most prevalent on carcinoma cell lines, was also present on hepatoma and fibroblast cell lines, and was absent from lymphoblastoid cells and platelets.     

The amino acid sequence of the 24-kDa subunit, an iron-sulfur protein, of rat liver mitochondrial NADH dehydrogenase deduced from cDNA sequence

Antiserum directed against bovine heart mitochondrial NADH dehydrogenase has been used to screen a rat liver cDNA expression library in lambda gt11. The insert cDNA of a positive clone was found to represent the 24-kDa subunit of NADH dehydrogenase by epitope selection using nitrocellulose filter containing the expressed proteins. The amino acid sequence deduced from the nucleotide sequence of the cloned cDNA indicated that the 24-kDa subunit is produced as a precursor with an amino-terminal extension, and that its mature form consists of 217 amino acid residues with a molecular weight of 23,933.     

Isolation and sequence analysis of cDNA clones for the small subunit of rabbit calcium-dependent protease

We have isolated and sequenced cDNA clones for the small subunit (30-kDa subunit) of rabbit calcium-dependent protease (Ca2+-protease) using synthesized oligodeoxynucleotide probes based on the partial amino acid sequence of the protein. A nearly full-length cDNA clone containing the total amino acid coding sequence was obtained. From the deduced sequence, the following conclusions about possible functions of the protein are presented. The kDa subunit comprises 266 residues (Mr = 28,238). The N-terminal region (64 residues) is mainly composed of glycine (37 residues) and hydrophobic amino acids and may interact with the cell membrane or an organelle. The sequence of the C-terminal 168 residues is highly homologous to the corresponding C-terminal region of the large subunit (80-kDa subunit) which has been identified as the calcium-binding domain. This region of the 30-kDa subunit contains four E-F hand structures and presumably binds Ca2+, as in the case of the 80-kDa subunit. Thus, the 30-kDa subunit may play important roles in regulating enzyme activity and/or possibly in determining the location of the Ca2+-protease. The marked sequence homology of the C-terminal regions of the two subunits may indicate that the calcium-binding domains have evolved from the same ancestral gene.     

cDNA sequence of the small subunit of the hamster ribonucleotide reductase.

Ribonucleotide reductase activity is markedly elevated in cell lines selected for resistance to hydroxyurea, a cytotoxic drug known specifically to inhibit ribonucleotide reductase. From a cDNA library constructed from a highly hydroxyurea-resistant hamster lung cell line, 600H in which the activity is elevated more than 80-fold, we have isolated a full length cDNA for the small subunit of the reductase. The cDNA is 3.48 kb long with an open reading frame of 1158 nucleotides and a long 3' flanking region of 2169 nucleotides from the termination codon. The derived polypeptide sequence is closely similar to the small subunit of the mouse, differing from it in 20 amino acid positions. Most of these replacements occur in the N-terminal segment of the protein. The hamster subunit does not contain 4 amino acid residues found in the mouse small subunit near the C-terminal end. RNA blots probed with the cDNA show two poly(A)+ RNA species which are elevated in hydroxyurea-resistant cells.     

Cloning and characterization of the P subunit of glycine decarboxylase from pea (Pisum sativum).

A pea leaf cDNA library constructed in lambda gt11 was screened with an antibody raised to the P subunit of glycine decarboxylase. One of the positive clones isolated was sequenced and shown to contain an open reading frame, which encoded the entire P subunit polypeptide. Aligning the deduced amino acid sequence with the amino acid sequence determined directly from the NH2 terminus of the mature P subunit shows the presence of a putative 86 amino acid leader sequence, presumably required for import into the mitochondria, and gives a Mr of the mature protein of 105,000. Comparison of this deduced amino acid sequence with the sequence of a pyridoxal phosphate-containing peptide isolated from the P subunit of chicken liver glycine decarboxylase shows remarkable conservation. The P subunit, however, shows little sequence homology with other published amino acid decarboxylases. Expression of the P subunit mRNA shows a pattern very similar to that of the corresponding polypeptide: it is strongly light induced and is expressed at a much higher level in leaves than in other tissues. Southern blot analysis suggests that the P subunit is encoded by a small multigene family.     

Sequence of the precursor to rat ornithine aminotransferase deduced from a cDNA clone

The nucleotide sequence of ornithine aminotransferase mRNA from rat liver, including the entire coding and 3' untranslated regions, was determined from two overlapping cDNA clones. The mRNA encodes a precursor polypeptide of 439 amino acid residues with a molecular weight of 48,332. The deduced amino acid composition of the proposed mature enzyme sequence (residues 35 through 439) was in good agreement with that reported for the purified protein. The amino-terminal segment of the precursor corresponding to residues 1 through 34 has an overall positive charge, containing 6 basic residues and only a single acidic residue, and is postulated to be the mitochondrial leader sequence. The first 22 amino acid residues of the proposed leader sequences share 54% homology with the leader peptide of rat ornithine transcarbamylase precursor and more limited homology to the leader peptides of other nuclear-encoded mitochondrial matrix proteins. Homology was also observed between residues 286 through 362 ornithine aminotransferase precursor and a region containing the pyridoxyl phosphate binding domain of mitochondrial aspartate aminotransferase.     

The complete amino acid sequence of human complement factor H.

The complete amino acid sequence of the human complement system regulatory protein, factor H, has been derived from sequencing three overlapping cDNA clones. The sequence consists of 1213 amino acids arranged in 20 homologous units, each about 60 amino acids long, and an 18-residue leader sequence. The 60-amino-acid-long repetitive units are homologous with those found in a large number of other complement and non-complement proteins. Two basic C-terminal residues, deduced from the cDNA sequence, are absent from factor H isolated from outdated plasma. A tyrosine/histidine polymorphism was observed within the seventh homologous repeat unit of factor H. This is likely to represent a difference between the two major allelic variants of factor H. The nature of the cDNA clones indicates that there is likely to be an alternative splicing mechanism, resulting in the formation of at least two species of factor H mRNA.     

Isolation and expression in Escherichia coli of a cDNA clone encoding porcine pancreatic elastase

We have cloned a DNA that is complementary to the messenger RNA that encodes porcine pancreatic elastase 1 from pancreas using rat pancreatic elastase 1 cDNA as a probe. This complementary DNA contains the entire protein coding region of 798 nucleotides which encodes an elastase of 266 amino acids, and 22 and 136 nucleotides of the 5' and 3'-untranslated sequences. When this deduced amino acid sequence was compared with known amino acid sequences, a carboxy-terminal 240 amino acids long peptide was found to be identical with a mature form of porcine pancreatic elastase 1, except for two amino acids. The porcine enzyme contains the same number of amino acid residues as the rat enzyme, and their amino acid sequences are 85% homologous. Taking the above findings together, we conclude that the cloned cDNA encodes a mature enzyme of 240 amino acids including a leader and activation peptide of 26 amino acids. We expressed the cloned porcine pancreatic elastase 1 cDNA in E. coli as a lac-fused protein. The resulting fused protein showed enzymatic activity and immunoreactivity toward anti-elastase serum.