Primary structure of human preangiotensinogen deduced from the cloned cDNA sequence

Cloned cDNA sequences for human preangiotensinogen have been isolated from a human liver cDNA library by hybridization with a restriction fragment derived from a previously cloned cDNA for rat preangiotensinogen. Analyses by nucleotide sequence determination, S1 nuclease mapping, and RNA blot hybridization indicate that human preangiotensinogen is encoded by two mRNAs that differ only in the length of the 3'-untranslated region. The deduced amino acid sequence shows that the mature angiotensinogen consists of 452 amino acid residues with the angiotensin sequence at its amino-terminal portion. Two potential initiation sites have been discussed. These are the methionine codon located at the position exactly corresponding to the initiation site of rat preangiotensinogen mRNA and an additional methionine codon positioned nearest the 5' end of the mRNA. The amino acid sequences starting at either of the initiation sites and preceding the angiotensin sequence constitute a large number of hydrophobic amino acid residues, thus representing the signal peptide characteristic of the secretory proteins. Human and rat preangiotensinogens show that 63.6% of the amino acid positions of the two proteins are identical. However, the amino-terminal portions directly distal to angiotensin I diverge markedly between the two proteins and differ in their possible glycosylation sites. These structural differences may contribute to the known species specificity exhibited by renin.     

Complete amino acid sequence of the human progesterone receptor deduced from cloned cDNA

A lambda gt10 library containing DNAs complementary to messenger RNAs from human breast cancer T47-D cells was constructed and screened with a cDNA probe encoding the rabbit progesterone receptor. Four overlapping clones have been sequenced. The open reading frame corresponds to a protein of 933 amino acids with a molecular weight of 98,868 Da. The cysteine rich basic region supposed to be involved in DNA binding is completely homologous in the human and rabbit receptors, whereas the C-terminal end, where hormone binding is thought to take place, differs by a single amino acid change. The human progesterone receptor is characterized, as is the rabbit receptor, by the very high proline content of its N-terminal region. When mRNAs from either human breast cancer cell lines T47-D and MCF-7 or from normal human uterus tissue were blotted and probed with the cloned cDNA, four main bands were observed (5100, 4300, 3700, and 2900 nucleotides).     

The amino acid sequence of rat liver glucokinase deduced from cloned cDNA

Rat liver glucokinase (ATP:D-hexose 6-phosphotransferase, EC 2.7.1.1) was purified to homogeneity, cleaved, and subjected to amino acid sequence analysis. Forty-five percent of the protein sequence was obtained, and this information was used to design oligonucleotide probes to screen a rat liver cDNA library. A 1601-base pair cDNA (GK1) contained an open reading frame that encoded the amino acid sequences found in the peptides used to generate the oligonucleotide probes. A second cDNA was subsequently identified (GK.Z2), which is 2346 base pairs long and corresponds to nearly the entire glucokinase mRNA. Blot transfer analysis of hepatic RNA showed that glucokinase mRNA exists as a single species of about 2400 nucleotides. Four hours of insulin treatment of diabetic rats resulted in a 30-fold induction of this mRNA. GK.Z2 has a long open reading frame which, with the known partial peptide sequence, allowed us to deduce the primary structure of glucokinase. The enzyme is composed of 465 amino acids and has a mass of 51,924 daltons. Glucokinase has 53 and 33% amino acid sequence identities with the carboxyl-terminal domains of rat brain hexokinase I and yeast hexokinase, respectively. If conservative amino acid replacements are also considered, glucokinase is similar to these two enzymes at 75 and 63% of positions, respectively. The putative glucose- and ATP-binding domains of glucokinase were identified, and these regions appear to be highly conserved in the hexokinase family of enzymes.     

Complete amino acid sequence of human intestinal aminopeptidase N as deduced from cloned cDNA

The complete primary structure (967 amino acids) of an intestinal human aminopeptidase N (EC 3.4.11.2) was deduced from the sequence of a cDNA clone. Aminopeptidase N is anchored to the microvillar membrane via an uncleaved signal for membrane insertion. A domain constituting amino acid 250-555 positioned within the catalytic domain shows very clear homology to E. coli aminopeptidase N and contains Zn2+ ligands. Therefore these residues are part of the active site. However, no homology of the anchor/junctional peptide domain is found suggesting that the juxta- and intra-membraneous parts of the molecule have been added/preserved during development. It is speculated that this part carries the apical address.     

Amino acid sequence of rat apolipoprotein A-II deduced from the nucleotide sequence of cloned cDNA

A rat apolipoprotein A-II cDNA clone was isolated from a rat liver cDNA library by in situ hybridization of bacteriophage plaques using a 32P-labeled human apoA-II cDNA as a probe. The cDNA insert from this clone was characterized by DNA sequencing. The amino acid composition derived from the DNA sequence data matched well with that of rat apoA-II reported earlier (Herbert et al. 1974. J. Biol Chem. 249: 5718-5724), indicating that the cDNA insert coded for rat apoA-II. Further evidence was provided by a comparison of the amino acid sequence of rat apoA-II obtained here with that of human apoA-II (Brewer et al. 1972. Proc. Natl. Acad. Sci. USA. 69: 1304-1308). While the rat apoA-II cDNA insert did not code for the entire presegment, it had the same COOH-terminal residues of the presegment as well as the same prosegment (Ala-Leu-Val-Arg-Arg) as in human preproapoA-II, suggesting that rat apoA-II was also synthesized initially as preproapoA-II. Mature rat apoA-II contains 79 amino acids. Residue 6 of mature rat apoA-II is Asp, while it is Cys in human apoA-II, and this would account for the absence of dimeric forms of rat apoA-II in plasma. While the overall amino acid sequence homology between rat and human apoA-II is about 50%, the amphipathic alpha-helical structures, which are responsible for lipid-binding, seem to be conserved in the two proteins. The size of rat apoA-II mRNA was estimated to be about 600 nucleotides.(ABSTRACT TRUNCATED AT 250 WORDS)     

The nucleotide sequence of a cloned human leukocyte interferon cDNA

We have determined the nucleotide sequence of the human leukocyte interferon cDNA carried in hybrid plasmid Z-pBR322(Pst)/HcIF-2h, which has been shown to direct the formation of a polypeptide with human leukocyte interferon activity (Nagata et al., 1980). The 910 base pair insert contains a 567 (or 543) base pair coding sequence, which determines a putative preinterferon polypeptide consisting of a signal peptide of 23 (or less likely 15) amino acids, followed by an interferon polypeptide of 166 amino acids (calculated molecular weight, 19 390). The coding sequence is preceded by a (most likely incomplete) 56 bp leader and followed by a 242 bp trailer and seven A residues from the poly(A) tail: A comparison of the sequence of 35 amino terminal amino acids of lymphoblastoid interferon (Zoon et al., 1980; M. Hunkapiller and L. Hood, personal communication) and the corresponding sequence deducted for leukocyte interferon revealed 9 differences. This suggests that these two interferons are encoded by two non-allelic genes.     

The gamma-subunit of ATP synthase from spinach chloroplasts. Primary structure deduced from the cloned cDNA sequence

cDNA clones encoding the gamma-subunit of chloroplast ATP synthase were isolated from a spinach library using synthetic oligonucleotide probes. The predicted amino acid sequence indicated that the mature chloroplast gamma-subunit consists of 323 amino acid residues and is highly homologous (55% identical residues) with the sequence of the cyanobacterial subunit. The positions of the four cysteine residues were identified. The carboxyl-terminal region of the chloroplast gamma-subunit is highly homologous with those of the gamma-subunits from six other sources (bacteria and mitochondria) sequenced thus far.     

Structure and evolution of human α-fetoprotein deduced from partial sequence of cloned cDNA

The nucleotide sequence of a recombinant DNA clone, containing a partial mRNA sequence for human α-fetoprotein (AFP) in the plasmid vector pBR322, has been determined. Two regions of the cloned nucleotide sequence were found to agree with published amino acid sequences of two cyanogen bromide peptides derived from human AFP. Examination of the amino acid sequence, deduced from the cloned portion of the mRNA coding region, reveals extensive homology with the third domain of the human serum albumin molecule. A total of 44% ( ) amino acids and 54% ( ) nucleotides are identical in the two structures. The landmark cysteine residues are found in the same positions in both polypeptide chains, presumably forming the same disulfide bridges in AFP as those found in the albumin. The sequence homology reinforces the evidence that human AFP and albumin constitute a gene family, in analogy to the same family found in rodents. A comparison of the human and rodent sequence data suggests that the rate of molecular evolution has been faster for AFP than for albumin.     

Prothymosin alpha and parathymosin: amino acid sequences deduced from the cloned rat spleen cDNAs

A rat spleen cDNA library was screened for clones carrying the cDNAs for prothymosin alpha and parathymosin. Sequence analysis of a clone carrying the entire coding region for prothymosin alpha confirmed and completed the amino acid sequence for this polypeptide and established the number of amino acid residues as 111. Rat prothymosin alpha differs from human prothymosin alpha at six positions, including four substitutions and two insertions. The nucleotide sequences of the cDNAs for the rat and human polypeptides are more than 90% identical in the open reading frames, with significant homology extending into the 5' and 3' flanking regions. From the same library, we also isolated a clone carrying 80% of the coding region for rat parathymosin. The number of amino acid residues in rat parathymosin is 101, based on the sequence deduced from the cDNA insert and earlier information on the sequence in the amino-terminal portion of this polypeptide. Despite their similarity in size and amino acid composition, rat prothymosin alpha and rat parathymosin show only limited sequence homology, primarily in the segment including residues 14 through 25, where 10 of 12 positions are identical in the two polypeptides. this is also the region of significant sequence similarity to a 12-amino-acid segment in the p17 protein of the human immunodeficiency disease associated virus (HTLV-IIIB).     

Primary structure of human proacrosin deduced from its cDNA sequence

cDNA clones encoding proacrosin, the zymogen of acrosin, were isolated from a human testis cDNA library by using a fragment of boar acrosin cDNA as a probe. Nucleotide sequencing of the longest cDNA clone has predicted that human proacrosin is synthesized with a 19-amino-acid signal peptide at the N-terminus. The cleavable signal sequence is followed by a 23-residue segment corresponding to the light chain and then by a 379-residue stretch that constitutes the heavy chain containing the catalytic site of the mature protease. The C-terminal portion of the deduced sequence for the heavy chain is very rich in proline residues, most of which are encoded by a unique repeat of CCCCCA. The active-site residues including histidine, aspartic acid, and serine are also predicted to be located at residues 69, 123, and 221, respectively.     

Amino acid sequence of alpha-subunit in hen egg white ovomucin deduced from cloned cDNA.

The primary amino acid sequence of alpha-subunit in ovomucin (OVM) from hen thick egg white was determined. The 2087 amino acid residues with a relative molecular mass of 230.9 kDa along the full length of the alpha-subunit were represented. The alpha-subunit contains domains, arranged from the N- to C-terminals in the following order: D1-D2-D'-D3-R (central region)-D4-C1-CK (Cystine-knot), in a manner similar to the arrangement of D, C and CK domains in human pre-pro-von Willebrand factor (hpp-vWF) and hMUC2. The alpha-subunit showed identities on amino acid sequences with hpp-vWF and hMUC2 at 33 and 41% in the N-terminal region and 30 and 38% in the C-terminal region, respectively. The numbers and positions of cysteine residues were highly conserved among alpha-subunit, hpp-vWF and hMUC2. However, R showed no virtual sequence homology with the corresponding regions in two proteins. It was estimated that alpha-subunit was not part of a large peptide of OVM, but was independently synthesized from beta-subunit.     

The primary sequence of the PFK-1 inactivating zinc-binding protein as deduced from cDNA sequencing. Identity of the zinc-binding protein with rat parathymosin

We have recently described the sequence of the Zn2+-binding domain (43 amino acid residues) of a newly detected Zn2+-binding protein which reversibly inactivates phosphofructokinase-1 in a Zn2+-dependent manner [(1986) J. Biol. Chem. 269, 5895-5900; (1988) Eur. J. Biochem. 177, 561-568]. Here, we describe the primary sequence of this protein based on a full-length cDNA. A sequence comparison reveals the identity of the Zn2+-binding protein with a protein called parathymosin-alpha.     

Structure of human alpha 2-plasmin inhibitor deduced from the cDNA sequence

We have isolated three cDNA clones for human alpha 2-plasmin inhibitor (alpha 2-PI). Two clones are from human hepatoma cell line, Hep G2, and cover the entire protein coding region plus the 3'-flanking region up to the poly(A) sequence, and the other clone is from human liver and contains the carboxyl-terminal half. The total length of the cDNAs is 2.29 kb, corresponding to more than 95% of the full-length mRNA. alpha 2-PI seems to consist of 452 amino acid residues plus 39 amino acid residues for the signal peptide. The amino acid sequence shows 23 to 28% homology to those of five other protease inhibitors, plasminogen activator inhibitor (PAI), protein C inhibitor (PCI), alpha 1-antitrypsin (alpha 1-AT), antithrombin III (AT III), and alpha 1-antichymotrypsin (alpha 1-AC). alpha 2-PI seems to be the most distantly related among these inhibitors. Comparison of the phylogenetic trees of proteases and their inhibitors indicates that four proteases, namely elastase (or trypsin), chymotrypsin, plasminogen activator, and thrombin, may have evolved concurrently with the corresponding inhibitors. However, alpha 2-PI and PCI seem to have evolved asynchronously from their substrates. The data suggest that alpha 2-PI may originally have inhibited some protease other than plasmin, and protein C may have had an inhibitor different from the present one early in its evolutionary history.     

The complete amino acid sequence of the catalytic domain of rat brain hexokinase, deduced from the cloned cDNA

The complete amino acid sequence of the catalytic domain of rat brain hexokinase (ATP:D-hexose 6-phosphotransferase, EC 2.7.1.1) has been deduced from the nucleotide sequence of cloned cDNA. Extensive similarity in sequence, taken to indicate similarity in secondary and tertiary structure, is seen between the mammalian enzyme and yeast hexokinase isozymes A and B. All residues critical for binding glucose to the yeast enzyme are conserved in brain hexokinase. A location for the substrate ATP binding site is proposed based on relation of structural features in the yeast enzyme to characteristics commonly observed in other nucleotide binding enzymes; sequences in regions proposed to be important for binding of ATP to the yeast enzyme are highly conserved in brain hexokinase.     

Structure of dog and rabbit precursors of atrial natriuretic polypeptides deduced from nucleotide sequence of cloned cDNA

The structure of precursors of dog and rabbit atrial natriuretic polypeptides was determined by nucleotide sequence analysis of cloned cDNA of mRNA encoding the peptides. The dog and rabbit precursors are 149 and 153 residues long having 23- and 25-residue putative signal peptides at their N-termini respectively. The 28-residue peptide with identical sequence to that of human, which has potent natriuretic activity, is located at the C-terminus of the dog precursor. The 28-residue peptide of identical sequence to that of mouse/rat is located at C-terminus of rabbit precursor followed by additional Arg-Arg sequence which is also found in rat/mouse precursors and is apparently removed during processing.     

Amino acid sequence deduced from a rat kidney cDNA suggests it encodes the Zn-peptidase aminopeptidase N

We have isolated and characterized rat kidney cDNA clones encoding a 140-kDa glycoprotein that exhibits characteristics of a cell surface Zn-peptidase. Structural features predicted for this putative kidney Zn-peptidase (KZP) are most consistent with properties previously determined for the Zn-peptidase aminopeptidase N. The deduced amino acid sequence of rat KZP is almost identical to the NH2-terminal sequence of aminopeptidase N purified from rabbit. The overall amino acid composition predicted for rat KZP is remarkably similar to that previously determined for rabbit and pig aminopeptidase N. The predicted Mr of rat kidney KZP approximates the Mr of the unglycosylated form of aminopeptidase N. The topology predicted for KZP is identical to that observed for aminopeptidase N: a short cytoplasmic domain at the NH2 terminus immediately precedes an uncleaved signal/anchor domain; a stalk region connects this membrane anchor to the extracellular, hydrophilic bulk of the protein containing catalytic sites required for Zn-peptidase activity. In addition, mRNA encoding KZP is present in tissues known to exhibit aminopeptidase N activity. Taken together with the observation that only a single gene homologous to KZP DNA is present in the rat and human genomes, these results suggest that we have established the primary structure of rat kidney aminopeptidase N.     

Internal quadruplication in the structure of human interstitial retinol-binding protein deduced from its cloned cDNA

Interstitial retinol-binding protein (IRBP) is a glycoprotein that shuttles retinoids between the retina and pigment epithelium and is secreted by the photoreceptor cells of the vertebrate eye. Human retina cDNA libraries in lambda gt10 were screened with a previously isolated human IRBP probe (H.4 IRBP), yielding five overlapping cDNA clones generating a 4223-base sequence. A 17-kilobase pair clone (HGL.3) isolated by screening a human genomic library in EMBL3 with H.4 IRBP yielded a 2.5-kilobase pair SstI fragment that overlapped the 5' end of the cDNA sequence by 329 nucleotide residues. An open reading frame encoded the N-terminal sequence of human IRBP and predicted a protein consisting of 1262 amino acids with a molecular mass of 136,600. Two putative N-linked glycosylation sites were identified. The translated sequence suggests that there is a 16-amino acid presumptive signal peptide rich in hydrophobic residues and with a high alpha-helix probability preceding the N terminus of the mature protein. The amino acid sequence of human IRBP could be aligned with 87% identity with the amino acid sequences of 31 peptides (605 residues) purified from a tryptic digest of bovine IRBP. The protein sequence of human IRBP contains four duplicated segments (302-310 residues in length) with 33-38% identity. From the degree of identity between the bovine and human sequences, it is possible that IRBP evolved by several gene duplications that occurred 600-800 million years ago, before the emergence of the vertebrates.     

Primary structure of human carcinoembryonic antigen (CEA) deduced from cDNA sequence

The cDNAs corresponding to the mRNA encoding a polypeptide which is immunoreactive with the antisera specific to carcinoembryonic antigen (CEA) (1) are cloned. The amino acid sequences deduced from the nucleotide sequences of the cDNAs show that it is synthesized as a precursor with a signal peptide followed by 668 amino acids of the putative mature CEA peptide, whose N-terminal 24 amino acids and amino acids 286 to 295 exactly coincide with those known for N-terminal sequences of CEA (2) and NFA-1 (3), respectively. The first 108 N-terminal residues are followed by three very homologous repetitive domains of 178 residues each and then by 26 mostly hydrophobic residues which probably comprise a membrane anchor. Each repetitive domains contains 4 cysteines at precisely the same positions and as many as 28 possible N-glycosylation sites are found in the CEA peptide region agreeing with high carbohydrate content of purified CEA.