Differential expression of the multiple forms of rat prekininogen mRNAs after acute inflammation

Responses of the rat liver prekininogen mRNAs after induction of acute inflammation were examined by blot-hybridization and S1 nuclease protection analyses with the aid of cDNA probes specific for rat kininogens. Marked changes in the relative levels of the low molecular weight (LMW) prekininogen mRNAs were observed after administration of Escherichia coli lipopolysaccharide, and the mRNA levels increased with a half-maximal dose of approximately 100 ng of lipopolysaccharide/100 g body weight. At maximum level of induction, the LMW prekininogen mRNAs comprised about 1% of total liver mRNA, thus representing a major component of the liver mRNA in the acutely inflamed rat. Differences in the inflammatory responses of various forms of the prekininogen mRNAs were then investigated by S1 nuclease protection analysis with the use of three different cDNA probes, each specific for either K-prekininogen or two types of T-prekininogens. Both of the T-prekininogen mRNAs increased progressively during the first 24 h after induction of inflammation, and at maximum level of induction, these two mRNAs increased about 10- and 13-fold over their normal level. In contrast, neither of the high molecular weight and LMW K-prekininogen mRNAs exhibited such an increase after induction of inflammation. Thus, the expressions of the rat T- and K-prekininogen mRNAs are differentially regulated in response to the induction of acute inflammation.     

The relationship between rat major acute phase protein and the kininogens

The rat major acute phase protein (alpha 1-MAP) is a cysteine protease inhibitor. The stoichiometry of the interaction between the inhibitor and enzyme was shown to be 1:2. A cDNA clone specific for rat alpha 1-MAP was isolated from a cDNA library prepared from an inflamed rat liver RNA template. The 1458-base pair insert was sequenced and positively identified by alignment with a partial amino acid sequence obtained by radiosequence analysis of the primary translation product for alpha 1-MAP. Complete sequence analysis determined the alpha 1-MAP cDNA coded for the entire protein with the exception of the first four amino acids of the signal peptide, all of which were identified by radiosequencing. The coding sequence spans 1282 nucleotides, followed by 115 base pairs of a 3' untranslated region. Two putative active sites, suggested by the enzyme-inhibitor ratio, have been identified by analysis of internal duplications of the alpha 1-MAP sequence and the alignment of these regions with the sequences of several low molecular weight cysteine protease inhibitors. A computer homology analysis of the protein sequence revealed a 59.3% overall identity between rat alpha 1-MAP and bovine low molecular weight (LMW) kininogen. The homology included the signal peptide regions. LMW kininogen is a precursor of bradykinin. alpha 1-MAP does contain a bradykinin sequence; the flanking amino acids are different, however. Evidence for the expression of the LMW and a high molecular weight kininogen from the same gene, and the high degree of homology between these proteins and the rat acute phase protein suggest that all three proteins belong to a precisely regulated gene family.     

Complete sequence of the lamprey fibrinogen alpha chain

The complete amino acid sequence of the lamprey fibrinogen alpha chain has been determined by a combination of peptide sequencing and cDNA and genomic cloning. The chain, which has an apparent molecular weight by dodecyl sulfate-polyacrylamide gel electrophoresis of ca. 100,000, is composed of 961 amino acid residues and has a calculated molecular weight of 96,722. It is distinguished by a large number of 18-residue repeats in a region where mammalian fibrinogens have 13-residue repeats. The data are in accord with our previous finding that the lamprey alpha chain has a distinctive amino acid composition, almost half the residues being glycine, serine, or threonine. The chain differs from mammalian alpha chains in that there are no cysteines in the carboxy-terminal half, and thus no intrachain loop, nor are there any RGD sequences in the lamprey alpha chain. Taken together with previous data on the sequences of the beta and gamma chains, the findings bear significantly on our understanding of fibrin formation. The alpha chain also provides an interesting case of structural convergence during evolution.     

Purification and characterization of two kinds of low molecular weight kininogens from rat (non-inflamed) plasma. One resistant and the second sensitive to rat glandular kallikreins

Two kinds of low molecular weight kininogens (identified as A and B) were isolated from pooled plasma of Sprague-Dawley rats. They show a single band on sodium dodecyl sulfate-polyacrylamide gel electrophoresis in the presence and absence of 2-mercaptoethanol, and the molecular weights are 68,000 for low Mr kininogen A and 73,000 for low Mr kininogen B. Although the molecular weights and amino acid compositions of the low Mr kininogens are similar, rat submaxillary and urinary kallikreins released bradykinin from low Mr kininogen B, whereas low Mr kininogen A was resistant to these enzymes. The COOH-terminal portion of low Mr kininogen A was isolated after cyanogen bromide treatment, and the amino acid sequence of the COOH-terminal 55 residues including the T-kinin (Ile-Ser-bradykinin) was determined. The COOH-terminal portion consists of two sequences with substitution of 4 residues. One peptide corresponds to alpha 1-major acute phase protein (Cole, T., Inglis, A. S., Roxburgh, C. M., Howlett, G. J., and Schreiber, G. (1985) FEBS Lett. 182, 57-61) and the other to the TI-kininogen predicted from a cDNA study (Furuto-Kato, S., Matsumoto, A., Kitamura, N., and Nakanishi, S. (1985) J. Biol. Chem. 260, 12054-12059). The results demonstrate that there exist at least two kinds of low Mr kininogens with clearly different function in rat plasma: one of them, low Mr kininogen A, is a precursor of T-kinin and is resistant to kallikreins, and the second, low Mr kininogen B, is sensitive to tissue kallikreins and shares properties with bovine and human low Mr kininogens. The results also demonstrate that T-kininogen is a mixture of two isoproteins which correspond to alpha 1-major acute phase protein or TI-kininogen, respectively. We could not detect the low Mr kininogen corresponding to the TII-kininogen predicted from the cDNA study of Furuto-Kato et al.     

Isolation and characterization of a cDNA clone for the amino-terminal portion of the pro-alpha 1(II) chain of cartilage collagen

We have isolated a cDNA clone (pRcol 2) which is complementary to the 5'-terminal portion of the rat pro-alpha 1(II) chain mRNA. A synthetic oligonucleotide was used both as a primer for cDNA synthesis and as a probe for screening a cDNA library. The probe was a mixture of sixteen 14-mers deduced from an amino acid sequence present in the amino-terminal telopeptide of the rat cartilage alpha 1(II) chain. This primer was chosen so that the resulting cDNA would contain the sequence of the 5' end of the mRNA. The nucleotide sequences of the cDNA were determined and compared with that of three other interstitial procollagen chain mRNAs (pro-alpha 1(I), pro-alpha 2(I), and pro-alpha 1(III) chain mRNA). pRcol 2 contains a 521-base pair (bp) insert, including 153 bp of the 5' untranslated region plus 368 bp coding for the signal peptide, the amino-terminal propeptide, and a part of the telopeptide. The signal peptide of the type II collagen chain is composed of about 20 amino acids. There is little homology between the amino acid sequence of the signal peptide in the pro-alpha 1(II) chain and that of three other interstitial procollagen chains. The NH2-terminal propeptide is deduced to contain short nonhelical sequences at its amino and carboxyl ends and an internal helical collagenous domain comprising 25 repeats of Gly-X-Y with one interruption. There is a strong conservation of the amino acid sequence of the carboxyl-terminal part of the NH2-terminal propeptide in the pro-alpha 1(II), pro-alpha 1(I), and pro-alpha 2(I) chains. Type II collagen mRNA does not contain a sequence corresponding to a uniquely conserved nucleotide sequence around the translation initiation site which occurs in mRNA for other procollagen chains.     

Sequence and acute phase regulation of rat alpha 1-inhibitor III messenger RNA

cDNA clones coding for the plasma proteinase inhibitor alpha 1-inhibitor III were isolated from an acute phase rat liver library. The isolates could be divided into four groups with characteristic BamHI restriction fragment patterns. The identity of the prototype clone pRLA1I3/2J was established by comparison with the published amino acid sequence of the purified protein. It codes for a 1477-amino acid precursor polypeptide with a 24-residue signal peptide. The mature protein shares 58% overall sequence identity with rat alpha 2-macroglobulin and contains a typical internal thiolester sequence. Twenty-two of its twenty-three cysteinyl residues are conserved with alpha 2-macroglobulin implying similar tertiary structure. However, the prototype alpha 1-inhibitor III sequence differed significantly from the rat and human alpha 2-macroglobulin sequences in its bait region suggesting alpha 1-inhibitor III possesses proteinase inhibitory specificities different from those of alpha 2-macroglobulin. The variant alpha 1-inhibitor III clone pRLA1I3/2J from a second cDNA group also differed from the prototype in the bait region coding sequence, although both specify similar signal peptides and NH2 termini. The observation of variant cDNA classes suggests that acute phase rat livers produce a heterogeneous mixture of alpha 1-inhibitor III mRNA molecules. Evidence was obtained for the presence of at least four different alpha 1-inhibitor III-related genes in the rat genome. During the first 24 h of an acute phase response the abundance of hepatic alpha 1-inhibitor III mRNA was decreased 3-4-fold. This decrease was of the same order of magnitude as the reported reduction of the corresponding plasma protein concentration, suggesting that in the early phase of the acute inflammatory response the plasma concentration of this protein is mainly controlled through the abundance of its hepatic mRNA.     

Construction of DNA sequences complementary to rat alpha 1 and alpha 2 collagen mRNA and their use in studying the regulation of type I collagen synthesis by 1,25-dihydroxyvitamin D

Type I collagen mRNA from fetal rat calvaria was used as a template for the synthesis of a cDNA that was subsequently inserted in the PstI site of the plasmic vector pBR322 and cloned. Three recombinant plasmids containing type I collagen specific sequences were characterized: p alpha 1R1 is 1600 bp and spans approximately 500 amino acid residues within the triple helical region of alpha 1(I) and p alpha 1R2 is 900 bp in size and covers the entire 3' noncoding and about half of the C-terminal propeptide region of alpha 1(I) collagen mRNA. The third recombinant p alpha 2R2 is 1500 bp and contains alpha 2(I) sequences specific for the entire 3' noncoding and C-terminal propeptide region. Partial nucleic acid sequence data revealed that the decreasing order of amino acid and nucleotide homology to similar regions of the rat cDNA was mouse greater than human greater than chick. Northern hybridization of mRNA after electrophoresis in 0.8% agarose revealed two distinctly different molecular weight patterns characteristic of alpha 1(I) (4.7 and 5.7 kb) and alpha 2(I) (4.2 and 4.5 kb) collagen mRNA when hybridized with the corresponding cDNA probe. Despite the high degree of sequence homology, DNA probes from rat or human produced a significantly reduced hybridization signal when used as an interspecies hybridization probe than to its corresponding mRNA. The rat cDNA probes were used in a dot hybridization assay to measure the type I collagen mRNA content in the fetal rat calvaria.(ABSTRACT TRUNCATED AT 250 WORDS)     

Major acute phase alpha 1-protein of the rat is homologous to bovine kininogen and contains the sequence for bradykinin: its synthesis is regulated at the mRNA level

The complete amino acid sequence of major acute phase alpha 1-protein of the rat (MAP) was derived from the nucleotide sequence of cloned cDNA. Amino acid analysis and partial sequencing supported the predicted sequence. The amino acid compositions of MAP and rat low-Mr kininogen are identical within experimental variation. The sequence is homologous (60%) to that of bovine low-Mr kininogen and both proteins carry the sequence for the vasoactive nonapeptide bradykinin in their C-terminal region. The rate of synthesis of MAP and the levels of MAP mRNA change coordinately during the acute phase response to inflammation.     

Molecular cloning of the cDNA encoding the third polypeptide (gamma) of brain calmodulin-dependent protein kinase II

cDNA clones for three distinct types of rat brain calmodulin-dependent protein kinase II have been isolated. Two of them were identified as cDNA clones for the alpha and beta subunits of this kinase. The other showed a nucleotide sequence similar but, not identical, to that encoding either the alpha or beta subunit. The cDNA sequence encoded a polypeptide, designated gamma, consisting of 527 amino acid residues with a molecular weight of 59,038. The deduced amino acid sequence of gamma was 84 and 87% homologous to those of alpha and beta, respectively. Higher homologies of the sequences were found in the amino-terminal halves of the three species, alpha, beta, and gamma. RNA blot analysis revealed that the mRNAs for alpha, beta, and gamma were expressed in rat brain with different regional specificities.     

Molecular cloning of cDNAs for two subunits of rat multicatalytic proteinase. Existence of N-terminal conserved and C-terminal diverged sequences among subunits

cDNA clones for two subunits (designated subunits K and L) of rat liver multicatalytic proteinase (MCP) were isolated using oligonucleotide probes synthesized according to their partial amino acid sequences. The encoded polypeptides of subunits K and L consisted of 255 and 261 amino acid residues with calculated molecular mass of 28.3 kDa and 29.5 kDa, respectively. Northern blot analysis revealed that subunits K and L were expressed in all tissues examined and their expression patterns were almost identical. The deduced amino acid sequences showed no similarities to known protein sequences other than the recently reported sequences of rat and Drosophila MCP subunits. Sequence comparison of MCP subunits of rat and Drosophila revealed that the N-terminal two-thirds of the sequence (especially the N-terminal approximately 20 residues) is conserved, but the C-terminal third of the sequence shows no similarity, suggesting functional and structural roles for both regions. Implications for the structural and functional aspects of MCP subunits are discussed based on the sequence similarity.     

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.     

Cloning and characterization of cDNA encoding 3-methylcholanthrene inducible rat mRNA for UDP-glucuronosyltransferase

We have isolated cDNA clones of the mRNA for rat UDP-glucuronosyltransferase that catalyzes the glucuronidation of 4-nitrophenol, by using synthetic oligonucleotides as hybridization probes. The complete nucleotide sequence of the 1,927-base pairs cDNA insert has been determined. With untranslated sequences of 124 and 216 base pairs in the 5'- and 3'-terminal regions, respectively, the cDNA insert contained 1,587 base pairs that encode a complete primary structure of a putative precursor form of 4-nitrophenol UDP-glucuronosyltransferase with a calculated molecular weight of 60,114. The cDNA sequence also indicates the presence of 25 amino acids preceding the sequence determined by microsequence of the isolated protein. This extrapeptide, for the most part, consists of hydrophobic amino acids which are characteristic of the signal peptides as found for secretory proteins and most transmembrane proteins. Furthermore, the deduced amino acid sequence contains a putative halt transfer signal of a hydrophobic segment (residues 487-510), which is flanked on both sides by the peptide segments of highly charged amino acid residues (residues 463-486 and 511-529). These features are consistent with the properties of transmembrane proteins. Specific cDNA probes were used to analyze the induction of the enzyme in rat tissues by treatment with 3-methylcholanthrene. RNA blot analysis showed that 3-methylcholanthrene increased 10- to 15-fold the amount of hybridizable mRNA in liver. The livers and kidneys from 3-methylcholanthrene-treated rats were found to contain almost the same amount of hybridizable mRNA, although the basal level in the kidney was much higher than that of the liver, and the amounts in the lung were much lower than that of the liver and kidney.     

Primary structure of human plasma fibronectin. Characterization of a 31,000-dalton fragment from the COOH-terminal region containing a free sulfhydryl group and a fibrin-binding site

The 31-kDa domain of human plasma fibronectin has been completely characterized. This fragment is located at the COOH-terminal end of the molecule immediately preceding the 3-kDa interchain disulfide-containing peptide. The 31-kDa domain was obtained after trypsin digestion of fibronectin and purified by affinity chromatography on gelatin- and heparin-Sepharose columns. The fragment eluted in the heparin-unbound fraction and was further purified by DEAE-cellulose and high performance liquid chromatography. The 31-kDa fragment contained a fibrin-binding site (fibrin II site) which was only active at physiological NaCl concentrations and therefore differed from that located in the NH2-terminal domain which also bound at lower NaCl concentrations. The 31-kDa domain bound to thiopropyl-Sepharose and was shown to contain a free sulfhydryl group located at position 35 in the sequence. To determine the complete amino acid sequence of this fragment, a trypsin digestion was performed on the reduced and alkylated 31-kDa domain, and the 17 resulting peptides were isolated by high performance liquid chromatography; their amino acid compositions and amino acid sequences have been determined, and the arrangement of peptides was achieved by comparison with the sequences deduced from human and rat cDNA clones and with a related plasmic fragment from bovine fibronectin. Comparison of these three sequences showed 23 amino acid differences between human and rat fibronectin and 16 between human and bovine fibronectin. This represents a 91 and 94% homology, respectively. An interesting finding is that the 31-kDa fragment contains a deletion of 31 residues when compared to the rat cDNA sequence. This deletion appears to represent a species difference since it is due to a shorter mRNA in the case of human fibronectin.     

Molecular cloning and sequence analysis of cDNA for the catalytic subunit 1 alpha of rat kidney type 1 protein phosphatase, and detection of the gene expression at high levels in hepatoma cells and regenerating livers as compared to rat livers.

A cDNA clone containing the full coding sequence of a type 1 protein phosphatase catalytic subunit 1 alpha has been isolated from a rat kidney lambda gt 10 library. The protein sequence deduced from the cDNA contains 330 amino acid residues with a molecular mass of 38 kDa. The cDNA clone from rat kidney was 89% identical at the nucleotide level in the coding region to type 1 protein phosphatase 1 alpha from rabbit skeletal muscle. However, the two protein sequences were completely identical. The type 1 alpha protein phosphatase from rat kidney shows 49% homology of amino acid sequence to the rat type 2A alpha protein phosphatase. Thus, the protein sequence of type 1 alpha protein phosphatase was completely conserved between rat and rabbit. The mRNA levels of type 1 protein phosphatase were determined in rat liver, AH13, a strain of rat hepatoma, and regenerating rat liver by Northern blot analysis using the cDNA fragment as a probe, under which conditions a single mRNA of 1.5 kb was detected. The mRNA levels of AH13 were remarkably increased when compared to those of normal ivers, whereas the mRNA levels of regenerating livers were slightly but significantly increased. These results demonstrate a marked increase in gene expression of type 1 protein phosphatase in hepatoma cells, suggesting an important role of the type 1 protein phosphatase in hepatocarcinogenesis.     

Structural studies on bovine spleen heme oxygenase. Immunological and structural diversity among mammalian heme oxygenase enzymes.

Heme oxygenase is an Mr 32,000 microsomal enzyme which catalyzes the rate-limiting step in the oxidative catabolism of heme to yield equimolar quantities of biliverdin IX alpha, carbon monoxide, and iron. In the present investigation, evidence is presented suggesting that immunochemical and structural differences exist between bovine spleen heme oxygenase and heme oxygenase enzymes from other mammalian species. Using an antibody directed against bovine spleen heme oxygenase, enzyme-linked immunosorbent assays, Western blotting experiments, and cell-free translation immunoprecipitation studies showed that bovine spleen heme oxygenase is only weakly immunochemically related to heme oxygenase from rat spleen. This observation was supported by the fact that a rat spleen heme oxygenase cDNA probe did not hybridize significantly to bovine spleen heme oxygenase mRNA in Northern analyses nor to restriction fragments containing the bovine heme oxygenase gene in Southern analyses. Tryptic peptides were prepared from bovine spleen heme oxygenase and the amino acid sequences of nine peptides comprising 94 amino acid residues were determined, providing the first information on the primary structure of bovine spleen heme oxygenase. Comparison of the sequences of these tryptic peptides with regions of the deduced amino acid sequences of rat spleen and human macrophage heme oxygenase revealed sequence similarities ranging from 55 to 100%. Several peptides displaying the highest degree of sequence similarity were found to occur in regions of the heme oxygenase molecule postulated to contain the heme binding site, indicating that despite the immunochemical and apparent structural differences between bovine spleen heme oxygenase and the rat and human enzymes, functionally important amino acid residues have been conserved in the evolution of mammalian heme oxygenase genes.     

Molecular cloning and primary structure of rat testes metalloendopeptidase EC 3.4.24.15

The complete amino acid sequence of rat testes metalloendopeptidase (EC 3.4.24.15) was deduced from the nucleotide sequence of a cDNA clone isolated by screening a rat testes library with a polyclonal antibody raised against a homogeneous preparation of the rat testes enzyme. The correctness of the sequence was verified by N-terminal amino acid sequence analysis of the isolated enzyme and by partial amino acid sequence analysis of three tryptic peptides located near the N-terminus, the middle, and C-terminus of the native protein. The enzyme is composed of 645 amino acids with a molecular weight of 72,985. This value is close to that of the purified rat testes and brain enzyme as determined by polyacrylamide gel electrophoresis under denaturing and reducing conditions and by molecular sieving chromatography. The enzyme contains the putative active-site sequence -H-E-F-G-H- that is homologous to the sequence in the active site of thermolysin and several other related bacterial enzymes, as well as to active-site sequences of several mammalian zinc metallopeptidases. No amino acid sequence homology, beyond this active site, was found with thermolysin, a bacterial zinc metalloendopeptidase, nor with several mammalian zinc metallopeptidases. Northern blot hybridization analyses showed the presence of mRNA encoding the enzyme in rat testes, but not in other rat tissues in spite of the finding that enzyme activity is widely distributed in all tissues and that relatively high activities are present in rat brain and pituitary.     

Structure and regulation of rat long-chain acyl-CoA synthetase

Complementary DNAs encoding rat long-chain acyl-CoA synthetase have been isolated. The cDNAs were identified using synthetic oligonucleotide probes based on partial amino acid sequences of lysyl endopeptidase peptides of the purified enzyme. Rat long-chain acyl-CoA synthetase is predicted to contain 699 amino acid residues and to have a calculated molecular weight of 78,177. Significant sequence similarity was found between parts of long-chain acyl-CoA synthetase and firefly luciferase. Based on the similarity of the reaction mechanisms of the two enzymes, we propose a function for the similar region. The long-chain acyl-CoA synthetase mRNA is expressed in liver, heart, and epididymal adipose tissues and, to a much lesser extent, in brain, small intestine, and lung. The level of long-chain acyl-CoA synthetase mRNA is increased 7-8-fold in rat liver by feeding a diet high in carbohydrate or fat, consistent with the physiological significance of the enzyme in fatty acid metabolism.     

Coding nucleotide sequence of rat liver malic enzyme mRNA

The nucleotide sequence of the mRNA for malic enzyme ((S)-malate NADP+ oxidoreductase (oxaloacetate-decarboxylating, EC 1.1.1.40) from rat liver was determined from three overlapping cDNA clones. Together, these clones contain 2078 nucleotides complementary to rat liver malic enzyme mRNA. The single open reading frame of 1761 nucleotides codes for a 585-amino acid polypeptide with a calculated molecular mass of about 65,460 daltons. The cloned cDNAs contain the complete 3'-noncoding region of 301 nucleotides for the major mRNA species of rat liver and 16 nucleotides of the 5'-noncoding region. Amino acid sequences of seven tryptic peptides (67 amino acids) from the purified protein are distributed through the single open reading frame and show excellent correspondence with the translated nucleotide sequence. The putative NADP-binding site for malic enzyme was identified by amino acid sequence homology with the NADP-binding site of the enoyl reductase domain of fatty acid synthetase.