Structure and bactericidal activity of an antibiotic dodecapeptide purified from bovine neutrophils

Cytoplasmic granules of neutrophils store a variety of cationic polypeptides, which exert in vitro a potent antibacterial action and are potentially involved in host defense mechanisms. From an acid extract of bovine neutrophil granules we have purified over 2,000-fold a dodecapeptide exhibiting bactericidal activity against both Escherichia coli and Staphylococcus aureus at 10(-7)-10(-5) M concentration. The purification procedure involved only two steps of ion-exchange and reversed-phase chromatography. The peptide, named bactenecin, has the amino acid sequence, Arg-Leu-Cys-Arg-Ile-Val-Val-Ile-Arg-Val-Cys-Arg, maintained in a cyclic structure by a disulfide bond between the two cysteine residues. Computer modeling of the dodecapeptide resulted in a conformation in which the chain adopts an antiparallel extended structure forming a gamma turn at residue 7.     

Primary amino acid sequence of bovine dopamine beta-hydroxylase

Fifty-eight tryptic and Staphylococcus aureus V8 protease generated peptides from bovine dopamine beta-hydroxylase were isolated by reverse-phase high pressure liquid chromatography and sequenced. These peptide sequences were compared with the deduced amino acid sequences of bovine and human dopamine beta-hydroxylase obtained from the cloned cDNAs. Bovine peptide sequences had five differences with the sequence derived from the bovine cDNA, and four of the changes could be accounted for by a single base change in the DNA. N-terminal sequence analysis of the bovine enzyme indicated that it contained two N termini, one of which is 3 amino acids longer than the other and begins with the sequence Ser-Ala-Pro. The amino acid sequences deduced from the bovine and human cDNAs are 19 and 25 amino acids longer, respectively, and these additional amino acids represent leader peptide sequences. Two bovine peptide sequences contained glycosylation sites and gave positive tests for carbohydrate residues, and two others contained the consensus sequence for a glycosylation site but were negative in the carbohydrate test. The bovine enzyme contains 6 Trp, as compared with 7 in the bovine cDNA and 8 in the human cDNA. The protein and bovine cDNA contain 24 Tyr each, as compared with 26 in the human cDNA. These numbers indicate that the true epsilon 1% 280 = 8.95, and, therefore, that it is 28% lower than the previously determined value. The data also identify 5 His-containing regions that may be involved in Cu2+ coordination at the active site.     

Peptide alignment of the porcine 21-hydroxylase cytochrome P-450 using a cDNA sequence of the corresponding bovine enzyme

The amino acid sequence deduced from a cDNA clone of the bovine adrenal steroid 21-hydroxylase cytochrome P-450 has been utilized to align peptide sequences derived from the corresponding porcine enzyme. Homology analysis revealed that only fifty percent of the amino acid sequence predicted by the cDNA clone overlapped with peptide sequences from the porcine enzyme. The homology in the remaining portions of the bovine sequence was restored by considering amino acid sequences predicted by the two additional DNA reading frames of the cDNA sequence. Forty eight percent of the bovine sequences predicted by the two alternate reading frames showed strong homology with the porcine peptide sequences. A minimum of 4 nucleotide sequencing errors account for the observed reading frame alterations and the approximate position of each error in the bovine cDNA sequence has been established.     

Cloning of a full-length complementary DNA for fatty-acid-binding protein from bovine heart

A full-length cDNA for bovine heart fatty-acid-binding protein (H-FABP) was cloned from a lambda gt11 cDNA library established from bovine heart muscle. The cDNA sequence shows an open reading frame coding for a protein with 133 amino acids. Colinearity with the amino acid sequences of four tryptic peptides was asserted. H-FABP isolated from bovine heart begins with an N-acetylated valine residue, however, as derived from analysis of the tryptic, amino-terminal-blocked peptide and the molecular mass of the peptide obtained via secondary-ion mass spectrometry. The molecular mass of the total protein is 14673 Da. Bovine H-FABP is 89% homologous to rat H-FABP and 97% homologous to the bovine mammary-derived growth-inhibition factor described recently by B?hmer et al. [J. Biol. Chem. 262, 15137-15143 (1987)]. Significant homologies were also found with bovine myelin protein P2 and murine adipocyte protein p422. Secondary-structure predictions were proposed for these proteins, based on computer analysis, which reveal striking similarities.     

Antipeptide antibody against the human low-density-lipoprotein receptor. Characterization and cross-reactivity with bovine lymphocytes.

A dodecapeptide corresponding to the external N-terminal sequence of the human low-density-lipoprotein (LDL) receptor was synthesized. Antibodies raised in rabbits against the peptide were purified and were shown to react specifically with the peptide and with human LDL receptor of fibroblasts, HeLa cells and lymphocytes using binding studies and immunoblotting. By indirect immunogold analysis, antibodies bound to the LDL receptor of human lymphocytes could be revealed as clusters. Anti-receptor peptide immunoglobulins specifically bound to the human HeLa cell's LDL receptor with a lower affinity than LDL (Kd x 3). The anti-receptor peptide immunoglobulins and 125I-labelled-LDL competed with each other for the LDL-receptor sites. Antibodies failed to react with lymphocytes of subjects with the homozygous form of familial hypercholesterolaemia. Cross-reactivity with the dodecapeptide of the bovine LDL receptor was limited, but this cross-reactivity was confirmed by the binding of anti-receptor peptide immunoglobulins to the LDL receptor from bovine lymphocytes.     

Molecular cloning and primary structure of human chromogranin A (secretory protein I) cDNA

Chromogranin A (CGA), also referred to as secretory protein I, is an acidic protein that has been detected in all neuroendocrine cell types examined and is often present in large amounts relative to other secreted proteins. For example, CGA comprises at least 40% of the soluble protein of the adrenal chromaffin granule, and it appears to be the major secretory protein in the parathyroid secretory granules. CGA complementary DNAs (cDNAs) from bovine adrenal and pituitary have recently been cloned and sequenced and found to be nearly identical. A region of bovine CGA has a high degree of amino acid sequence identity to pancreastatin, a recently isolated porcine peptide that inhibits glucose-induced insulin secretion. This suggests that CGA may be a prohormone. We have cloned and sequenced a human cDNA encoding CGA. This human CGA cDNA has an overall 86% nucleic acid identity to the bovine cDNA. Like the bovine CGA cDNA, the human cDNA has little homology to pancreastatin at the 5' region of this peptide but significant amino acid homology to the carboxyl-terminal portion of pancreastatin where the biologic activity resides. There is an area within the pancreastatin region of human CGA and porcine pancreastatin with a 70% amino acid identity to the calcium-binding moiety of the E-F hand proteins such as parvalbumin and oncomodulin. These data suggest that CGA and pancreastatin may both be members of a larger family of calcium-binding proteins.     

Sequence of membrane-associated thyroid hormone binding protein from bovine liver: its identity with protein disulphide isomerase

The complementary DNAs of the bovine liver membrane-associated 3,5,3'-triiodo-L-thyronine binding protein with 55 k-dalton (T3BP) were cloned and the nucleotide sequences were determined. Monospecific antibodies against T3BP were used to screen a bovine liver cDNA library in lambda gtll. We analyzed the sequences of two cloned T3BP cDNAs. The clones encoded a polypeptide of 510 amino acid residues, including a signal peptide of 20 amino acid. Northern blot analysis of bovine and human RNA showed that the mRNAs encoding T3BP are 2.7 kilobase in length. Amino acid sequence of N-terminal and other three peptides isolated from purified T3BP were found in the predicted amino acid sequence from the cDNA sequence. The predicted amino acid sequence is closely homologous (93%) with that of rat protein disulphide isomerase (EC 5.3.4.1), which catalyzes the isomerization of the protein disulphide bonds and has been shown to play an important role in post-translational regulation. The results suggest that T3BP and protein disulphide isomerase should be the same protein.     

Molecular Cloning and Expression of Yak (Bos grunniens) Lactoferrin cDNA in Pichia pastoris

cDNA encoding lactoferrin from yak was isolated by RT-PCR and then sequenced. The cloned cDNA (2127 bp) encodes a 709 amino acid precursor molecule of yak lactoferrin with a signal peptide of 19 amino acids. The yak lactoferrin cDNA was expressed in Pichia pastoris. The recombinant protein, purified by Ni-NTA affinity column, had a molecular weight of 76 kDa and reacted with an antibody raised against native bovine lactoferrin. The iron-binding behavior and antimicrobial activity of the purified protein indicated that it was correctly folded and functional.     

Isolation and sequence of rat testis cDNA for a calcium binding polypeptide similar to the regulatory subunit of calcineurin.

We have cloned and sequenced rat testis cDNAs coding for a calcium binding polypeptide similar to calcineurin beta subunit, the Ca(2+)-binding subunit of the Ca2+/calmodulin stimulated protein phosphatase. Rat testis cDNA library was screened with a monoclonal antibody Va1 raised against bovine brain calcineurin beta subunit. The deduced amino acid sequence is similar to that of human brain calcineurin beta subunit with respect to containing four putative calcium binding sites. However, distinct differences were found: 1) The cloned cDNA had six amino acids polypeptide tail at carboxy-terminal which is absent in human brain calcineurin beta subunit. This amino acids tail makes the carboxy-terminal highly hydrophilic in contrast to the human brain beta subunit which is hydrophobic at carboxy-terminal; 2) eleven amino acids at the N terminal of the cloned cDNA were completely different from the corresponding region of the brain calcineurin beta subunit.     

Molecular characterization of cDNA encoding B. taurus cathelicidin-7 like antibiotic peptide from bone marrow cells of Bubalus bubalis.

Cathelicidins represent a diverse family of endogenous cationic antibiotic peptide present in all mammalian species. In the present study, a novel cathelicidin cDNA was identified and characterized from bone marrow cells of buffalo (Bubalus bubalis) using RT-PCR based approach. The cDNA encodes a propeptide of 1.18 kDa with net positive charge at neutral pH. The precursor peptide possesses a signal peptide of 29 amino acids and a biologically active peptide of 34 residues. Comparison of sequences indicates only 66.1 and 64.1% identity at nucleotides and amino acids level respectively, with the already reported cathelicidin congener from the same species. However, high degree of similarity (92.8% nucleotides and 81.9% amino acids) was noticed with cathelicidin 7 sequence of Bos taurus suggesting interspecies conservation of cathelicidin peptides rather than intra-species within bovidae family. Phylogenetic trees analyses also support these data. Our findings, further justify the cloned cDNA as a unique cathelicidin member of B. bubalis, and may reasonably considered to be another example of structural diversity exhibited by cathelicidin-derived peptides as reported from other mammals.     

Rat pancreatic colipase mRNA: nucleotide sequence of a cDNA clone and nutritional regulation by a lipidic diet

A cDNA clone encoding rat pancreatic colipase was isolated using as a probe a synthetic deoxyoligonucleotide corresponding to a highly conserved amino acid sequence region in colipases from other species. The cloned messenger codes for a protein of 95 amino acids plus a signal peptide of 17 amino acids. The structure of the full-length cDNA was also determined and the corresponding amino acid sequence showed a high degree of homology with those of other known colipases. Quantification of the homologous mRNA in the pancreas of animals fed a high-lipid diet was consistent with a specific though moderate induction of colipase messenger by the nutritional manipulation.     

Strategy for selection of internal, low redundancy probes for identification of specific cDNA

Internal peptide fragments containing tryptophan residues are useful predictors of coding sequence for selection of restriction enzyme fragments or synthetic oligonucleotides to use in isolation of a cDNA or genomic clone. We describe a strategy to identify such fragments which uses an on-line photodiode array spectrophotometric analysis of tryptic fragment elution from an HPLC system to select peptides for amino acid sequence analysis. We applied this information to the identification and subsequent isolation of a cDNA corresponding to bovine placental lactogen from a bovine placental cDNA library which contains numerous closely related gene products.     

Isolation and nucleotide sequence of cDNA clone for bovine pancreatic anionic trypsinogen. Structural identity within the trypsin family.

A cDNA clone encoding an anionic form of bovine trypsinogen was isolated from a pancreatic cDNA library. The corresponding 855-nucleotide mRNA contains a short 5' noncoding region of 8 nucleotides and a long 3' noncoding region of 56 nucleotides in addition to a poly(A) tail of at least 50 nucleotides. The deduced amino acid sequence for the anionic pretrypsinogen (247 residues) includes the N-terminal 15-amino-acid signal peptide followed by an 8-amino-acid activation peptide. The zymogen (232 residues) contains an additional C-terminal serine, compared with the amino acid sequence of bovine cationic trypsinogen. The identity between the anionic and cationic forms of bovine trypsinogen (65%) is lower than that existing between the anionic protein and other mammalian anionic trypsinogens (73-85%), suggesting that trypsin gene duplication in mammals occurred prior to the evolutionary events responsible for the species divergence. Bovine pancreatic anionic trypsin possesses all the key amino acids characteristic of the serine protease family.     

Molecular cloning of cDNA encoding the C subunit of H(+)-ATPase from bovine chromaffin granules

A cDNA encoding subunit C of the V-ATPase from bovine chromaffin granules was cloned and sequenced. The gene encodes a hydrophilic protein of 382 amino acids with a calculated molecular weight of 43,989. Hydropathy plots revealed no apparent transmembrane segments and a rather high helix content was detected. A cDNA encoding most of the C subunit of the V-ATPase of human brain was also cloned and sequenced. The deduced amino acid sequence of this gene is almost identical to the bovine polypeptide with only one change of tyrosine 336 that was replaced by histidine in the human gene. Two polypeptide fragments derived from subunit E of V-ATPase from chromaffin granules were sequenced and found to be identical to the predicted amino acid sequence of this subunit from bovine kidney. These observations support the idea that the amino acid sequences of corresponding subunits from different V-ATPases are highly conserved. Unlike the A and B subunits of V-ATPases, that are homologous to the beta and alpha subunits of F-ATPases, subunits C and E showed no homology with analogous subunits of the F-ATPase family. It is proposed that the addition of the C and gamma subunits to the respective V- and F-ATPases during evolution defined them as two separate families of H(+)-ATPases.     

Characterization and Expression of the cDNA Coding for the Human Myelin/Oligodendrocyte Glycoprotein

Abstract: We report here the characterization of a full-length cDNA encoding the human myelin/oligodendrocyte glycoprotein (MOG). The sequence of the coding region of the human MOG cDNA is highly homologous to that of other previously cloned mouse, rat, and bovine MOG cDNAs, but the 3' untranslated region differs by an insertion of an Alu sequence between nucleotides 1,590 and 1,924. Accordingly, northern blot analyzes with cDNA probes corresponding to the coding region or the 3' untranslated Alu-containing sequence revealed a single band of 2 kb, rather than the 1.6 kb of bovine, rat, or mouse MOG cDNA(s). Immunocytochemical analysis of HeLa cells transfected with human MOG cDNA, which was performed using a specific antibody raised against whole MOG, clearly indicated that MOG is expressed at the cell surface as an intrinsic protein. These data are in accordance with the predicted amino acid sequence, which contains a signal peptide and two putative transmembrane domains. The knowledge of the human MOG sequence should facilitate further investigations on its potential as a target antigen in autoimmune demyelinating diseases like multiple sclerosis.     

Primary structure of the beta-subunit of bovine transducin deduced from the cDNA sequence

DNA complementary to the bovine retinal mRNA coding for the beta-subunit of transducin has been cloned by screening a cDNA library with oligodeoxyribonucleotide probes. Nucleotide sequence analysis of the cloned cDNA has revealed that this polypeptide consists of 340 amino acid residues (including the initiating methionine). Furthermore, cDNA hybridizable with a transducin beta-subunit cDNA probe has been cloned from a library derived from bovine brain poly(A)+ RNA. Comparison of the cloned cDNAs, in conjunction with blot hybridization analysis and S1 nuclease mapping of poly(A)+ RNA from bovine retina, brain and liver, suggests that the mRNAs coding for the beta-subunits of transducin and other guanine nucleotide binding proteins have the same protein-coding sequence but partly different 5'-noncoding sequences.     

Cloning and sequencing of a copper-containing,topa quinone-containing monoamine oxidase from human placenta

A 4040-bp cDNA was cloned from a human placenta library by screening with a polymerase chain reaction-amplified fragment. The fragment was generated from the library using primers corresponding to conserved sequences encompassing the topa quinone (TPQ) cofactor sites of the copper-containing proteins, bovine serum amine oxidase (BSAO) and human kidney diamine oxidase (DAO). The cloned cDNA contains a coding sequence from positions 161 to 2449. Between bases 2901 and 2974, in a very long 1591-bp 3′-untranslated region, there is a G/A-rich region in the minus strand, which contains a (AGG)₅ tandem repeat. The human placenta cDNA sequence and its translated amino acid sequence are 84% and 81% identical to the corresponding BSAO sequences, while the identities for the placenta sequences and those for human kidney DAO are 60% and 41%, respectively. The TPQ consensus nucleotide and protein sequences are identical for the placenta enzyme and BSAO, but the corresponding sequences for human kidney DAO are nonidentical. Three His residues that have been identified as Cu(II) ligands in other amine oxidases are conserved in the human placenta amine oxidase protein sequence. It was concluded that the placenta cDNA open-reading frame codes for a copper-containing, TPQ-containing monoamine oxidase. A putative 19-amino acid signal peptide was identified for human placenta amine oxidase. The resulting mature protein would be composed of 744 amino acids, and would have a M_r of 82 525. Comparison of the human placenta amine oxidase with DNA sequences found in GenBank suggests that the gene for this enzyme is located in the q21 region of human chromosome 17, near the BRCA1 gene.     

Isolation and Identification of Novel Terpene Lactones from Quince Fruit (Cydonia oblonga Mill., Marmelo)

A glucoamylase gene has been cloned from a Rhizopus genomic DNA library using synthetic oligonucleotides corresponding to the amino acid sequence of the glucoamylase. Since this glucoamylase gene was not expressed in yeast cells, we have cloned a glucoamylase gene from a cDNA library prepared from Rhizopus mRNA. Sequence analysis of both glucoamylase genes revealed that the genomic gene contained 4 intervening sequences and the cDNA gene lacked 145 nucleotides corresponding to the N-terminal region. The glucoamylase consists of 604 amino acids including a putative signal peptide and its molecular weight was calculated to be 65,000. The glucoamylase gene to be expressed in yeast cells was constructed by recombination of both genes. The yeast cells containing this constructed glucoamylase gene secreted the glucoamylase into the culture fluid and grew at almost the normal rate on a medium containing starch as the sole carbon source.     

Sequencing of the chicken non-erythroid spectrin cDNA reveals an internal repetitive structure homologous to the human erythrocyte spectrin.

Immunological screening of a chicken gizzard cDNA expression library was used to isolate two clones encoding a part of the non-erythroid spectrin-like protein. Clones were identified by immunoblotting of the polypeptides synthesized in Escherichia coli cells transformed with cDNA cloned in the pUC8 plasmid vector using polyclonal rabbit antibodies raised against bovine non-erythroid spectrin. The sequence of an approximately 1.5-kb cDNA insert of one clone was determined. Analysis of the predicted amino acid sequence reveals that, despite differences in immunological cross-reactivity and peptide maps, the chicken non-erythroid and the human erythrocyte spectrins are highly homologous proteins. Like the human erythrocyte spectrin, the chicken smooth muscle spectrin appears also to be constructed from repeated, homologous structures of 106 amino acid residues. This is probably a universal structure motif of spectrins.