Cloning of complementary DNA encoding the pB1 component of the 54-kilodalton glycoprotein of boar seminal plasma

Complementary DNA (cDNA) encoding a protein component pB1 (also pAIF-1 and DQH) of the 54-kilodalton glycoprotein of boar seminal plasma was cloned and its nucleotide sequence was determined (Gene Bank accession no. AF047026). The pB1 precursor protein is a 130-amino-acid-long polypeptide containing a 25-amino-acid-long signal peptide. The amino acid sequence of the pB1 is homologous to that of SFP1_BOVIN (named also BSP-A1/A2, PDC-109/ major protein and SVSp109), SFP3_BOVIN (BSP-A3), SFP4 BOVIN (BSP-30 KD), and SP1_HORSE (HSP-1) seminal plasma proteins. The homology extends also for the signal peptide of SFP1_BOVIN protein. All these seminal plasma proteins contain two fibronectin type-II domains that differ from those found in other proteins such as colagenases, fibronectins, and mannose receptors. The first domain located in the N-terminal region of pB1 is four amino acids shorter than those present in other proteins. High homology is also observed between 3' noncoding regions of the nucleotide sequences of cDNAs of pB1_PIG and SFP1_BOVIN (Gene Bank accession nos. AF047026 and P02784, respectively).     

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.     

A method for the evaluation of the efficiency of signal sequences for secretion and correct N-terminal processing of human parathyroid hormone produced in Escherichia coli.

Expression plasmids have been constructed for evaluation of different signal sequences for secretion and correct amino terminal processing of foreign proteins expressed in Escherichia coli. cDNA representing the N-terminal region (1-37) of human parathyroid hormone was inserted between DNA coding for two different forms of the signal sequence and two IgG binding domains (ZZ) derived from Staphylococcal protein A. The expression products were secreted to the periplasm and even to the growth medium and were easily purified by affinity chromatography using the ZZ part as a specific handle. Further analyses showed that the expression products were correctly processed to the mature protein hPTH(1-37)ZZ in a construct where the wild type signal sequence of Staphylococcus protein A was used. When a mutated signal sequence which lacks the normal cleavage site was employed, the fusion protein was not cleaved. Since signal sequences seem to be processed in the correct way in this system, we conclude that the general design of this type of expression vector is well suited for studying the N-terminal processing and secretion of heterologous proteins in E. coli.     

The nucleotide sequence of the yeast MEL1 gene.

The complete nucleotide sequence of the MEL1 gene of the yeast, Saccharomyces cerevisiae, encoding alpha-galactosidase was determined. The nucleotide sequence contains an open reading frame of 1413 bp encoding a protein of 471 amino acids. Comparison with the known N-terminal amino acid sequence of the mature secreted protein indicated that alpha-galactosidase is synthesized as a precursor with an N-terminal signal sequence of 18 amino acids. The general features of this signal peptide resemble those of other yeast signal peptides. Molecular weight of the mature alpha-galactosidase polypeptide deduced from the nucleotide sequence is 50.049 kd. The 5' regulatory region has sequences in common with other yeast genes regulated by the GAL4-protein.     

Amino acid sequence of the signal peptide of mitochondrial nicotinamide nucleotide transhydrogenase as determined from the sequence of its messenger RNA

The amino acid sequence of the bovine mitochondrial nicotinamide nucleotide transhydrogenase was recently deduced from isolated cDNAs and reported [Yamaguchi, M., Hatefi, Y., Trach, K., and Hoch, J.A. (1988) J. Biol. Chem. 263, 2761-2767]. The cDNAs lacked the N-terminal coding region, however, and the 8 N-terminal residues were determined by protein sequencing. In the present study, the nucleotide sequence of the 5' upstream region was determined by dideoxynucleotide sequencing of the transhydrogenase messenger RNA, and amino acid sequences of the N-terminal region and the signal peptide of the enzyme were deduced from the nucleotide sequence. The N-terminal sequence of the enzyme as deduced from the mRNA sequence is the same as that determined by protein sequencing, with one difference. Protein sequencing showed Ser as the N-terminal residue. The mRNA sequence indicated that Ser is the second N-terminal residue, and the first is Cys. That preparations of the enzyme are mixtures of two polypeptides, one polypeptide being one residue shorter at the N terminus than the other, has been pointed out in the above reference. The signal peptide consists of 43 residues, is rich in basic (4 Lys, 2 Arg) and hydroxylated (4 Thr, 3 Ser) amino acids, and lacks acidic residues.     

Novel multigene families encoding highly repetitive peptide sequences. Sequence analyses of rat and mouse proline-rich protein cDNAs

Multigene families encode the proline-rich proteins that are so prominent in human saliva and are dramatically induced in mouse and rat salivary glands by isoproterenol treatment and by feeding tannins. A cDNA encoding an acidic proline-rich protein of rat has been sequenced (Ziemer, M. A., Swain, W. F., Rutter, W. J., Clements, S., Ann, D. K., and Carlson D. M. (1984) J. Biol. Chem. 259, 10475-10480). This study presents the nucleotide sequences of five additional proline-rich protein cDNAs complementary to both mouse and rat parotid and submandibular gland mRNAs. Amino acid compositions deduced from the nucleotide sequences are typical for proline-rich proteins: 25-45% proline, 18-22% glycine, and 18-22% glutamine and generally an absence of sulfur-containing amino acids except for the initiator methionine. These proline-rich proteins display unusual repeating peptide sequences of 14-19 amino acids. The derived amino acid sequence of the cDNA insert of plasmid pMP1 from mouse has a 19-amino acid sequence which is repeated four times. The inserts of plasmids pUMP40 and pUMP4 also from mouse encode for 12 and 11 repeats of a 14-amino acid peptide, respectively. These repetitive sequences, and others from rat and mouse cDNAs and from human genomic clones, all show very high homologies and likely evolved from duplication of internal portions of an ancestral gene. Gene conversion could account for the high degree of conservation of nucleotide sequences of the repeat regions. Protein derived from the nucleotide sequences are all characterized by four general regions: a putative signal peptide, a transition region, the repetitive region, and a carboxyl-terminal region. The 5'-flanking sequences and sequences encoding the putative signal peptides are highly conserved (greater than 94%) in all six cDNAs. This sequence conservation may be important in the regulation of the biosynthesis of these unusual proteins.     

Cloning and sequence analysis of cDNA for rat liver uricase

We have isolated cDNA clones for rat liver uricase using an oligonucleotide corresponding to the N-terminal sequence of 8 amino acids. The nucleotide sequences of the cDNAs have been determined, and the amino acid sequence of the protein deduced. A 867-base open reading frame coding for 289 amino acids, corresponding to a molecular mass of 33,274 daltons, was confirmed by matching eight sequences of a total of 53 amino acids from peptide sequence analyses of the fragments generated by lysyl endopeptidase digestion of purified rat liver uricase. The deduced amino acid sequence of rat liver uricase shares 40% homology with that of soybean nodulin-specific uricase and has an N-terminal extension of 7 amino acids. In contrast, soybean uricase has a C-terminal extension of 12 amino acids, which is presumably the result of local gene duplication. Completely different N- and C-terminal structures of the two uricases suggest that the signals for targeting the proteins to the peroxisome are not located on the terminal continuous stretches of amino acids.     

The primary sequence of Ricinus communis agglutinin. Comparison with ricin

A mixture of synthetic oligonucleotides representing all possible sequences of a peptide present in the ricin B chain has been used to screen a cDNA library constructed using ripening castor bean seed poly(A+) RNA. The eight largest recombinant plasmids selected, by hybridization, a single mRNA species whose translational product was identified as preprolectin by immunoprecipitation. Restriction enzyme analysis of these clones demonstrated that two classes were present representing sequences complementary to two distinct but closely related preprolectin mRNA species. The nucleotide sequence of the cloned cDNA from one of these classes encodes preproricin and has been presented elsewhere (Lamb, F. I., Roberts, L. M., and Lord, J. M., (1985) Eur. J. Biochem. 148, 265-270). The nucleotide sequence of the second class is presented here and shown to represent prepro-Ricinus communis agglutinin. The entire coding sequence was deduced from two overlapping cDNA clones having inserts of 1668 and 1151 base pairs. The coding region defines a preproprotein with a 24-amino acid N-terminal signal sequence preceding the A chain (266 amino acids) which is joined to the B chain (262 amino acids) by a 12-amino acid linking peptide. The protein was confirmed as R. communis agglutinin since the deduced B chain N-terminal sequence corresponds exactly with that determined for purified R. communis agglutinin B chain over a region where several residue differences occur in the ricin B chain. The nucleotide and deduced amino acid sequences of the R. communis agglutinin precursor are compared with those of the ricin precursor.     

Characterization of two isoforms of a human DnaJ homologue,HSJ2

Two cDNA forms were characterized for a human dnaJ homologue, HSJ2. Nucleotide sequencing showed that the gene product HSJ2 was longer than previously reported, extending its homology to other human DnaJ paralogues, and that the two cDNAs encoded two proteins as a result of alternative splicing. The products were 326 amino acids (designated as HSJ2a) and 241 amino acids (HSJ2b) in length, sharing the N-terminal 231 amino acids including the DnaJ homology region. When fused to green fluorescent protein and expressed in HeLa cells, HSJ2a was found to be localized to the nucleus, indicating that HSJ2a is a nuclear co-chaperone. HSJ2b, however, was observed throughout the cell, consistent with the elimination of a putative nuclear localization signal sequence as a result of the alternative splicing.     

Isolation of cDNA clones coding for spinach nitrite reductase: Complete sequence and nitrate induction

Summary The main nitrogen source for most higher plants is soil nitrate. Prior to its incorporation into amino acids, plants reduce nitrate to ammonia in two enzymatic steps. Nitrate is reduced by nitrate reductase to nitrite, which is further reduced to ammonia by nitrite reductase. In this paper, the complete primary sequence of the precursor protein for spinach nitrite reductase has been deduced from cloned cDNAs. The cDNA clones were isolated from a nitrate-induced cDNA library in two ways: through the use of oligonucleotide probes based on partial amino acid sequences of nitrite reductase and through the use of antibodies raised against purified nitrite reductase. The precursor protein for nitrite reductase is 594 amino acids long and has a 32 amino acid extension at the N-terminal end of the mature protein. These 32 amino acids most likely serve as a transit peptide involved in directing this nuclearencoded protein into the chloroplast. The cDNA hybridizes to a 2.3 kb RNA whose steady-state level is markedly increased upon induction with nitrate.     

Comparative sequence analysis of the mRNAs coding for mouse and rat whey protein

Whey acidic protein (WAP) is a major milk protein found in mouse and rat. Cloned WAP cDNAs from both species have been sequenced and the respective protein sequences have been deduced. Mouse and rat WAP (134 and 137 amino acids respectively) are acidic, cysteine rich proteins which contain a N-terminal signal peptide of 19 amino acids. Most of the cysteines are located in two clusters containing six cysteine residues each, arranged in an identical pattern. Comparison of the mouse and rat WAPs show that the signal peptide and the first cysteine domain are conserved to a greater extent than the rest of the protein. This result is reflected in the nucleotide sequence homology, where the regions coding for the signal peptide and cysteine domain I are the only regions where the rate of replacement substitution is lower than the rate of silent substitution. The 3' non-coding regions show a 91% conservation which is half the substitution rate for the coding region. This low rate of sequence divergence in the 3' non-translated region of the mRNA may indicate a functional importance for this region.     

Autoantigen 1 of the guinea pig sperm acrosome is the homologue of mouse Tpx-1 and human TPX1 and is a member of the cysteine-rich secretory protein (CRISP) family

We have cloned and sequenced cDNAs encoding autoantigen 1 (AA1), a testis-specific protein and the major autoantigen of the guinea pig sperm acrosome. The cDNA predicts a precursor protein of 244 amino acids including a 21 amino acid hydrophobic, secretory signal sequence. The mature polypeptide is predicted to have a molecular mass of 24,891 Daltons which agrees with the experimentally determined molecular weight of 25,000. Consistent with previous studies demonstrating that AA1 is not a glycoprotein, the predicted amino acid sequence contained no canonical sites for N-linked glycosylation. Comparison with other sequences showed that AA1 is the guinea pig homologue of the testis-specific protein Tpx-1 in mice and TPX1 in humans. AA1 also showed significant amino acid sequence homology with other cysteine-rich secretory proteins (CRISP's): rat and mouse acidic epididymal glycoproteins (AEG; also known as proteins D/E in rats) and helothermine, a toxin from the Mexican beaded lizard. In addition, AA1 had a lesser degree of homology with antigen 5 (vespid wasp venom), PR-1 (a plant pathogenesis related protein), and GliPR (a protein identified in human gliomas). Northern analysis of RNA from purified guinea pig spermatogenic cells showed that a 1.5 kb message was first detected in pachytene spermatocytes, was strongest in round spermatids, and was detected at a low level in condensing spermatids. Immunoblot analysis and metabolic labeling data of AA1 in spermatogenic cells showed that the protein was synthesized as early as the pachytene spermatocyte stage of spermatogenesis. Thus, the patterns of AA1 mRNA and protein expression during spermatogenesis are similar to the expression of other acrosomal mRNAs and proteins that are first detected meiotically. © 1996 Wiley-Liss, Inc.     

Mapping of export signals of Pseudomonas aeruginosa pilin with alkaline phosphatase fusions.

Pili of Pseudomonas aeruginosa are assembled from monomers of the structural subunit, pilin, after secretion of this protein across the bacterial membrane. These subunits are initally synthesized as precursors (prepilin) with a six-amino-acid leader peptide that is cleaved off during or after membrane traversal, followed by methylation of the amino-terminal phenylalanine residue. This report demonstrates that additional sequences from the N terminus of the mature protein are necessary for membrane translocation. Gene fusions were made between amino-terminal coding sequences of the cloned pilin gene (pilA) and the structural gene for Escherichia coli alkaline phosphatase (phoA) devoid of a signal sequence. Fusions between at least 45 amino acid residues of the mature pilin and alkaline phosphatase resulted in translocation of the fusion proteins across the cytoplasmic membranes of both P. aeruginosa and E. coli strains carrying recombinant plasmids, as measured by alkaline phosphatase activity and Western blotting. Fusion proteins constructed with the first 10 amino acids of prepilin (including the 6-amino-acid leader peptide) were not secreted, although they were detected in the cytoplasm. Therefore, unlike that of the majority of secreted proteins that are synthesized with transient signal sequences, the membrane traversal of pilin across the bacterial membrane requires the transient six-amino-acid leader peptide as well as sequences contained in the N-terminal region of the mature pilin protein.     

Proteolytic processing of the vitellogenin precursor in the boll weevil,Anthonomus grandis

The soluble proteins of the eggs of the coleopteran insect Anthonomus grandis Boheman, the cotton boll weevil, consist almost entirely of two vitellin types with M_rs of 160,000 and 47,000. We sequenced their N-terminal ends and one internal cyanogen bromide fragment of the large vitellin and compared these sequences with the deduced amino acid sequence from the vitellogenin gene. The results suggest that both the boll weevil vitellin proteins are products of the proteolytic cleavage of a single precursor protein. The smaller 47,000 M vitellin protein is derived from the N-terminal portion of the precursor adjacent to an 18 amino acid signal peptide. The cleavage site between the large and small vitellins at amino acid 362 is adjacent to a pentapeptide sequence containing two pairs of arginine residues. Comparison of the boll weevil sequences with limited known sequences from the single 180,000 M_r honey bee protein show that the honey bee vitellin N-terminal exhibits sequence homology to the N-terminal of the 47,000 M_r boll weevil vitellin. Treatment of the vitellins with an N-glycosidase results in a decrease in molecular weight of both proteins, from 47,000 to 39,000 and from 160,000 to 145,000, indicating that about 10–15% of the molecular weight of each vitellin consists of N-linked carbohydrate. The molecular weight of the deglycosylated large vitellin is smaller than that predicted from the gene sequence, indicating possible further proteolytic processing at the C-terminal of that protein. © 1993 Wiley-Liss, Inc.

1 This article is a US Government work and, as such, is in the public domain in the United States of America.

     

The adenine nucleotide translocator of higher plants is synthesized as a large precursor that is processed upon import into mitochondria.

Two maize genes and cDNAs encoding the mitochondrial adenine nucleotide translocator (ANT), a nuclear-encoded inner mitochondrial membrane carrier protein, have previously been isolated in this laboratory. Sequence analysis revealed the existence of much longer open reading frames than the corresponding fungal and mammalian ANT genes. Potato ANT cDNAs have subsequently been isolated and sequenced and alignment of the deduced plant amino acid sequences with the equivalent fungal and mammalian polypeptides indicated that the plant proteins contain N-terminal extensions. When the plant cDNA clones are expressed in vitro they direct the synthesis of precursor proteins that are specifically processed at the N-terminus upon import into isolated mitochondria. N-terminal amino acid sequence data obtained from the native proteins purified from both maize and potato mitochondria has allowed identification of the putative processing sites. Further import analysis has shown that two distinct regions of the maize precursor protein contain targeting information, the 97 amino acids at the N-terminus and the 267 C-terminal amino acids. This is the first report that provides experimental evidence that the adenine nucleotide translocator of higher plants is synthesized as a large precursor protein that is specifically cleaved upon import into mitochondria. Import of ANT into higher plant mitochondria therefore appears to be different to the corresponding process in fungal and mammalian systems where targeting of ANT to mitochondria is mediated by internal signals and there is no N-terminal processing.     

Efficient function of signal peptidase 1 of Escherichia coli is partly determined by residues in the mature N-terminus of exported proteins

Exported proteins require an N-terminal signal peptide to direct them from the cytoplasm to the periplasm. Once the protein has been translocated across the cytoplasmic membrane, the signal peptide is cleaved by a signal peptidase, allowing the remainder of the protein to fold into its mature state in the periplasm. Signal peptidase I (LepB) cleaves non-lipoproteins and recognises the sequence Ala-X-Ala. Amino acids present at the N-terminus of mature, exported proteins have been shown to affect the efficiency at which the protein is exported. Here we investigated a bias against aromatic amino acids at the second position in the mature protein (P2′). Maltose binding protein (MBP) was mutated to introduce aromatic amino acids (tryptophan, tyrosine and phenylalanine) at P2′. All mutants with aromatic amino acids at P2′ were exported less efficiently as indicated by a slight increase in precursor protein in vivo. Binding of LepB to peptides that encompass the MBP cleavage site were analysed using surface plasmon resonance. These studies showed peptides with an aromatic amino acid at P2′ had a slower off rate, due to a significantly higher binding affinity for LepB. These data are consistent with the accumulation of small amounts of preMBP in purified protein samples. Hence, the reason for the lack of aromatic amino acids at P2′ in E. coli is likely due to interference with efficient LepB activity. These data and previous bioinformatics strongly suggest that aromatic amino acids are not preferred at P2′ and this should be incorporated into signal peptide prediction algorithms.     

Molecular characterization, nucleotide sequence, and expression of the fliO, fliP, fliQ, and fliR genes of Escherichia coli.

The fliL operon of Escherichia coli contains seven genes that are involved in the biosynthesis and functioning of the flagellar organelle. DNA sequences for the first three genes of this operon have been reported previously. A 2.2-kb PstI restriction fragment was shown to complement known mutant alleles of the fliO, fliP, fliQ, and fliR genes, the four remaining genes of the fliL operon. Four open reading frames were identified by DNA sequence analysis and correlated to their corresponding genes by complementation analysis. These genes were found to encode very hydrophobic polypeptides with molecular masses of 11.1, 26.9, 9.6, and 28.5 kDa for FliO, FliP, FliQ, and FliR, respectively. Analysis of recombinant plasmids in a T7 promoter-polymerase expression system enabled us to identify three of the four gene products. On the basis of DNA sequence analysis and in vivo protein expression, it appears that the fliP gene product is synthesized as a precursor protein with an N-terminal signal peptide of 21 amino acids. The FliP protein was homologous to proteins encoded by a DNA sequence upstream of the flaA gene of Rhizobium meliloti, to a gene involved in pathogenicity in Xanthomonas campestris pv. glycines, and to the spa24 gene of the Shigella flexneri. The latter two genes encode proteins that appear to be involved in protein translocation, suggesting that the FliP protein may have a similar function.     

cDNA cloning and expression of ghrelin in giant panda (<Emphasis Type="Italic">Ailuropoda melanoleuca</Emphasis>)

Ghrelin is an endogenous ligand for the growth hormone secretagogue receptor. It plays an important role in stimulating growth hormone secretion, food intake, body weight gain and gastric motility. cDNA sequences coding for ghrelin precursor protein (prepro-ghrelin) were isolated from the stomach of a giant panda. Two different mRNA sequences of ghrelin were obtained. The long open reading frame of ghrelin (354 bp) encodes a precursor protein of 117 amino acids with a 23 amino acid signal peptide. The short one (351 bp) encodes a precursor protein of 116 amino acids with the same 23 amino acid signal peptide. The presumed giant panda mature ghrelin proteins also had two forms. Comparative analysis showed that the first and the fourth amino acids (Gly and Phe) were completely conserved and the third amino acid (Ser) was also highly conserved in the mature ghrelin. RT-PCR analysis of giant panda ghrelin mRNA in various tissues revealed high level of expression in stomach, relative lower levels of expression in small intestine, liver and kidney, and no expression in thymus, spleen and heart.     

Molecular cloning of a human co-beta-glucosidase cDNA: evidence that four sphingolipid hydrolase activator proteins are encoded by single genes in humans and rats

Authentic cDNAs encoding the activator protein for acid beta-glucosidase (EC3.2.1.45), co-beta-glucosidase, were cloned from the pCD and lambda gt11 human cDNA libraries. Initial screening with oligonucleotide mixtures encoding amino acid sequences of co-beta-glucosidase identified partial cDNAs which were used to obtain a potentially full-length cDNA from the lambda gt11 library. This clone (2767 bp), EGTISI, contained 5' (38 bp) and 3' (1157 bp) noncoding sequences, a translation initiation site, and an open reading frame encoding 524 amino acids which included a typical hydrophobic signal sequence (16 amino acids). Computer analyses identified three regions of high similarity to co-beta-glucosidase encoded by tandem sequences in EGTISI. Searches revealed that two of these regions encoded peptides of known function; SAP1 (sphingolipid activator protein 1) and protein C (a new sphingolipid activator protein) were encoded by EGTISI sequences 5' and 3', respectively, to those for co-beta-glucosidase. The third region of similarity, encoding a theoretical peptide (undefined function), was located most 5' in the cDNA. EGTISI and its encoded polypeptide had high similarity (77% nucleotide identity and about 80% amino acid similarity) to a rat Sertoli cell cDNA and its encoded sulfated glycoprotein-1. These results indicate that a single highly conserved gene encodes the precursor for four potential sphingolipid activator proteins in rat and man.