MAJOR SPERM PROTEIN, BETA ISOFORM, ENGINEERED C59S/T90C MUTANT, PUTATIVE SUBFILAMENT STRUCTURE, PH 8.5
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Calcium binding S100A1 protein consists of two S100 alpha subunits. On the basis of sequence homology to other S100 proteins it is believed that the binding loops are formed by amino-acid residues 19-32 and 62-73 of S100 alpha polypeptide chain. In the oxidized form of the protein the subunits are linked covalently with each other by a disulphide bond between their Cys85 residues. A synthetic gene coding for bovine S100 alpha subunit was constructed and cloned into a derivative of pAED4 plasmid. The gene was expressed in Escherichia coli utilizing the T7 expression system. The expression products were purified and identified using mass spectrometry and by sequencing of their N- and C-termini. Three different forms (a, b, and c) of S100 alpha were produced: with the native sequence, with the initiator methionine at the N-terminus, and with an additional alanine at the C-terminus as well as with the initiator methionine. The material was partly oxidized. Interestingly, only the homodimers of a, b, and c species were formed. The total yield of the protein was about 50 mg/l of culture. Genes coding for Glu32-->Gln and Glu73-->Gln mutants of S100 alpha were obtained by site-directed mutagenesis and expressed in the same system. In both cases similar mixtures of oxidized and reduced a, b, and c species have been obtained. The total yield of E73Q mutant is similar to that of the native protein and that of E32Q lower by about a half. As expected, the mutants of S100 alpha subunits bind only one calcium ion.
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Site-directed mutagenesis
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Site-directed mutagenesis
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Phospholipase A
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Mouse anticoagulant protein C (461 residues) shares 69% sequence identity with its human ortholog. Interspecies experiments suggest that there is an incompatibility between mouse and human protein C, such that human protein C does not function efficiently in mouse plasma, nor does mouse protein C function efficiently in human plasma. Previously, we described a series of human activated protein C (APC) Gla domain mutants (e.g. QGNSEDY‐APC), with enhanced membrane affinity that also served as superior anticoagulants. To characterize these Gla mutants further in mouse models of diseases, the analogous mutations were now made in mouse protein C. In total, seven mutants (mutated at one or more of positions P 10 S 12 D 23 Q 32 N 33 ) and wild‐type protein C were expressed and purified to homogeneity. In a surface plasmon resonance‐based membrane‐binding assay, several high affinity protein C mutants were identified. In Ca 2+ titration experiments, the high affinity variants had a significantly reduced (four‐fold) Ca 2+ requirement for half‐maximum binding. In a tissue factor‐initiated thrombin generation assay using mouse plasma, all mouse APC variants, including wild‐type, could completely inhibit thrombin generation; however, one of the variants denoted mutant III (P10Q/S12N/D23S/Q32E/N33D) was found to be a 30‐ to 50‐fold better anticoagulant compared to the wild‐type protein. This mouse APC variant will be attractive to use in mouse models aiming to elucidate the in vivo effects of APC variants with enhanced anticoagulant activity.
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The rat brain Na+-Ca2+ exchanger (RBE) gene, as well as other isoforms of this protein family, can be organized into 12 transmembrane α helices, the first of which was proposed by Durkin et al.(14Durkin J.T. Ahrens D.C. Pan Y.-C.E. Reeves J.P. ArchBiochem. Biophys. 1991; 290: 369-375Crossref PubMed Scopus (64) Google Scholar) to constitute a cleavable signal peptide. We have prepared three amino-terminal mutants, in which 21, 26, and 31 amino acids beyond the initiating methionine were deleted. The deletions include the hydrophobic core of the putative signal peptide(N21), the entire putative signal peptide and parts of the putative signal peptidase cleavage site(N26), and the entire putative signal peptide and putative signal peptidase cleavage site(N31). All three mutant clones were transiently expressed in HeLa cells. The average Na+ gradient-dependent Ca2+ transport activity of the mutant exchangers was 108%(N21), 37.2% (N26), and 60.06%(N31) of the wild-type clone. Mutation of the putative cleavage site by an exchange of Ala-32 → Asp, resulted in a decrease in Na+-Ca2+ exchange activity to 7.7%, relative to the wild-type exchanger. Functional reconstitution of the proteins that were expressed in the transfected cells, resulted in transport activities of: 60.1%(N21), 26.75%(N26), 85.36%(N31), and 31% (Ala-32 → Asp) relative to the wild-type exchanger. Western blot analysis of the protein profile of RBE-1, N21, N26, N31 and Ala-32 → Asp-transfected HeLa cells was carried out by using an antipeptide antibody directed against a pentadecapeptide segment derived from the large putative cytoplasmic loop of the cloned rat exchanger gene. In the total cell extract and in the plasma membrane-enriched fraction, in addition to a major protein band of about 125 kDa, which corresponds to the molecular mass of the mature fully processed Na+-Ca2+ exchanger, an additional protein of about 135 kDa is revealed in the profile of N21- and N26-transfected cells. This band is not detected in the protein profile of RBE-1, N31, or Ala-32 → Asp. The amino-terminal truncated mutants of the cloned Na+-Ca2+ exchanger could be expressed and processed also in a reticulocyte lysate supplemented with dog microsomes. Our results suggest that the putative signal peptide of the cloned Na+-Ca2+ exchanger gene does not play a mandatory role in functional expression of the protein in HeLa cells. The rat brain Na+-Ca2+ exchanger (RBE) gene, as well as other isoforms of this protein family, can be organized into 12 transmembrane α helices, the first of which was proposed by Durkin et al.(14Durkin J.T. Ahrens D.C. Pan Y.-C.E. Reeves J.P. ArchBiochem. Biophys. 1991; 290: 369-375Crossref PubMed Scopus (64) Google Scholar) to constitute a cleavable signal peptide. We have prepared three amino-terminal mutants, in which 21, 26, and 31 amino acids beyond the initiating methionine were deleted. The deletions include the hydrophobic core of the putative signal peptide(N21), the entire putative signal peptide and parts of the putative signal peptidase cleavage site(N26), and the entire putative signal peptide and putative signal peptidase cleavage site(N31). All three mutant clones were transiently expressed in HeLa cells. The average Na+ gradient-dependent Ca2+ transport activity of the mutant exchangers was 108%(N21), 37.2% (N26), and 60.06%(N31) of the wild-type clone. Mutation of the putative cleavage site by an exchange of Ala-32 → Asp, resulted in a decrease in Na+-Ca2+ exchange activity to 7.7%, relative to the wild-type exchanger. Functional reconstitution of the proteins that were expressed in the transfected cells, resulted in transport activities of: 60.1%(N21), 26.75%(N26), 85.36%(N31), and 31% (Ala-32 → Asp) relative to the wild-type exchanger. Western blot analysis of the protein profile of RBE-1, N21, N26, N31 and Ala-32 → Asp-transfected HeLa cells was carried out by using an antipeptide antibody directed against a pentadecapeptide segment derived from the large putative cytoplasmic loop of the cloned rat exchanger gene. In the total cell extract and in the plasma membrane-enriched fraction, in addition to a major protein band of about 125 kDa, which corresponds to the molecular mass of the mature fully processed Na+-Ca2+ exchanger, an additional protein of about 135 kDa is revealed in the profile of N21- and N26-transfected cells. This band is not detected in the protein profile of RBE-1, N31, or Ala-32 → Asp. The amino-terminal truncated mutants of the cloned Na+-Ca2+ exchanger could be expressed and processed also in a reticulocyte lysate supplemented with dog microsomes. Our results suggest that the putative signal peptide of the cloned Na+-Ca2+ exchanger gene does not play a mandatory role in functional expression of the protein in HeLa cells. The presence of an amino-terminal signal peptide earmarks the protein for insertion into the endoplasmic reticulum from where it is targeted to the plasma membrane via the Golgi apparatus. Many polytopic membrane proteins, however, do not contain cleavable amino termini that can be identified as a signal peptide, yet they are correctly targeted to the plasma membrane(1von Heijne G. J. Membr. Biol. 1990; 115: 195-201Crossref PubMed Scopus (866) Google Scholar, 2Guan X.-M. Kobilka T.S. Kobilka B.K. J. Biol. Chem. 1992; 267: 21995-21998Abstract Full Text PDF PubMed Google Scholar, 3Tam L.Y. Loo T.W. Clarke D.M. Reithmeier R.A.F. J. Biol. Chem. 1994; 269: 32542-32550Abstract Full Text PDF PubMed Google Scholar, 4von Heijne G. Subcell. Biochem. 1994; 22: 1-19Crossref PubMed Scopus (39) Google Scholar). It is thought that transmembrane segments in these proteins have targeting information. The Na+-Ca2+ exchanger is a major Ca2+-regulating protein present in all excitable and many nonexcitable cells(5Allen T.J.A. Noble D. Reuter H. Sodium-Calcium Exchange. Oxford University Press, Oxford1989Google Scholar, 6Blaustein M.P. DiPolo R. Reeves J.P. Ann. N. Y. Acad. Sci. 1991; 639PubMed Google Scholar). The protein has been cloned(7Nicoll D.A. Longoni S. Philipson K.D. Science. 1990; 250: 562-565Crossref PubMed Scopus (628) Google Scholar, 8Low W. Kasir J. Rahamimoff H. FEBS Lett. 1992; 316: 63-67Crossref Scopus (69) Google Scholar, 9Furman I. Cook O. Kasir J. Rahamimoff H. FEBS Lett. 1993; 319: 105-109Crossref PubMed Scopus (78) Google Scholar, 10Reilly R.F. Shugrue C.A. Am. J. Physiol. 1992; 262: F1105-F1109PubMed Google Scholar, 11Lee S.-L. Yu A.S.L. Lytton J. J. Biol. Chem. 1994; 269: 14849-14852Abstract Full Text PDF PubMed Google Scholar, 12Kofuji P. Lederer W.J. Schulze D.H. J. Biol. Chem. 1994; 269: 5145-5149Abstract Full Text PDF PubMed Google Scholar) and functionally expressed, and the presence of multiple isoforms, which are the product of two different genes(12Kofuji P. Lederer W.J. Schulze D.H. J. Biol. Chem. 1994; 269: 5145-5149Abstract Full Text PDF PubMed Google Scholar, 13Li Z. Matsuoka S. Hryshko L.V. Nicoll D.A. Bersohn M.M. Burke E.P. Lifton R.P. Philipson K.D. J. Biol. Chem. 1994; 269: 17434-17439Abstract Full Text PDF PubMed Google Scholar), was established. Hydropathy analysis using a window of 20 amino acids suggested (7Nicoll D.A. Longoni S. Philipson K.D. Science. 1990; 250: 562-565Crossref PubMed Scopus (628) Google Scholar, 8Low W. Kasir J. Rahamimoff H. FEBS Lett. 1992; 316: 63-67Crossref Scopus (69) Google Scholar, 13Li Z. Matsuoka S. Hryshko L.V. Nicoll D.A. Bersohn M.M. Burke E.P. Lifton R.P. Philipson K.D. J. Biol. Chem. 1994; 269: 17434-17439Abstract Full Text PDF PubMed Google Scholar) that the cloned Na+-Ca2+ exchanger proteins can be organized into 12-transmembrane α helices. Partial sequencing, however, of the amino terminus of the purified bovine cardiac Na+-Ca2+ exchanger indicated that the first amino acid of this protein corresponds to amino acid number 33 of the cloned gene(14Durkin J.T. Ahrens D.C. Pan Y.-C.E. Reeves J.P. ArchBiochem. Biophys. 1991; 290: 369-375Crossref PubMed Scopus (64) Google Scholar). Hence it was suggested that the first putative transmembrane α helix (amino acids 1-24) and the next eight amino acids (25-32) that precede the amino terminus (amino acid 33), constitute a signal peptide that is presumably cleaved and hence not detectable in the "mature" protein. In this work, we have examined the importance of the putative signal peptide of the cloned Na+-Ca2+ exchanger gene in functional expression of the transporter. Our studies indicate that neither the hydrophobic core of the putative signal peptide nor the following eight amino acids, which were suggested to be part of the putative signal peptidase cleavage site, are mandatory for functional expression of Na+-Ca2+ exchange activity. rat brain exchanger endoplasmic reticulum. rat brain exchanger endoplasmic reticulum. Truncation of clone RBE-1 (9Furman I. Cook O. Kasir J. Rahamimoff H. FEBS Lett. 1993; 319: 105-109Crossref PubMed Scopus (78) Google Scholar) has been carried out by the method of Kunkel(15Kunkel T.A. Proc. Natl. Acad. Sci. U. S. A. 1985; 82: 488-492Crossref PubMed Scopus (4903) Google Scholar). Appropriate phosphorylated antisense primers were annealed to the uracil-containing single-stranded DNA derived from RBE-1. The primers were typically 30-40-mer long and flanking (about half of the nucleotides upstream and the other half downstream) the desired deletion. Synthesis of second strand was carried out in vitro using T4 DNA polymerase and T4 DNA ligase. Selection of mutant plasmids was based on sequencing of the segment containing the mutation. To ensure that no other mutations beyond the planned occurred, either a 0.4-kilobase pair HindIII-NcoI fragment (N21, N26, or N31) or a 0.4-kilobase pair SalI-NcoI fragment (Ala-32 → Asp) containing the desired mutation was subcloned into a corresponding HindIII-NcoI- or SalI-NcoI-digested wild-type RBE-1. The entire subcloned fragment including the ligation sites were fully sequenced. The following antisense primers (5′to3′) were used: CAACATGGGTAAACAACATGTTGTACAATGAG(N21); CTGCAGTTATATGGTCCATGTTGTACAATGAG(N26); TTTCTGCCTCTGTATCCATGTTGTACAATGAG(N31); TTCTGCCTCTGTATCATCAGTTATATGGTCAAC (Ala-32 → Asp). Plasmid preparations were carried out by standard procedures (16Maniatis T. Fritsch E.F. Sambrook J. Molecular Cloning: A Laboratory Manual. Cold Spring Harbor Laboratory, Cold Spring Harbor, NY1989: 1.38-1.41-7.43-7.45Google Scholar) or with the Wizard®174; Promega plasmid preparation kit. Sequencing was carried out by the dideoxy method (17Sanger F. Nicken S. Coulson A.R. Proc. Natl. Acad. Sci. U. S. A. 1977; 74: 5463-5467Crossref PubMed Scopus (52756) Google Scholar) using the Sequenase II kit (U. S. Biochemical Corp.). Transient expression of the wild-type cloned (in pBluescript) rat brain Na+-Ca2+ exchanger gene RBE-1 and its mutants in HeLa or L-cells was done essentially as described in (8Low W. Kasir J. Rahamimoff H. FEBS Lett. 1992; 316: 63-67Crossref Scopus (69) Google Scholar) and (9Furman I. Cook O. Kasir J. Rahamimoff H. FEBS Lett. 1993; 319: 105-109Crossref PubMed Scopus (78) Google Scholar). 2.5 × 105 cells/well in a 24-well culture plate or 5 × 106 cells/90-mm Petri dish or 12.5 × 106 cells/135-mm Petri dish were transfected with 1.5, 15, or 37.5 μg of plasmid DNA, respectively. Dotap (Boehringer Mannheim, Germany) was used for transfection. Prior to transfection, the cells were infected with the recombinant vaccinia virus VTF-7(18Blakely R.D. Clark J.A. Rudnik G. Amara S. Anal. Biochem. 1991; 194: 302-308Crossref PubMed Scopus (151) Google Scholar). Expression of transport activity was determined 16-17 h following transfection as described previously (8Low W. Kasir J. Rahamimoff H. FEBS Lett. 1992; 316: 63-67Crossref Scopus (69) Google Scholar, 9Furman I. Cook O. Kasir J. Rahamimoff H. FEBS Lett. 1993; 319: 105-109Crossref PubMed Scopus (78) Google Scholar). Culture media were obtained from Biological Industries, Beit Haemek, Israel. For determination of transport activity in whole cells, these were preincubated for 10 min with 0.14 M NaCl, 0.01 M Tris-HCl, pH 7.4, after which they were exposed to 45Ca2+ in either 0.01 M Tris-HCl-buffered 0.14 M NaCl or 0.14 M KCl. The reactions were stopped by aspiration of the uptake media and two washes with 0.14 M KCl at 4°C, after which the cells were solubilized with 0.3 N NaOH, neutralized with 0.2 M NaPi, pH 4.5, and counted in a liquid scintillation counter. Net Na+ gradient-dependent Ca2+ uptake was calculated by subtraction of the amount of 45Ca2+ associated with the cells in the presence of external NaCl (no gradient) from the amount of 45Ca2+ associated with the cells in the presence of external KCl. Each measurement was done in triplicate. For reconstitution experiments, cells were harvested and dissolved in a solution containing 0.2 M NaPi, pH 7.4, 2% cholate, and 15-20 mg/ml brain phospholipids as described previously(8Low W. Kasir J. Rahamimoff H. FEBS Lett. 1992; 316: 63-67Crossref Scopus (69) Google Scholar, 9Furman I. Cook O. Kasir J. Rahamimoff H. FEBS Lett. 1993; 319: 105-109Crossref PubMed Scopus (78) Google Scholar). Reconstitution and determination of Na+ gradient-dependent Ca2+ uptake was done as in (19Barzilai A. Rahamimoff H. Biochemistry. 1987; 26: 6113-6118Crossref PubMed Scopus (22) Google Scholar). Protein was determined by the method of Lowry et al.(20Lowry O.H. Rosebrough V.J. Farr A.L. Randall R.J. J. Biol Chem. 1951; 193: 265-275Abstract Full Text PDF PubMed Google Scholar). The TNT®174; mRNA-free combined transcription/translation reticulocyte lysate system (Promega) was used. To detect protein synthesis, [35S]methionine (Amersham Corp., SJ1515) was added to the lysate. The manufacturer's instructions were followed with fidelity, except that the K+ concentration was increased by 40 mM. When stated, dog microsomes (Promega) were added to the lysate (2 μl of microsomes/25 μl of final volume of lysate reaction mixture). The proteins synthesized were analyzed on SDS-containing polyacrylamide gels(21Laemmli U.K. Nature. 1970; 227: 680-685Crossref PubMed Scopus (207487) Google Scholar). The acrylamide and N,N′-methylenebisacrylamide concentrations were reduced to 5 and 0.125%, respectively. 8 M urea was always added to the sample buffer, otherwise the proteins synthesized in the lysate aggregated and did not enter the gel. To evaluate the extent of the glycosylation by the microsomes, translation products were treated with peptide N-glycosidase F (New England BioLabs), and the size of the proteins synthesized before and after the treatment was determined by SDS-polyacrylamide gel electrophoresis (see above). Peptide N-glycosidase treatment was carried out as specified by the manufacturer, and it involved boiling for 10 min in 0.5% SDS, 1%β-mercaptoethanol and addition of Nonidet P-40 to a final concentration of 1%. Total RNA was extracted from 17 h postinfected/transfected HeLa cells, using the TRI®174; reagent (Molecular Research Center, Inc. Cincinnati OH). RNA separation using formaldehyde-containing 1% agarose gels was carried out by standard procedures(16Maniatis T. Fritsch E.F. Sambrook J. Molecular Cloning: A Laboratory Manual. Cold Spring Harbor Laboratory, Cold Spring Harbor, NY1989: 1.38-1.41-7.43-7.45Google Scholar). Following electrophoresis, the gels were soaked in 0.5 N NaOH and then in 20 × SSC, after which the denatured RNA was transferred to nitrocellulose by capillary elution. Hybridization was carried out in 50% formamide buffer overnight at 53°C. As probes, we have used either a [α-32P]dCTP-labeled polymerase chain reaction-amplified DNA fragment of 863 base pairs corresponding to nucleotides 1936-2799 of the cloned exchanger gene or a [γ-32P]ATP-labeled 53-mer synthetic antisense oligonucleotide corresponding to nucleotides 14-67 (the numbering refers to the open reading frame of the cloned exchanger RBE-1) of the following sequence: 5′-AGAGAGCCACCAGAGTTACCAGACGAAATCCCATTGAAACATTGGGTGGGAGAC-3′. Labeling of the 863-base pair probe was done by random priming using the Klenow fragment of DNA polymerase, and that of the oligonucleotide with T4 polynucleotide kinase. Quantitative densitometric analysis of the Northern blots was done with the Fuji thermal imager FTI 500 using the Macintosh program Image. Transfected HeLa cells were rinsed twice in phosphate-buffered saline, scraped from the 135-mm culture dish with a rubber policeman, and divided into three parts. One part was used for determination of expression of Na+ gradient-dependent Ca2+ transport activity. The second part was pelleted, suspended in a minimal volume of phosphate-buffered saline, clarified by a 10-s sonication, and used without further treatment. This total cell extract contained the entire repertoire of proteins produced in the transfected HeLa cells. The third part was suspended in 5 mM Tris-HCl, pH 7.4, 1 mM EDTA, 0.02 mM phenylmethylsulfonyl fluoride and subfractionated by differential centrifugation into a plasma membrane-enriched fraction P30, sedimenting at 30,000 × g and an endoplasmic reticulum-enriched fraction P100 sedimenting at 100,000 × g. From each fraction an aliquot was kept for the determination of protein. The different fractions were separated by SDS-containing gel electrophoresis (21Laemmli U.K. Nature. 1970; 227: 680-685Crossref PubMed Scopus (207487) Google Scholar) and transfered electrophoretically by standard procedures to nitrocellulose(22Harlow E. Lane D. Antibodies: A Laboratory Manual. Cold Spring Harbor Laboratory, Cold Spring Harbor, NY1988: 490-491-4978-79Google Scholar). As primary antibody, we have used antiserum (AbO-8) obtained from a rabbit immunized with a synthetic pentadecapeptide derived from amino acids 645-659 (9Furman I. Cook O. Kasir J. Rahamimoff H. FEBS Lett. 1993; 319: 105-109Crossref PubMed Scopus (78) Google Scholar) of the cloned rat brain exchanger RBE-1. The peptide was coupled to large nonimmunogenic carriers keyhole limpets hemocyanin, bovine serum albumin, and thyroglobulin using glutaraldehyde(22Harlow E. Lane D. Antibodies: A Laboratory Manual. Cold Spring Harbor Laboratory, Cold Spring Harbor, NY1988: 490-491-4978-79Google Scholar). Each subsequent injection of the peptide was done with a different carrier. Antibody production was tested by enzyme-linked immunosorbent assay first against the peptide and then against transfected HeLa cells. Mock transfected cells, with pBluescript were used as controls. For Western blots, the antiserum was purified by preadsorption on HeLa cells. This was done by incubating the antiserum with the cells at a dilution of 1:20 (in 3% bovine serum albumin, 0.02% NaN3 in phosphate-buffered saline) for 60 min at 25°C, after which the cells were sedimented and discarded. Usually, confluent cells harvested from a 25-ml culture flask were used for each ml of diluted antiserum. Before use, the purified antiserum was further diluted in 3% bovine serum albumin, 0.02% NaN3 in phosphate-buffered saline to a final dilution of 1:500. 125I-Protein A (DuPont NEN) was used as a secondary antibody. Quantitative analysis of the protein profile was carried out with the Fujix Bas 1000 PhosphorImager using the Tina 2.06 analysis program. The plasma membrane fraction was characterized by measuring the specific activity of 5′-AMP nucleotidase. In the P30 fraction, the specific activity of the enzyme increased between 3- and 6-fold in different preparations over the total cell extract and about 15-fold over the P100 fraction. In order to investigate the role of the amino-terminal segment of the cloned exchanger gene RBE-1 in functional expression of Na+-Ca2+ exchange activity, we have prepared three amino-terminal deletion mutants of the protein: N21, N26, and N31 (see Table1). All of the deletions conserved the initiating methionine. Mutant N21, in which we have deleted the initial 21 amino acids beyond the initiating methionine, was designed to test the role of the hydrophobic core of the putative signal peptide in functional expression. The deletion of amino acids 2-21, removed most of the hydrophobic core of the amino-terminal segment and the two positive charges within it that are thought to be important in the interaction with the negatively charged surface of the inner membrane of the endoplasmic reticulum(24Jain R.G. Rusch S.L. Kendall D.A. J. Biol. Chem. 1994; 269: 16305-16310Abstract Full Text PDF PubMed Google Scholar). In mutant N26, we had deleted amino acids 2-27. This deletion included the entire hydrophobic segment of the putative signal peptide (amino acids 2-24), and three of the amino acids that presumably form part of the putative signal peptidase recognition and cleavage site(24Jain R.G. Rusch S.L. Kendall D.A. J. Biol. Chem. 1994; 269: 16305-16310Abstract Full Text PDF PubMed Google Scholar, 25von Heijne G. J. Mol. Biol. 1992; 225: 487-494Crossref PubMed Scopus (1403) Google Scholar). The longest truncation was done in mutant N31, in which all of the amino acids between the initiating methionine and the putative amino-terminal aspartic acid were deleted. This mutant lacks the entire putative signal peptide and the putative signal peptidase cleavage site.TABLE I Open table in a new tab All of the amino-terminal mutants were expressed transiently in HeLa cells using the recombinant vaccinia virus VTF-7 expression system (18Blakely R.D. Clark J.A. Rudnik G. Amara S. Anal. Biochem. 1991; 194: 302-308Crossref PubMed Scopus (151) Google Scholar). The wild-type exchanger (clone RBE-1) was always expressed in parallel to the mutant exchangers. Na+-Ca2+ exchange activity was determined in "whole cells" exactly as described previously(8Low W. Kasir J. Rahamimoff H. FEBS Lett. 1992; 316: 63-67Crossref Scopus (69) Google Scholar, 9Furman I. Cook O. Kasir J. Rahamimoff H. FEBS Lett. 1993; 319: 105-109Crossref PubMed Scopus (78) Google Scholar). Fig. 1 summarizes the results obtained. In Fig. 1A, Na+ gradient dependent Ca2+ uptake of the three amino-terminal mutants N21, N26, and N31 in whole cells is shown. Since the expression of transport activity varied in different transfection experiments, the numerical value of the average steady state rate of Na+ gradient-dependent Ca2+ uptake (nmol of Ca2+ transported/mg of HeLa cell protein) of the wild-type exchanger RBE-1 in each experiment has been defined as 100%, and the transport activity of the mutant clones is presented in relative values. Fig. 1B, shows the numerical value of the average Ca2+ transport activity in these experiments (n = 12), of the wild-type exchanger RBE-1 in the presence (dottedbar) and in the absence (clearbar) of a driving Na+ gradient (for details, see "Experimental Procedures"). Net Na+ gradient-dependent Ca2+ uptake is calculated by subtracting the amount of Ca2+ transported in the absence of a driving Na+ gradient from that obtained in its presence. It should be also noted, that no endogenous Na+-Ca2+ exchange activity could be detected in infected/nontransfected HeLa cells (8Low W. Kasir J. Rahamimoff H. FEBS Lett. 1992; 316: 63-67Crossref Scopus (69) Google Scholar, 9Furman I. Cook O. Kasir J. Rahamimoff H. FEBS Lett. 1993; 319: 105-109Crossref PubMed Scopus (78) Google Scholar) or infected/control plasmid (pBluescript SK) (mock) transfected cells. From the data presented in Fig. 1A, it can be seen that the expression of transport activity of N21 is similar to that of the wild-type clone RBE-1, suggesting that the hydrophobic core of the putative signal peptide does not seem to play a significant role in functional expression of the Na+-Ca2+ exchanger. Determination of the transport activity of whole HeLa cells transfected with N26 and N31 revealed that these mutants are functional as well. Na+ gradient-dependent Ca2+ transport activity of these mutants was 35.4% (S.D. = 5.3) and 60.06% (S.D. = 8.48) of that of the wild-type exchanger. Although the expression of transport activity of these amino-terminally truncated mutants is somewhat lower than that of the wild-type clone, the results suggest that mutant protein is synthesized and is inserted in functional form into the plasma membrane. Functional expression of amino-terminal truncated mutants of the cloned Na+-Ca2+ exchanger gene is not restricted to HeLa cells, since transfection of L-cells with the same mutant exchangers resulted in functional expression similar to that obtained in HeLa cells. Relative to the wild-type exchanger RBE-1, 65, 43, and 38.3% of the Na+-Ca2+ exchange activity was obtained when L-cells were transfected with the truncated mutants N21, N26, and N31, respectively (data not shown). There are several ways to explain the somewhat lower transport activity of the truncated mutants N26 and N31 relative to the wild-type exchanger. One explanation could be that these mutants have an impaired trafficking machinery to the plasma membrane. To test this hypothesis, we reconstituted into brain phospholipids the proteins expressed in wild-type and mutant Na+-Ca2+ exchanger-transfected HeLa cells and examined their transport activity. Our rationale was that by solubilization of the transfected cells and reconstitution of the proteins into proteoliposomes, the presence of intracellular Na+-Ca2+ exchangers that were completed but not targeted to the plasma membrane would be revealed. If the proportion of these nontargeted Na+-Ca2+ exchangers within HeLa cells transfected with the mutant exchangers, N26 and N31 is significant, transport activity following reconstitution, should increase and result in values similar to those detected in cells transfected with the wild-type exchanger or mutant N21. The results of these experiments are shown in Fig. 2A. It can be seen, that the transport activity of the reconstituted amino-terminal truncated Na+-Ca2+ exchangers relative to the transport activity of the wild-type exchanger, is in principle similar to the transport activity obtained in the whole cell experiments. Fig. 2B shows the average Ca2+ transport activity (n = 9) of the reconstituted wild-type exchanger RBE-1 in the presence (dottedbar) and in the absence (clearbar) of a driving Na+ gradient. These experiments suggest that truncation of the entire putative signal peptide of the cloned Na+-Ca2+ exchanger gene RBE-1 including the signal peptidase cleavage site (mutant N31) do not result in an accumulation of significant amounts of intracellular Na+-Ca2+ exchangers. To try and elucidate the possible role of the signal peptide and that of the signal peptidase cleavage site in functional expression of the Na+-Ca2+ exchanger, a mutant exchanger was prepared in which the putative cleavage site Ala-32 was changed to Asp. We chose this exchange since analysis of the patterns of amino acids near the cleavage site (23von Heijne G. Eur. J. Biochem. 1983; 133: 17-21Crossref PubMed Scopus (1606) Google Scholar) indicated, that Asp was not found in position −1. Transfection of HeLa cells with this mutant clone indicated that its Na+-Ca2+ exchange activity was only 7.7% (S.D. = 4.4; n = 5) when compared with the wild-type exchanger RBE-1 (see Fig. 3), which was tested in parallel, suggesting that the amino acids in the vicinity of cleavage of the signal peptide might be of importance in functional expression. It was interesting to note that reconstitution of the proteins synthesized in the Ala-32 → Asp-transfected HeLa cells led to an increase in transport activity. Compared with the 7.7% relative to the wild-type exchanger in whole cells, following reconstitution, the transport activity increased to 31% (S.D. = 12.07; n = 5). This would suggest that either some protein was not targeted to the plasma membrane or it acquired functional conformation only following reconstitution. To test this possibility, we have isolated RNA from HeLa cells infected with the recombinant vaccinia virus VTF-7 and mock transfected with the plasmid pBluescript KS and from HeLa cells transfected with the wild-type and the three mutant cloned Na+-Ca2+ exchanger genes. Northern blot analysis (Fig. 4A) using a 863-base pair probe (spanning nucleotides 1936-2799 of the cloned rat Na+-Ca2+ exchanger gene) revealed that 1) the probe did not hybridize to RNA isolated from infected HeLa cells (laneA) or infected and mock-transfected (with pBluescript KS) HeLa cells (laneB); 2) the probe hybridized to RNA isolated from HeLa cells transfected with either wild-type (laneC) or the mutant exchangers (lanesD-F); 3) in most experiments (as also in that shown in Fig. 4A), we did not detect significant differences between the amount of the wild-type and mutant mRNAs, although their transport activity was somewhat lower (Fig. 1 and 2). Hence, there is no clear correlation in most experiments between the expression of Na+-Ca2+ exchange activity, which usually was somewhat lower when the cells were transfected with N26 and N31 and the amount of mutant-derived mRNA. We have also hybridized to these RNA preparations an antisense 53-mer-long oligonucleotide derived from the truncated amino-terminal region of the cloned Na+-Ca2+ exchanger genes (see "Experimental Procedures"). Fig. 4B shows that the oligonucleotide hybridized only to RNA isolated from HeLa cells transfected with the wild-type Na+-Ca2+ exchanger (laneB). This rules out any possibility that a contaminant signal peptide containing plasmid, either in the transfecting DNAs or in the cells, was responsible for the transport activity of the truncated mutants. We have also tested directly the correlation between the functional expression of Na+-Ca2+ exchange activity, the length of the putative signal peptide, and the nature of the signal peptidase cleavage site with the amount of Na+-Ca2+ exchanger protein synthesized and targeted to the plasma membrane. Western blot analysis was carried out to detect Na+-Ca2+ exchanger-derived protein in a total extract obtained from tr
Amino terminal
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Chaperone (clinical)
Wild type
Mutant protein
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Prolyl 4-hydroxylase (EC 1.14.11.2) catalyzes the posttranslational formation of 4-hydroxyproline in collagens. The vertebrate enzyme is an alpha 2 beta 2 tetramer, the beta subunit of which is a highly unusual multifunctional polypeptide, being identical to protein disulfide-isomerase (EC 5.3.4.1). We report here the cloning of a second mouse alpha subunit isoform, termed the alpha (II) subunit. This polypeptide consists of 518 aa and a signal peptide of 19 aa. The processed polypeptide is one residue longer than the mouse alpha (I) subunit (the previously known type), the cloning of which is also reported here. The overall amino acid sequence identity between the mouse alpha (II) and alpha (I) subunits is 63%. The mRNA for the alpha (II) subunit was found to be expressed in a variety of mouse tissues. When the alpha (II) subunit was expressed together with the human protein disulfide-isomerase/beta subunit in insect cells by baculovirus vectors, an active prolyl 4-hydroxylase was formed, and this protein appeared to be an alpha (II) 2 beta 2 tetramer. The activity of this enzyme was very similar to that of the human alpha (I) 2 beta 2 tetramer, and most of its catalytic properties were also highly similar, but it differed distinctly from the latter in that it was inhibited by poly(L-proline) only at very high concentrations. This property may explain why the type II enzyme was not recognized earlier, as an early step in the standard purification procedure for prolyl 4-hydroxylase is affinity chromatography on a poly(L-proline) column.
Tetramer
BETA (programming language)
Alpha (finance)
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Protein kinase domain
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Tetramer
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The microsomal triglyceride transfer protein (MTP) is a heterodimer composed of the ubiquitous multifunctional protein, protein disulfide isomerase, and a unique 97-kDa subunit. Mutations that lead to the absence of a functional 97-kDa subunit cause abetalipoproteinemia, an autosomal recessive disease characterized by a defect in the assembly and secretion of apolipoprotein B (apoB) containing lipoproteins. Previous studies of abetalipoproteinemic patient, C.L., showed that the 97-kDa subunit was undetectable. In this report, [35S]methionine labeling showed that this tissue was capable of synthesizing the 97-kDa MTP subunit. Electrophoretic analysis showed two bands, one with a molecular mass of the wild type 97-kDa subunit and the other with a slightly lower molecular weight. Sequence analysis of cDNAs from additional intestinal biopsies showed this patient to be a compound heterozygote. One allele contained a perfect in-frame deletion of exon 10, explaining the lower molecular weight band. cDNAs of the second allele were found to contain 3 missense mutations: His297→ Gln, Asp384→ Ala, and Arg540→ His. Transient expression of each mutant showed that only the Arg540→ His mutant was non-functional based upon its inability to reconstitute apoB secretion in a cell culture system. The other amino acid changes are silent polymorphisms. High level coexpression in a baculovirus system of the wild type 97-kDa subunit or the Arg540→ His mutant along with human protein disulfide isomerase showed that the wild type was capable of forming an active MTP complex while the mutant was not. Biochemical analysis of lysates from these cells showed that the Arg to His conversion interrupted the interaction between the 97-kDa subunit and protein disulfide isomerase. Replacement of Arg540 with a lysine residue maintained the ability of the 97-kDa subunit to complex with protein disulfide isomerase and form the active MTP holoprotein. These results indicate that a positively charged amino acid at position 540 in the 97-kDa subunit is critical for the productive association with protein disulfide isomerase. Of the 13 mutant MTP 97-kDa subunit alleles described to date, this is the first encoding a missense mutation. The microsomal triglyceride transfer protein (MTP) is a heterodimer composed of the ubiquitous multifunctional protein, protein disulfide isomerase, and a unique 97-kDa subunit. Mutations that lead to the absence of a functional 97-kDa subunit cause abetalipoproteinemia, an autosomal recessive disease characterized by a defect in the assembly and secretion of apolipoprotein B (apoB) containing lipoproteins. Previous studies of abetalipoproteinemic patient, C.L., showed that the 97-kDa subunit was undetectable. In this report, [35S]methionine labeling showed that this tissue was capable of synthesizing the 97-kDa MTP subunit. Electrophoretic analysis showed two bands, one with a molecular mass of the wild type 97-kDa subunit and the other with a slightly lower molecular weight. Sequence analysis of cDNAs from additional intestinal biopsies showed this patient to be a compound heterozygote. One allele contained a perfect in-frame deletion of exon 10, explaining the lower molecular weight band. cDNAs of the second allele were found to contain 3 missense mutations: His297→ Gln, Asp384→ Ala, and Arg540→ His. Transient expression of each mutant showed that only the Arg540→ His mutant was non-functional based upon its inability to reconstitute apoB secretion in a cell culture system. The other amino acid changes are silent polymorphisms. High level coexpression in a baculovirus system of the wild type 97-kDa subunit or the Arg540→ His mutant along with human protein disulfide isomerase showed that the wild type was capable of forming an active MTP complex while the mutant was not. Biochemical analysis of lysates from these cells showed that the Arg to His conversion interrupted the interaction between the 97-kDa subunit and protein disulfide isomerase. Replacement of Arg540 with a lysine residue maintained the ability of the 97-kDa subunit to complex with protein disulfide isomerase and form the active MTP holoprotein. These results indicate that a positively charged amino acid at position 540 in the 97-kDa subunit is critical for the productive association with protein disulfide isomerase. Of the 13 mutant MTP 97-kDa subunit alleles described to date, this is the first encoding a missense mutation.
Mutant protein
Wild type
Compound heterozygosity
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Mouse C127 epithelioid cells were genetically engineered to produce biologically active gamma-carboxylated human protein S. A full length human protein S cDNA was cloned into a bovine papilloma virus (BPV) based shuttle vector under the transcriptional control of the Moloney murine sarcoma virus enhancer and the mouse metallothionein promoter. Stable expression was obtained in transfected C127 cells. Expression of gamma-carboxylated protein S was dependent on the presence of vitamin K in the culture medium. Protein sequence analysis showed that recombinant and plasma protein S have the same amino terminal sequence. Analysis of specific post-translationally modified amino acids shows that recombinant protein S is fully gamma-carboxylated and fully beta-hydroxylated. Immunoblotting analysis using polyclonal and monoclonal antibodies shows that recombinant protein S has a slightly higher molecular weight than plasma protein S. After N-Glycanase treatment, identical molecular weights are observed for recombinant and plasma protein S, indicating that the difference is caused by differences in the N-linked carbohydrate side chains. Recombinant protein S also demonstrates normal cofactor activity for activated protein C in a clotting assay. Binding studies with the complement component, C4b-binding protein (C4BP), shows that recombinant protein S binds to C4BP with the same apparent affinity as plasma protein S. Two variant molecules are also tested for their binding to C4BP. The first variant has a replacement of amino acid residue leu-608 by val and was designated B variant. The second variant has three alterations, at positions 609, 611 and 612 where the acidic amino acid residues asp, asp and glu were replaced by asn, asn and gln, respectively and this variant was designated C variant.(ABSTRACT TRUNCATED AT 250 WORDS)
Protein A/G
Myc-tag
FLAG-tag
Protein G
Polyclonal antibodies
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