Molecular Cloning and Expression of a Mouse Brain cDNA Encoding a Novel Protein Target of Calcyclin

Abstract: A protein target of mouse calcyclin, p30, which we call calcyclin-binding protein (CacyBP), was identified in mouse brain and Ehrlich ascites tumor (EAT) cells. The amino acid sequence of the CacyBP chymotryptic peptide was used to prepare synthetic oligonucleotides that served as a probe to screen the mouse brain cDNA library. A 1.4-kb positive clone was detected, isolated, and sequenced. The analyzed clone contains an open reading frame encoding a protein of a molecular mass of ～26 kDa. The nucleotide and predicted amino acid sequences indicate that CacyBP is a novel protein. The results obtained from northern blots show that the CacyBP gene is expressed predominantly in mouse brain and EAT cells. Using a pGEX vector the recombinant CacyBP was expressed in Escherichia coli, and its properties were analyzed. The recombinant protein interacts with calcyclin at a physiologically relevant range of Ca²⁺ in solution during affinity chromatography and on blots. Because CacyBP, like calcyclin, is present in the brain, the interaction of these two proteins might be involved in calcium signaling pathways in neuronal tissue.     

A calcyclin-associated protein is a newly identified member of the Ca2+/phospholipid-binding proteins, annexin family.

A calcyclin-associated protein with an apparent molecular weight of 50,000 (CAP-50) was purified from rabbit lung. The procedure included ammonium sulfate precipitation, anion and cation ion-exchange, and calcyclin affinity chromatographies. Interestingly, partial amino acid sequences of lysyl-endpeptidase-digested fragments indicated that CAP-50 was a member of the Ca2+/phospholipid-binding proteins, the annexin family. The sequence of a proteolytic peptide with Staphylococcus aureus V8 protease on NH2-terminal region is not homologous with any other annexin family proteins. Phospholipid binding studies showed that CAP-50 bound to phosphatidylserine, phosphatidylethanolamine, phosphatidylinositol, and phosphatidic acid-containing vesicles, in a Ca(2+)-dependent manner. In the presence of Ca2+/calcyclin, CAP-50 formed a complex with calcyclin and bound to the PS-containing vesicles. The apparent Kd value of calcyclin for CAP-50 was calculated to be 1.61 x 10(-6) M. Zero-length cross-linking studies indicated that 1 mol of CAP-50 bound to an equimolar unit of calcyclin. CAP-50 inhibited the phospholipase A2 activity, dose-dependently (IC50 = 0.2 microM), however, calcyclin did not alter the inhibitory effect. With the 125I-calcyclin gel overlay method, calcyclin bound tightly to CAP-50 in a Ca(2+)-dependent manner after sodium dodecyl sulfate-polyacrylamide gel electrophoresis. These results suggest that rabbit lung CAP-50 is a newly identified member of the annexin family. Ca2+/calcyclin apparently regulates the function of CAP-50 on cytosolic face of the plasma membrane.     

Calcium-binding protein from mouse Ehrlich ascites-tumour cells is homologous to human calcyclin.

A Ca2+-binding protein was purified from mouse Ehrlich ascites-tumour cells. The protein forms monomers and disulphide-linked dimers, which can be separated by reverse-phase h.p.l.c. A partial amino acid sequence analysis demonstrated that the protein has an EF-hand structure. A striking homology was found to rat and human calcyclin (a member of the S-100 protein family), which is possibly involved in cell-cycle regulation.     

Chloroplast glyceraldehyde-3-phosphate dehydrogenase (NADP): amino acid sequence of the subunits from isoenzyme I and structural relationship with isoenzyme II

The structural relationship between isoenzymes I and II of chloroplast glyceraldehyde-3-phosphate dehydrogenase (D-glyceraldehyde-3-phosphate: NADP+ oxidoreductase (phosphorylating) EC 1.2.1.13) has been established at the protein level. The complete primary structure of subunits A and B of glyceraldehyde-3-phosphate dehydrogenase I from Spinacia oleracea has been determined by sequence analysis of the corresponding tryptic peptides, aligned by fragments derived from cyanogen bromide and Staphylococcus proteinase V8 digestions and by partially sequencing each intact subunit. Subunit A has an Mr of 36,225 and consists of 337 amino acid residues, whilst subunit B (Mr 39,355) consists of 368 residues. The amino acid sequence of subunit B, as determined through direct analysis of the protein, is identical to that recently deduced at cDNA level (Brinkmann et al. (1989) Plant Mol. Biol. 13, 81-94). The two subunits share a common portion of amino acid sequence which differs by 66 amino acid residues. Subunit B has an extra C-terminal sequence of 31 amino acid residues. Chloroplast glyceraldehyde-3-phosphate dehydrogenase II was partially characterized by sequencing the N-terminal portion of the intact protein and some of its tryptic peptides. The sequences of all the examined fragments fit precisely that of the corresponding regions of subunit A from glyceraldehyde-3-phosphate dehydrogenase I.     

Purification and properties of NAD+-dependent glyceraldehyde-3-phosphate dehydrogenase from spinach leaves.

An NAD+-dependent glyceraldehyde-3-phosphate dehydrogenase (D-glyceraldehyde-3-phosphate:NAD+ oxidoreductase (phosphorylating), EC. 1.2.1.12) has been purified from spinach leaves as a homogeneous protein of 150,000 daltons. Kinetic constants of 2.5 . 10(-4) M and 4 . 10(-4) M have been calculated for NAD+ and glyceraldehyde-3-phosphate, respectively. The amino acid composition is characterized by a cysteine content higher than that found in analogous enzymes. On sodium dodecyl sulphate gel electrophoresis, the native enzyme dissociates into two subunits of 37,000 and 14,000 daltons. The two subunits have been isolated in equimolar amounts by gel filtration; end-group analysis shows that alanine is the N-terminal residue of the large subunit, while serine is found at the N-terminus of the small subunit. Comparison of amino acid analysies and peptide maps shows that the two subunits have a different amino acid sequence. These results indicate that the NAD+-dependent glyceraldehyde-3-phosphate, dehydrogenase, isolated from spinach leaves has an atypical oligomeric structure, the protomer being formed by two different subunits.     

A calcium-binding protein from rabbit lung cytosol identified as the product of growth-regulated gene (2A9) and its binding proteins

Using Ca(2+)-dependent affinity chromatography on a synthetic compound (W-77)-coupled Sepharose 4B column, we purified two different Ca(2+)-binding proteins from rabbit lung extracts. The molecular weights of these proteins were estimated to be 17 kDa (calmodulin) and 10 kDa, respectively. The partial amino acid sequence of the 10-kDa protein revealed that it has two EF-hand structures. In addition, the 10-kDa protein was highly homologous (91%) to the product of growth-regulated gene, 2A9 (calcyclin). The Ca(2+)-binding property of the 10-kDa protein was observed by a change in the uv difference spectrum. Equilibrium dialysis showed that 1 mol of the 10-kDa protein bound to 2.04 +/- 0.05 mol of Ca2+ in the presence of 10(-4) M Ca2+. However, the protein failed to activate calmodulin-dependent enzymes such as Ca2+/CaM kinase II, myosin light chain kinase, and phosphodiesterase. We found that a 50-kDa cytosolic protein of the rabbit lung, intestine, and spleen bound to the 10-kDa protein, in a Ca(2+)-dependent manner. The distribution of calcyclin and calcyclin binding proteins was unique and seems to differ from that of calmodulin and calmodulin-binding proteins. Thus, calcyclin probably plays a physiological role through its binding proteins for the Ca(2+)-dependent cellular response.     

Molecular cloning of rabbit CAP-50, a calcyclin-associated annexin protein.

CAP-50 is a member of annexin family proteins which binds specifically to calcyclin in a Ca2+ dependent manner (Tokumitsu. H., Mizutani. A., Minami. H., Kobayashi. R., and Hidaka. H. (1992) J. Biol. Chem. 267,8919-8924). The cDNA representing the rabbit form of this protein has been cloned from rabbit lung cDNA library. Sequence analysis of two overlapping clones revealed a 81-nucleotides 5'-nontranslated region, 1512-nucleotides of open reading frame, a 672-nucleotides 3'-nontranslated region, and a poly(A) tail. Authenticity of the clones was confirmed by comparison of portions of the deduced amino acid sequence with eight sequences of proteolytic peptides obtained from rabbit lung protein. CAP-50 cDNA encodes a 503 residue protein with a calculated M(r) of 54,043 and shows that the protein is composed of four imperfect repeats and hydrophobic N-terminal region. C-terminal region including four imperfect repeats shows 58.1% identity with human synexin (annexin VII), 48.0% identity with annexin I, 47.4% identity with annexin II, 60.1% identity with annexin IV, 54.5% identity with annexin V. Hydrophobic N-terminal region composed of 202 amino acid residues is not homologous with other annexin proteins suggesting that CAP-50 is a novel member of annexin family proteins.     

Identification of the NAD(+)-binding fold of glyceraldehyde-3-phosphate dehydrogenase as a novel RNA-binding domain

There is growing evidence that metabolic enzymes may act as multifunctional proteins performing diverse roles in cellular metabolism. Among these functions are the RNA-binding activities of NAD(+)-dependent dehydrogenases. Previously, we have characterized the glycolytic enzyme glyceraldehyde-3-phosphate dehydrogenase (GAPDH) as an RNA-binding protein with preference to adenine-uracil-rich sequences. In this study, we used GST-GAPDH fusion proteins generated by deletion mutagenesis to search for the RNA binding domain. We established that the N-terminal 43 amino acid residues of GAPDH, which correspond to the first mononucleotide-binding domain of the NAD(+)-binding fold is sufficient to confer RNA-binding. We also provide evidence that this single domain, although it retains most of the RNA-binding activity, loses sequence specificity. Our results suggest a molecular basis for RNA-recognition by NAD(+)-dependent dehydrogenases and (di)nucleotide-binding metabolic enzymes that had been reported to have RNA-binding activity with different specificity. To support this prediction we also identified other members of the family of NAD(+)-dependent dehydrogenases with no previous history of nucleic acid binding as RNA binding proteins in vitro. Based on our findings we propose the addition of the NAD(+)-binding domain to the list of RNA binding domains/motifs.     

Isolation and characterization of a gene coding for glyceraldehyde-3-phosphate dehydrogenase from Saccharomyces cerevisiae.

A yeast glyceraldehyde-3-phosphate dehydrogenase gene has been isolated from a collection of Escherichia coli transformants containing randomly sheared segments of yeast genomic DNA. Complementary DNA, synthesized from partially purified glyceraldehyde-3-phosphate dehydrogenase messenger RNA, was used as a hybridization probe for cloning this gene. The isolated hybrid plasmid DNA has been mapped with restriction endonucleases and the location of the glyceraldehyde-3-phosphate dehydrogenase gene within the cloned segment of yeast DNA has been established. There are approximately 4.5 kilobase pairs of DNA sequence flanking either side of the glyceraldehyde-3-phosphate dehydrogenase gene in the cloned segment of yeast DNA. The isolated hybrid plasmid DNA has been used to selectively hybridize glyceraldehyde-3-phosphate dehydrogenase messenger RNA from unfractionated yeast poly(adenylic acid)-containing messenger RNA. The nucleotide sequence of a portion of the isolated hybrid plasmid DNA has been determined. This nucleotide sequence encodes 29 amino acids which are at the COOH terminus of the known amino acid sequence of yeast glyceraldehyde-3-phosphate dehydrogenase.     

Identification of the mammalian DNA-binding protein P8 as glyceraldehyde-3-phosphate dehydrogenase.

The DNA-binding protein P8 from transformed hamster fibroblasts (line NIL-1-hamster sarcoma virus) has been purified to homogeneity by DNA-cellulose and phosphocellulose chromatography. The molecular weight of dissociated P8 is 36000, the same as that reported for the subunits of glyceraldehyde-3-phosphate dehydrogenase, and the mobility of these proteins in polyacrylamide gels is identical. The amino acid composition of P8 is very similar to that of glyceraldehyde-3-phosphate dehydrogenase. When assayed for glyceraldehyde-3-phosphate dehydrogenase activity the P8 preparation had a specific activity of 54.6 units/mg, a value comparable to that of the crystalline enzyme from several sources. Furthermore, serum prepared against P8 crossreacts with glyceraldehyde-3-phosphate dehydrogenase from hamster muscle. These results show that P8 is glyceraldehyde-3-phosphate dehydrogenase. The interaction of P8 from transformed fibroblasts and glyceraldehyde-3-phosphate dehydrogenase from hamster and rabbit muscle with DNA has been studied using a Millipore filtration technique. These proteins have affinity for single-stranded DNA but not for double-stranded DNA.     

Cloning and sequence analysis of the glyceraldehyde-3-phosphate dehydrogenase gene from the zygomycetes fungus Rhizomucor miehei

Rhizomucor miehei is important from a biotechnological aspect in consequence of its content of aspartic proteinase, which has high milk-clotting activity. A genomic library of R. miehei NRRL 5901 has been constructed in a phage (Lambda Fix II) vector. The glyceraldehyde-3-phosphate dehydrogenase (gpd) gene was isolated from this library by hybridization of the recombinant phage clones with a gpd-specific gene probe generated by polymerase chain reaction. The complete nucleotide sequence encodes a putative polypeptide chain of 336 amino acids interrupted by 5 introns. The predicted amino acid sequence of this gene shows a high degree of sequence similarity to the glyceraldehyde-3-phosphate dehydrogenase proteins from yeast and filamentous fungi. The promoter region, containing a consensus TATA box, and 246-bp downstream from the putative stop codon were also determined. The possibility of using the gpd promoter in the construction of new transformation vectors is discussed.     

Identification of intracellular target proteins of the calcium-signaling protein S100A12.

In this report, we have focused our attention on identifying intracellular mammalian proteins that bind S100A12 in a Ca2+-dependent manner. Using S100A12 affinity chromatography, we have identified cytosolic NADP+-dependent isocitrate dehydrogenase (IDH), fructose-1,6-bisphosphate aldolase A (aldolase), glyceraldehyde-3-phosphate dehydrogenese (GAPDH), annexin V, S100A9, and S100A12 itself as S100A12-binding proteins. Immunoprecipitation experiments indicated the formation of stable complexes between S100A12 and IDH, aldolase, GAPDH, annexin V and S100A9 in vivo. Surface plasmon resonance analysis showed that the binding to S100A12, of S100A12, S100A9 and annexin V, was strictly Ca2+-dependent, whereas that of GAPDH and IDH was only weakly Ca2+-dependent. To localize the site of S100A12 interaction, we examined the binding of a series of C-terminal truncation mutants to the S100A12-immobilized sensor chip. The results indicated that the S100A12-binding site on S100A12 itself is located at the C-terminus (residues 87-92). However, cross-linking experiments with the truncation mutants indicated that residues 87-92 were not essential for S100A12 dimerization. Thus, the interaction between S100A12 and S100A9 or immobilized S100A12 should not be viewed as a typical S100 homo- or heterodimerization model. Ca2+-dependent affinity chromatography revealed that C-terminal residues 75-92 are not necessary for the interaction of S100A12 with IDH, aldolase, GAPDH and annexin V. To analyze the functional properties of S100A12, we studied its action in protein folding reactions in vitro. The thermal aggregation of IDH or GAPDH was facilitated by S100A12 in the absence of Ca2+, whereas in the presence of Ca2+ the protein suppressed the aggregation of aldolase to less than 50%. These results suggest that S100A12 may have a chaperone/antichaperone-like function which is Ca2+-dependent.     

Glyceraldehyde-3-phosphate dehydrogenase from Ehrlich ascites carcinoma cells its possible role in the high glycolysis of malignant cells.

Glyceraldehyde-3-phosphate dehydrogenase has been purified to apparent homogeneity from Ehrlich ascites carcinoma (EAC) cells. The enzyme is quite active over a pH range of 7.5-9.0 with an optimum pH of 8.4-8.7. The specific activity of the enzyme is much higher than that from other normal sources. In contrast to enzyme obtained from rabbit muscle, the EAC cell enzyme is not significantly inhibited by physiological concentrations of ATP at physiological pH. Kinetic studies using different substrates and inhibitors indicate that the properties of the EAC cell enzyme are significantly different from those of glyceraldehyde-3-phosphate dehydrogenase obtained from other normal sources. The striking dissimilarity of the malignant cell glyceraldehyde-3-phosphate dehydrogenase compared with this enzyme from other normal sources, particularly in respect to the interaction with ATP, may in part explain the high glycolysis of malignant cells.     

Glucose-dependent active ATP depletion by koningic acid kills high-glycolytic cells

Many types of cancer cells depend heavily on glycolysis for energy production even in aerobic conditions. We found that koningic acid (KA), an inhibitor of glyceraldehyde-3-phosphate dehydrogenase (GAPDH), selectively kills high-glycolytic cells through glucose-dependent active ATP deprivation. Out of seven tumor cell lines tested, growth of six cell lines, which had high glycolytic capacity, was inhibited by KA, whereas three normal cell lines, which had low glycolytic activity, were insensitive to KA. The growth inhibition and caspase-independent cell death in sensitive cells were related to severe ATP depletion that was promoted by glucose phosphorylation. Although GAPDH was completely inhibited in KA-insensitive CHO-K1 cells, KA-mediated ATP depletion was less extensive and transient, possibly due to utilization of ketogenic essential amino acids as energy source. KA suppressed Ehrlich ascites tumor growth in vivo and benefited the survival of the affected mice.     

Calcyclin and calvasculin exist in human platelets.

Using Ca(2+)-dependent affinity chromatography on a synthetic compound (W-7)-coupled Sepharose column, three distinct Ca(2+)-binding proteins have been identified in human platelets. The molecular mass of these three distinct proteins was estimated to be 10, 10.5, 17 kDa, respectively, by polyacrylamide gel electrophoresis in the presence of SDS. The partial amino acid sequence revealed these proteins have EF-hand structures and high homology to the predicted proteins, calcyclin, calvasculin, and calmodulin. Calcyclin and calvasculin have been considered as probably having roles in the control of cell proliferation, but the existence of these two proteins in platelets suggests that they have other intracellular functions related to the Ca(2+)-signal transduction system.     

Purification and characterization of annexin proteins from bovine lung

Calcium-dependent association with a detergent-extracted particulate fraction was used as the first step in the purification of a group of phospholipid binding proteins. Elution of the detergent-insoluble fraction with excess ethylene glycol bis(beta-aminoethyl ether)-N,N,N',N'-tetraacetic acid (EGTA) resulted in the release of several soluble proteins, termed calcium-activated proteins or CAPs. In the present paper, we describe the simultaneous purification of these CAPs and characterize their interaction with phospholipid, actin, and calmodulin. Partial sequence analysis has identified the majority of the CAPs as members of the annexin family of calcium and phospholipid binding proteins. Two additional CAPs may be novel proteins, one of which appears to be an annexin protein. All CAPs demonstrated Ca2(+)-dependent binding to phosphatidylserine vesicles but did not bind to phosphatidylcholine vesicles. The majority of CAPs exhibited Ca2(+)-dependent binding to F-actin; however, only CAP-III affected the rate of conversion of G-actin to F-actin. The interaction of CAP-III and lipocortin-85 with F-actin resulted in a Ca2(+)-dependent increase in both light scattering and sedimentation of F-actin under comparatively low centrifugal force. In contrast, only lipocortin-85 caused the formation of F-actin bundles. Although all of the CAPs bound to a calmodulin affinity column in a Ca2(+)-dependent manner, attempts to demonstrate binding of CAPs to native calmodulin were unsuccessful. These studies therefore document the similar behavior of the CAPs toward phospholipid and calmodulin but clearly show that F-actin binding or bundling is not a general property of these proteins. The reported purification procedure should allow further comparative studies of these proteins.     

Identification of the major annexins in Ehrlich ascites tumor cells

• 1.1. Three calcium-binding proteins have been purified from Ehrlich ascites tumor cells.
• 2.2. They were identified by amino acid sequence analysis on selected fragments obtained by tryptic digestion.
• 3.3. The proteins belong to the annexin family and were identified as annexins II, III and V.
• 4.4. Antibodies raised against the proteins were used to examine for their presence in a number of murine tissues.
• 5.5. The occurrence was found to be in reasonable accordance with earlier reports.

     

Molecular cloning, sequencing and expression in Escherichia coli of the 25-kDa growth-related protein of Ehrlich ascites tumor and its homology to mammalian stress proteins

The growth-related 25-kDa protein (p25) of Ehrlich ascites tumor (EAT) has been characterized by molecular cloning and sequencing of cDNA clones detected by hybridization with oligonucleotide probes synthesized according to the amino acid sequence of a tryptic peptide of p25. Detection of p25 mRNA in EAT of the exponential growth phase and of the stationary phase using cDNA-derived RNA probes demonstrated that the abundance of p25 mRNA is also growth-related. High-level expression of p25 in Escherichia coli has been established by oligonucleotide-directed mutagenesis of cDNA and insertion of the mutated cDNA into a T7-promoter expression vector. Recombinant p25 from the expressed cDNA sequence has been shown to comigrate with EAT p25 in electrophoresis and to react with antibodies against the EAT p25. On the amino acid level, p25 shows about 80% sequence homology to the human stress protein hsp27. Furthermore, p25 has similar isoforms of phosphorylation as demonstrated for small mammalian stress proteins from rat and human. From the results obtained, it is concluded that p25 is a mammalian stress protein, the abundance of which is related to growth characteristics of the Ehrlich ascites tumor.     

CacyBP/SIP,a calcyclin and Siah-1-interacting protein,binds EF-hand proteins of the S100 family

Recently, a human ortholog of mouse calcyclin (S100A6)-binding protein (CacyBP) called SIP (Siah-1-interacting protein) was shown to be a component of a novel ubiquitinylation pathway regulating beta-catenin degradation (Matsuzawa, S., and Reed, J. C. (2001) Mol. Cell 7, 915-926). In murine brain, CacyBP/SIP is expressed at a high level, but S100A6 is expressed at a very low level. Consequently we carried out experiments to determine if CacyBP/SIP binds to other S100 proteins in this tissue. Using CacyBP/SIP affinity chromatography, we found that S100B from the brain extract binds to CacyBP/SIP in a Ca2+-dependent manner. Using a nitrocellulose overlay assay with 125I-CacyBP/SIP and CacyBP/SIP affinity chromatography, we found that this protein binds purified S100A1, S100A6, S100A12, S100B, and S100P but not S100A4, calbindin D(9k), parvalbumin, and calmodulin. The interaction of S100 proteins with CacyBP/SIP occurs via its C-terminal fragment (residues 155-229). Co-immunoprecipitation of CacyBP/SIP with S100B from brain and with S100A6 from Ehrlich ascites tumor cells suggests that these interactions are physiologically relevant and that the ubiquitinylation complex involving CacyBP/SIP might be regulated by S100 proteins.     

Calcyclin is a calcium and zinc binding protein

Calcyclin, a cell cycle regulated protein, was recently purified from Ehrlich ascites tumour (EAT) cells and shown to be a calcium binding protein. Here we show that calcyclin monomer and dimer also bind zinc ions. Zinc binding sites seem to be different from calcium binding sites since: preincubation with Ca2+ lacks effect on the binding of Zn2+, and Ca2+ (but not Zn2+) increases tyrosine fluorescence intensity. Binding of Zn2+ reduces the extent of the conformational changes induced by Ca2+, and seems to affect Ca2(+)-binding. The data suggest that Ca2+ and Zn2+ might trigger the biological activity of calcyclin.