Cloning and expression of cDNA for human endonexin II, a Ca2+ and phospholipid binding protein

Endonexin II is a member of the family of Ca2+-dependent phospholipid binding proteins known as annexins. We cloned human endonexin II cDNA and expressed it in Escherichia coli. The apparent size and Ca2+-dependent phospholipid binding properties of purified recombinant endonexin II were indistinguishable from those of the placental protein. A single mRNA of approximately 1.6 kilobase pairs was found to be expressed in human cell lines and placenta and was in close agreement with the length of the cDNA clone (1.59 kilobase pairs). The cDNA predicted a 320-amino acid protein with a sequence that was in agreement with the previously determined partial amino acid sequence of endonexin II isolated from placenta. Endonexin II contained 58, 46, and 43% sequence identity to protein II, calpactin I (p36, protein I), and lipocortin I (p35), respectively. The partial sequence of bovine endonexin I was aligned with the sequence of endonexin II to give 63% sequence identity. Like these other proteins, endonexin II had a 4-fold internal repeat of approximately 70 residues preceded by an amino-terminal domain lacking similarity to the repeated region. It also had significant sequence identity with 67-kDa calelectrin (p68), a protein with an 8-fold internal repeat. Comparing the amino-terminal domains of these four proteins of known sequence revealed that, in general, only endonexin II and protein II had significant sequence identity (29%). Endonexin II was not phosphorylated by Ca2+/phospholipid-dependent enzyme (protein kinase C) even though it contained a threonine at a position analogous to the protein kinase C phosphorylation sites of lipocortin I, calpactin I, and protein II.     

The influence of Annexin32, a new Ca2+-dependent phospholipid-binding protein, on coagulation time and thrombosis

The pharmacodynamics of Annexin32, a new Ca2+-dependent phospholipid-binding protein, was studied by measuring coagulation time in rabbits and venous thrombosis in rabbits and rats. Rabbits and rats were given Annexin32 by intravenous administration. Then Kaolin partial thromboplastin time (KPTT), thrombosis in vitro and in vivo were assayed. The results showed that KPTT of rabbits was prolonged (p < 0.01), and the length and weight of thrombus in vitro were reduced (p < 0.01) after administration of Annexin32 at 1 mg/kg. It also inhibited thrombosis in vivo and reduced the weight of venous thrombus significantly in rats (p < 0.01). All these results suggested that Annexin32 possesses the characteristic of antithrombotic effect and fewer side effects on coagulation time.     

Characterizations of two distinct Ca2+-dependent phospholipid-binding proteins of 68-kDa isolated from human placenta

Two distinct 68-kDa proteins, named 68K-I (pI 6.4) and 68K-II (pI 5.6), were solubilized from human placenta by treatment with 5 mM EGTA. On DE52 cellulose column chromatography at pH 7.4, 68K-I in the EGTA eluate was recovered in the unadsorbed fractions, whereas 68K-II was retained on the column and eluted with 0.2 M NaCl. The 68K-I protein was obtained in more than 95% purity by further hydroxylapatite and cation exchange chromatographies, while the 68K-II protein was purified further by gel filtration and hydroxylapatite chromatographies. Partial amino acid sequence data showed that 68K-I protein was a novel protein which shared the same sequences as lipocortin I and that 68K-II was the same as human p68/67-kDa calelectrin (Crompton, M. R., Owens, R. J., Totty, N. F., Moss, S. E., Waterfield, M.D., and Crumpton, M. J. (1988) EMBO J. 7, 21-27; Südhof, T. C., Slaughter, C. A., Leznicki, I., Barjon, P., and Reynolds, G. A. (1988) Proc. Natl. Acad. Sci. U.S.A. 85, 664-668). The two proteins bound to acidic phospholipids, phosphatidylserine, and/or phosphatidylinositol, but not to phosphatidylcholine, in the presence of micromolar levels of Ca2+. 68K-I bound to phosphatidylinositol preferentially to phosphatidylserine, whereas 68K-II bound only to phosphatidylserine. Both 68K-I and 68K-II inhibited phospholipase A2 activity, and the inhibition by 68K-II was detectable only in the presence of 100 mM KCl. 68K-I, but not 68K-II, was found to bind to F-actin in a Ca2+-dependent (1 mM) manner. Moreover 68K-I, but not 68K-II, was phosphorylated in vitro at tyrosine residues by fps kinase and by epidermal growth factor receptor/kinase, the latter reaction being dependent on Ca2+ and epidermal growth factor. Western blot analysis with affinity purified anti-68K-I and anti-68K-II antibodies showed that 68K-I was located in only certain tissues, especially human placenta, whereas 68K-II was present in many human and rat tissues.     

Staurosporine, a protein kinase C inhibitor, attenuates Ca2+-dependent stretch-induced vascular tone

The effects of protein kinase C inhibition by staurosporine was studied on Ca-dependent tone of the rabbit facial vein. Tone was produced either by stretch or by readmission of Ca2+ in a non-depolarizing Ca2+-free salt solution. Stretch-induced tone was inhibited by staurosporine. When tissues were incubated in a Ca2+-free solution, staurosporine (50 nM) inhibited the contractile responses produced by readmission of Ca2+. These observations suggest that maintenance of stretch-induced extracellular Ca2+-dependent tone may be regulated by protein kinase C.     

Cloning and functional expression of human short TRP7, a candidate protein for store-operated Ca2+ influx.

The regulation and control of plasma membrane Ca(2+) fluxes is critical for the initiation and maintenance of a variety of signal transduction cascades. Recently, the study of transient receptor potential channels (TRPs) has suggested that these proteins have an important role to play in mediating capacitative calcium entry. In this study, we have isolated a cDNA from human brain that encodes a novel transient receptor potential channel termed human TRP7 (hTRP7). hTRP7 is a member of the short TRP channel family and is 98% homologous to mouse TRP7 (mTRP7). At the mRNA level hTRP7 was widely expressed in tissues of the central nervous system, as well as some peripheral tissues such as pituitary gland and kidney. However, in contrast to mTRP7, which is highly expressed in heart and lung, hTRP7 was undetectable in these tissues. For functional analysis, we heterologously expressed hTRP7 cDNA in an human embryonic kidney cell line. In comparison with untransfected cells depletion of intracellular calcium stores in hTRP7-expressing cells, using either carbachol or thapsigargin, produced a marked increase in the subsequent level of Ca(2+) influx. This increased Ca(2+) entry was blocked by inhibitors of capacitative calcium entry such as La(3+) and Gd(3+). Furthermore, transient transfection of an hTRP7 antisense expression construct into cells expressing hTRP7 eliminated the augmented store-operated Ca(2+) entry. Our findings suggest that hTRP7 is a store-operated calcium channel, a finding in stark contrast to the mouse orthologue, mTRP7, which is reported to enhance Ca(2+) influx independently of store depletion, and suggests that human and mouse TRP7 channels may fulfil different physiological roles.     

Cloning and characterization of human CADPS and CADPS2, new members of the Ca2+-dependent activator for secretion protein family

The recent identification of some of the components involved in regulated and constitutive exocytotic pathways has yielded important insights into the mechanisms of membrane trafficking and vesicle secretion. To understand precisely the molecular events taking place during vesicle exocytosis, we must identify all of the proteins implicated in these pathways. In this paper we describe the full-length cloning and characterization of human CADPS and CADPS2, two new homologs of the mouse Cadps protein involved in large dense-core vesicle (LDCV)-regulated exocytosis. We show that these two genes have disparate RNA expression patterns, with CADPS restricted to neural and endocrine tissues and CADPS2 expressed ubiquitously. We also identify a C2 domain, a known protein motif involved in calcium and phospholipid interactions, in both CADPS and CADPS2. We propose that CADPS functions as a calcium sensor in regulated exocytosis, whereas CADPS2 acts as a calcium sensor in constitutive vesicle trafficking and secretion. CADPS and CADPS2 were determined to span 475 kb and 561 kb on human chromosomes 3p21.1 and 7q31.3, respectively. The q31-q34 of human chromosome 7 has recently been identified to contain a putative susceptibility locus for autism (AUTS1). The function, expression profile, and location of CADPS2 make it a candidate gene for autism, and thus we conducted mutation screening for all 28 exons in 90 unrelated autistic individuals. We identified several nucleotide substitutions, including only one that would affect the amino acid sequence. No disease-specific variants were identified.     

Cloning,overexpression, and characterization of a bacterial Ca2+-dependent phospholipase D

Phospholipase D (PLD), an important enzyme involved in signal transduction in mammals, is also secreted by many microorganisms. A highly conserved HKD motif has been identified in most PLD homologs in the PLD superfamily. However, the Ca(2+)-dependent PLD from Streptomyces chromofuscus exhibits little homology to other PLDs. We have cloned (using DNA isolated from the ATCC type strain), overexpressed in Escherichia coli (two expression systems, pET-23a(+) and pTYB11), and purified the S. chromofuscus PLD. Based on attempts at sequence alignment with other known Ca(2+)-independent PLD enzymes from Streptomyces species, we mutated five histidine residues (His72, His171, His187, His200, His226) that could be part of variants of an HKD motif. Only H187A and H200A showed dramatically reduced activity. However, mutation of these histidine residues to alanine also significantly altered the secondary structure of PLD. Asparagine replacements at these positions yielded enzymes with structure and activity similar to the recombinant wild-type PLD. The extent of phosphatidic acid (PA) activation of PC hydrolysis by the recombinant PLD enzymes differed in magnitude from PLD purified from S. chromofuscus culture medium (a 2-fold activation rather than 4-5-fold). One of the His mutants, H226A, showed a 12-fold enhancement by PA, suggesting this residue is involved in the kinetic activation. Another notable difference of this bacterial PLD from others is that it has a single cysteine (Cys123); other Streptomyces Ca(2+)-independent PLDs have eight Cys involved in intramolecular disulfide bonds. Both C123A and C123S, with secondary structure and stability similar to recombinant wild-type PLD, exhibited specific activity reduced by 10(-5) and 10(-4). The Cys mutants still bound Ca(2+), so that it is likely that this residue is part of the active site of the Ca(2+)-dependent PLD. This would suggest that S. chromofuscus PLD is a member of a new class of PLD enzymes.     

Class-specific binding of two aminoacyl-tRNA synthetases to annexin, a Ca2+- and phospholipid-binding protein

Annexins are a family of Ca2+/phospholipid-binding proteins that have diverse functions. To understand the function of annexin in Physarum polycephalum, we searched for its binding proteins. Here we demonstrate the presence of two novel annexin-binding proteins. The homology search of partial amino acid sequences of these two proteins identified them as aminoacyl-tRNA synthetases (ARSs). Furthermore, antibody against aminoacyl-tRNA synthetases cross-reacted with one of two proteins. Our results imply the interaction between intracellular membrane dynamics and protein translation system, and may give a clue to understand the mechanism of some myositis diseases, which have been known to produce autoantibodies against ARSs.     

Isolation of a cDNA likely to encode a novel Ca2+-dependent/calmodulin-stimulated protein phosphatase

Complementary DNA encoding a novel protein phosphatase catalytic subunit has been isolated from a rabbit brain library. The deduced protein sequence is more similar to the major Ca2+-dependent/calmodulin-stimulated protein phosphatase (2B) in brain (55% identity) than to protein phosphatases 1 and 2A (38-39% identity). A putative calmodulin-binding domain is present C-terminal to the catalytic domain, which closely resembles that of the mouse brain enzyme. These findings represent the first indication that at least two distinct Ca2+-dependent/calmodulin-stimulated protein phosphatases are present in mammalian brain.     

Non-fusion expression in Escherichia coli, purification, and characterization of a novel Ca2+- and phospholipid-binding protein annexin B1

Annexin B1 is a novel member of the annexin family of Ca2+- and phospholipid-binding proteins from Cysticercus cellulosae. To obtain high quality annexin B1 for biochemical and biophysical analyses, its cDNA was cloned into the prokaryotic expression vector pJLA503 and the translation initiation codon was immediately under the control of the inducible bacteriophage lambda promoters P(R) and P(L). After induction by shifting temperature, large amounts of non-fusion protein were produced in Escherichia coli in a soluble form. The recombinant protein was purified to homogeneity by means of two subsequent ion-exchange chromatographic steps. The final yield was about 25 mg/L bacterial culture. Western blot analysis showed that recombinant annexin B1 was specifically recognized by serum of pigs infected with cysticercosis. Secondary structure predictions from circular dichroism spectroscopy indicated that alpha-helix is the main secondary structure of the protein. In anticoagulant assays, the recombinant non-fusion protein exhibited dose-dependent effects in modified kaolin partial thromboplastin time (KPTT) prolongation and doubled the clotting time of control human plasma at 60 microg/ml. The expression, purification, and initial characterization of annexin B1 set an important stage for further characterization of the protein.     

Isolation and characterization of three forms of 36-kDa Ca2+-dependent actin- and phospholipid-binding proteins from human placenta membrane

We purified three forms of 36-kDa proteins, two monomeric 36-kDa proteins, which had pIs of 7.5 (36K-I) and 6.4 (36K-II), and one 36-kDa complex (36K-C) consisting of two subunits, 36-kDa (pI 7.5) and 12-kDa (pI 5.8), from human placenta membrane. The 36-kDa subunit of 36K-C was identical to 36K-I as judged by pI, cyanogen bromide peptide mapping and immunological cross-reactivity. The three proteins showed F-actin- and phosphatidylserine-binding abilities dependent on Ca2+ concentrations at millimolar and micromolar levels, respectively. They all had phospholipase A2 inhibitory activity. Only 36K-II was phosphorylated extensively at tyrosine residue in Ca2+- and EGF- dependent manners in the membrane fraction of A431 cells. 36K-I was the best substrate for src kinase, whereas 36K-II was the best for fps kinase. However, 36K-C was not phosphorylated by any kinases used here.     

S100P, a novel Ca(2+)-binding protein from human placenta. cDNA cloning, recombinant protein expression and Ca2+ binding properties.

A novel member of the S100 protein family, present in human placenta, has been characterized by protein sequencing, cDNA cloning, and analysis of Ca(2+)-binding properties. Since the placenta protein of 95 amino acid residues shares about 50% sequence identity with the brain S100 proteins alpha and beta, we proposed the name S100P. The cDNA was expressed in Escherichia coli and recombinant S100P was purified in high yield. S100P is a homodimer and has two functional EF hands/polypeptide chain. The low-affinity site (Kd = 800 microM), which, in analogy to S100 beta, seems to involve the N-terminal EF hand, can be followed by the Ca(2+)-dependent decrease in tyrosine fluorescence. The high-affinity site, provided by the C-terminal EF hand, influences the reactivity of the sole cysteine which is located in the C-terminal extension (Cys85). Binding to the high-affinity site (Kd = 1.6 microM) can be monitored by fluorescence spectroscopy of S100P labelled at Cys85 with 6-proprionyl-2-dimethylaminonaphthalene (Prodan). The Prodan fluorescence shows a Ca(2+)-dependent red shift of the maximum emission wavelength from 485 nm to 502 nm, which is accompanied by an approximately twofold loss in integrated fluorescence intensity. This indicates that Cys85 becomes more exposed to the solvent in Ca(2+)-bound S100P, making this region of the molecule, the so-called C-terminal extension, an ideal candidate for a putative Ca(2+)-dependent interaction with a cellular target. In p11, a different member of the S100 family, the C-terminal extension which contains a corresponding cysteine (Cys82 in p11), is involved in a Ca(2+)-independent complex formation with the protein ligand annexin II. The combined results support the hypothesis that S100 proteins interact in general with their targets after a Ca(2+)-dependent conformational change which involves hydrophobic residues of the C-terminal extension.     

Ca2+-dependent binding of cytosolic components to insulin-secretory granules results in Ca2+-dependent protein phosphorylation

Interaction of Cu(II) and Gly-His-Lys, a growth-modulating tripeptide from plasma, was investigated by 13C- and 1H-n.m.r. and e.p.r. spectroscopy. The n.m.r. line-broadening was interpreted in terms of major and minor species formed as a function of pH. The results indicate that the n.m.r. line-broadening is due to the presence of minor species in rapid exchange and not due to the major species in solution, which has a large tau M. It is concluded that the technique of 13C- and 1H-n.m.r. line broadening, caused by paramagnetic Cu(II) ion, should be undertaken with caution, since the method may not be useful for obtaining structural information on the major species. The e.p.r. spectra over a wide pH range are almost entirely due to similarly co-ordinating species. Starting at pH 5.5, the narrowest absorption near 340 mT shows superhyperfine structure, which comes out sharply in the pH region 6.0-9.6. The spectra in this pH range showed the seven lines of nitrogen superhyperfine splitting, indicating clearly the co-ordination of three nitrogen atoms to Cu(II). The e.p.r. parameters in the medium pH range, A parallel = 19.5 mT and g parallel = 2.21, fit well with the contention that Cu(II) is ligated to Gly-His-Lys through one oxygen atom and three nitrogen atoms in a square-planar configuration.     

Phospholipid/Ca2+-dependent protein kinase activity in human parathyroid adenoma

We have examined the activities of phospholipid/Ca2+-dependent and cyclic AMP-dependent protein kinases of the parathyroid adenomas and the atrophic glands which were resected from three patients with primary hyperparathyroidism. Phospholipid/Ca2+-dependent protein kinase activity of atrophic parathyroid gland was exclusively present in cytosol fraction (90.7 +/- 12.3%). On the other hand, phospholipid/Ca2+-dependent protein kinase activity of parathyroid adenomas was 66.9 +/- 6.4% in cytosol and 33.1 +/- 6.4% in membrane fraction, suggesting a translocation of the enzyme from the cytosol to the membranes. Cyclic AMP-dependent protein kinase activity appeared to be higher in parathyroid adenoma than in atrophic parathyroid gland in both cytosol and membrane fractions.     

Cloning, expression and chromosomal localization of human Ca2+/calmodulin-dependent protein kinase kinase

A human cDNA clone encoding the calcium/calmodulin-dependent protein kinase kinase (CaMKK) was isolated by RT-PCR amplification of the fragment corresponding to the conserved kinase catalytic domain followed by rapid amplification of cDNA ends and cDNA library screening. Compilation of nucleotide sequencing data yielded a consensus cDNA sequence of 1.9 kb with an open reading frame of 1,251 nucleotides in length which translates to a polypeptide of 417 amino acids (47 kd). It showed significant homology to the rat brain CaMKK isozymes. The human CaMKK, which was expressed as a Flag-tagged protein in human non-small cell lung cancer H-1299 cells followed by immunoprecipitation with anti-Flag antibody, was shown to phosphorylate recombinant human CaMK I in a calcium/CaM-dependent fashion. Northern blot analysis revealed that human CaMKK is ubiquitously expressed, with brain showing the highest level of expression. The CaMKK gene is localized to human chromosome 12. The presence of cDNA clones with divergent 3' terminal sequences suggests a family of CaMKK variants which may arise from alternative splicing.     

Effect of L-alpha-phosphatidylinositol on a vascular smooth muscle Ca2+-dependent protease. Reduction of the Ca2+ requirement for autolysis

Vascular smooth muscle contains large amounts of a Ca2+-dependent protease. Similar to a Ca2+-dependent protease previously purified from chicken gizzard smooth muscle (Hathaway, D. R., Werth, D. K., and Haeberle, J. R. (1982) J. Biol. Chem. 257, 9072-9077), the mammalian vascular muscle protease is a heterodimer consisting of 76,000- and 30,000-dalton subunits (IIa). The enzyme can undergo autolysis in the presence of Ca2+ to produce a smaller species consisting of 76,000- and 18,000-dalton subunits (IIb). Autolysis greatly reduces the Ca2+ dependence of catalytic activity. The autolytic species, IIb, was approximately 23-fold more sensitive to Ca2+ (K0.5 = 39 microM) than the native enzyme, IIa (K0.5 = 891 microM). In this communication, we report that phosphatidylinositol and to a lesser extent one metabolic derivative, dioleoylglycerol, stimulate autolysis of the vascular Ca2+-dependent protease by reducing the Ca2+ for autolysis from K0.5 = 680 microM in the absence of lipid to K0.5 = 87 microM in the presence of both phosphatidylinositol and dioleoylglycerol. Moreover, the reduction in the Ca2+ requirement for autolysis produced by the phosphatidylinositol was antagonized by the phospholipid-binding drug, trifluoperazine. In addition, the effect of phosphatidylinositol was specific for autolysis, and none of several phospholipids or derivatives tested altered the Ca2+ dependence or maximal rate for protein degradation of the autolytic product, IIb. Our results suggest that autolysis may be an important initial step in the activation of the Ca2+-dependent protease in vascular smooth muscle and that this step may be regulated by a combination of Ca2+ and phosphatidylinositol.     

Cloning, sequencing, and expression of cDNA for human beta-galactosidase

We cloned and sequenced the full-length cDNA for human placental beta-galactosidase. The 2379-nucleotide sequence contains 2031 nucleotides which encode a protein of 677 amino acids. The amino acid sequence includes a putative signal sequence of 23 amino acids and 7 potential asparagine-linked glycosylation sites. The cDNA in the expression vector pSVL was used to transfect COS cells. Expression of the cDNA in transfected COS cells produced immunoprecipitable proteins and led to an increase in beta-galactosidase activity.