Cloning, characterization, and expression of calcyphosine 2, a novel human gene encoding an EF-hand Ca(2+)-binding protein

Calcyphosine is a calcium-binding protein containing four EF-hand domains, initially identified as thyroid protein p24. It was first cloned and its counterparts in rabbit, human, and mouse, crayfish and lobster of invertebrate were also cloned. Here we describe the cloning and characterization of a novel human calcyphosine gene. The 3829-bp cDNA encodes a EF-hand Ca(2+)-binding protein homologous to the dog calcyphosine. It also contains two EF-hand Ca(2+)-binding motif. It is abundantly expressed in many tissues including by RT-PCR analysis and believed to play important role in calcium signaling. It was mapped to human genome 12q15.     

Characterization and hetereologous expression of cDNAs encoding two novel closely related Ca(2+)-binding proteins in Dictyostelium discoideum

During a yeast two hybrid screen of a Dictyostelium cDNA library using the Ca(2+)-binding protein CBP1 as bait, we isolated a full-length cDNA encoding a novel Ca(2+)-binding protein (termed CBP4a). The protein is composed of 162 amino acids and contains four consensus EF-hands. PCR amplification of Dictyostelium genomic DNA using primers specific for the cDNA sequence resulted in the isolation of a gene encoding a different Ca(2+)-binding protein of 162 amino acids (designated CBP4b) with 90% amino acid sequence identity to CBP4a. Southern blot analysis confirmed the presence of two closely related genes in the Dictyostelium genome. CBP4a and CBP4b mRNAs are expressed at the same stages of development as CBP1 mRNA. In addition, both novel proteins bind (45)Ca(2+) and interact with CBP1 in vitro in a Ca(2+)-dependent manner.     

Ca2+-dependent binding of calcium-binding protein 1 to presynaptic group III metabotropic glutamate receptors and blockage by phosphorylation of the receptors

Presynaptic group III metabotropic glutamate receptors (mGluRs) and Ca(2+) channels are the main neuronal activity-dependent regulators of synaptic vesicle release, and they use common molecules in their signaling cascades. Among these, calmodulin (CaM) and the related EF-hand Ca(2+)-binding proteins are of particular importance as sensors of presynaptic Ca(2+), and a multiple of them are indeed utilized in the signaling of Ca(2+) channels. However, despite its conserved structure, CaM is the only known EF-hand Ca(2+)-binding protein for signaling by presynaptic group III mGluRs. Because the mGluRs and Ca(2+) channels reciprocally regulate each other and functionally converge on the regulation of synaptic vesicle release, the mGluRs would be expected to utilize more EF-hand Ca(2+)-binding proteins in their signaling. Here I show that calcium-binding protein 1 (CaBP1) bound to presynaptic group III mGluRs competitively with CaM in a Ca(2+)-dependent manner and that this binding was blocked by protein kinase C (PKC)-mediated phosphorylation of these receptors. As previously shown for CaM, these results indicate the importance of CaBP1 in signal cross talk at presynaptic group III mGluRs, which includes many molecules such as cAMP, Ca(2+), PKC, G protein, and Munc18-1. However, because the functional diversity of EF-hand calcium-binding proteins is extraordinary, as exemplified by the regulation of Ca(2+) channels, CaBP1 would provide a distinct way by which presynaptic group III mGluRs fine-tune synaptic transmission.     

Cloning, expression and characterization of a novel four EF-hand Ca(2+)-binding protein from olive pollen with allergenic activity

A novel allergenic member of the family of Ca(2+)-binding proteins has been cloned from olive tree pollen. The isolated DNA codes for a protein of 171 amino acid residues, which displays four EF-hand sequence motifs. The encoded protein was overproduced in Escherichia coli and purified. The protein (18? omitted?795 Da), which binds Ca(2+) and IgE antibodies from patients allergic to olive pollen, undergoes Ca(2+)-dependent conformational changes. It is retained on a phenyl-Sepharose column, which indicates the existence of regulatory EF-hand domains. This fact suggests its involvement in Ca(2+)-dependent signal transduction events of the pollen grain. This allergen could be considered as a member of a new subfamily of EF-hand Ca(2+)-binding proteins since it displays a low amino acid sequence similarity with the so far known proteins.     

Helper virus-independent trans-replication of hepatitis C virus-derived minigenome

In Paramecium, ciliary reversal is coupled with voltage-gated Ca(2+) channels on the ciliary membrane. We previously isolated a P. caudatum mutant, cnrC, with a malfunction of the Ca(2+) channels and discovered that the channel activity of cnrC was restored by transfection of the P. caudatum centrin (Pccentrin1p) gene, which encodes a member of the Ca(2+)-binding EF-hand protein family. In this study, we injected various mutated Pccentrin1p genes into cnrC and investigated whether these genes restore the Ca(2+) channel activity of cnrC. A Pccentrin1p mutant gene lacking Ca(2+) sensitivity of the third and fourth EF-hands lost the ability to restore the channel function of cnrC, and mutation of the fourth EF-hand caused more serious impairment than mutation of the third EF-hand. Moreover, a Pccentrin1p gene lacking the N-terminal 34-amino acid sequence also lost the ability to restore the channel activity. Native-PAGE analysis demonstrated that the N-terminal sequence is important for the Ca(2+)-dependent structural change of Pccentrin1p. These results demonstrate that Pccentrin1p Ca(2+)-dependently regulates the Ca(2+) channel activity in vivo.     

X-ray structures of the microglia/macrophage-specific protein Iba1 from human and mouse demonstrate novel molecular conformation change induced by calcium binding

The ionized calcium-binding adaptor molecule 1 (Iba1) with 147 amino acid residues has been identified as a calcium-binding protein, expressed specifically in microglia/macrophages, and is expected to be a key factor in membrane ruffling, which is a typical feature of activated microglia. We have determined the crystal structure of human Iba1 in a Ca(2+)-free form and mouse Iba1 in a Ca(2+)-bound form, to a resolution of 1.9 A and 2.1 A, respectively. X-ray structures of Iba1 revealed a compact, single-domain protein with two EF-hand motifs, showing similarity in overall topology to partial structures of the classical EF-hand proteins troponin C and calmodulin. In mouse Iba1, the second EF-hand contains a bound Ca(2+), but the first EF-hand does not, which is often the case in S100 proteins, suggesting that Iba1 has S100 protein-like EF-hands. The molecular conformational change induced by Ca(2+)-binding of Iba1 is different from that found in the classical EF-hand proteins and/or S100 proteins, which demonstrates that Iba1 has an unique molecular switching mechanism dependent on Ca(2+)-binding, to interact with target molecules.     

The novel murine Ca2+-binding protein,Scarf, is differentially expressed during epidermal differentiation

Calcium (Ca2+) signaling-dependent systems, such as the epidermal differentiation process, must effectively respond to variations in Ca2+ concentration. Members of the Ca2+-binding proteins play a central function in the transduction of Ca2+ signals, exerting their roles through a Ca2+-dependent interaction with their target proteins, spatially and temporally. By performing a suppression subtractive hybridization screen we identified a novel mouse gene, Scarf (skin calmodulin-related factor), which has homology to calmodulin (CaM)-like Ca2+-binding protein genes and is exclusively expressed in differentiating keratinocytes in the epidermis. The Scarf open reading frame encodes a 148-amino acid protein that contains four conserved EF-hand motifs (predicted to be Ca2+-binding domains) and has homology to mouse CaM, human CaM-like protein, hClp, and human CaM-like skin protein, hClsp. The functionality of Scarf EF-hand domains was assayed with a radioactive Ca2+-binding method. By Southern blot and computational genome sequence analysis, a highly related gene, Scarf2, was found 15 kb downstream of Scarf on mouse chromosome 13. The functional Scarf Ca2+-binding domains suggest a role in the regulation of epidermal differentiation through the control of Ca2+-mediated signaling.     

cDNA cloning of a calcineurin B homolog in Saccharomyces cerevisiae.

We have isolated a cDNA clone encoding a homolog of mammalian calcineurin B (the regulatory subunit of calmodulin-dependent protein phosphatase) by screening a cDNA expression library of Saccharomyces cerevisiae with antiserum against bovine calcineurin B. The yeast calcineurin B homolog (YCNB) is composed of 175 amino acids with a calculated molecular mass of 19,639 daltons and contains four putative Ca(2+)-binding domains. The amino-acid alignment of YCNB with human calcineurin B demonstrates 53% sequence identity and 82% homology. Southern blot analysis indicates that the gene for YCNB is a single-copy gene. Thus, yeast calmodulin-dependent protein phosphatase apparently has a heterodimeric structure similar to that of the enzyme in mammalians.     

LETM1, a novel gene encoding a putative EF-hand Ca(2+)-binding protein, flanks the Wolf-Hirschhorn syndrome (WHS) critical region and is deleted in most WHS patients.

Deletions within human chromosome 4p16.3 cause Wolf-Hirschhorn syndrome (WHS), which is characterized by severe mental and developmental defects. It is thought that haploinsufficiency of more than one gene contributes to the complex phenotype. We have cloned and characterized a novel gene (LETM1) that is deleted in nearly all WHS patients. LETM1 encodes a putative member of the EF-hand family of Ca(2+)-binding proteins. The protein contains two EF-hands, a transmembrane domain, a leucine zipper, and several coiled-coil domains. On the basis of its possible Ca(2+)-binding property and involvement in Ca(2+) signaling and/or homeostasis, we propose that haploinsufficiency of LETM1 may contribute to the neuromuscular features of WHS patients.     

Purification, properties, and molecular cloning of a novel Ca(2+)-binding protein in radish vacuoles

To understand the roles of plant vacuoles, we have purified and characterized a major soluble protein from vacuoles of radish (Raphanus sativus cv Tokinashi-daikon) taproots. The results showed that it is a novel radish vacuole Ca(2+)-binding protein (RVCaB). RVCaB was released from the vacuolar membrane fraction by sonication, and purified by ion exchange and gel filtration column chromatography. RVCaB is an acidic protein and migrated on sodium dodecyl sulfate-polyacrylamide gel with an apparent molecular mass of 43 kD. The Ca(2+)-binding activity was confirmed by the (45)Ca(2+)-overlay assay. RVCaB was localized in the lumen, as the protein was recovered in intact vacuoles prepared from protoplasts and was resistant to trypsin digestion. Plant vacuoles store Ca(2+) using two active Ca(2+) uptake systems, namely Ca(2+)-ATPase and Ca(2+)/H(+) antiporter. Vacuolar membrane vesicles containing RVCaB accumulated more Ca(2+) than sonicated vesicles depleted of the protein at a wide range of Ca(2+) concentrations. A cDNA (RVCaB) encoding a 248-amino acid polypeptide was cloned. Its deduced sequence was identical to amino acid sequences obtained from several peptide fragments of the purified RVCaB. The deduced sequence is not homologous to that of other Ca(2+)-binding proteins such as calreticulin. RVCaB has a repetitive unique acidic motif, but not the EF-hand motif. The recombinant RVCaB expressed in Escherichia coli-bound Ca(2+) as evidenced by staining with Stains-all and migrated with an apparent molecular mass of 44 kD. These results suggest that RVCaB is a new type Ca(2+)-binding protein with high capacity and low affinity for Ca(2+) and that the protein could function as a Ca(2+)-buffer and/or Ca(2+)-sequestering protein in the vacuole.     

Prediction of EF-hand calcium-binding proteins and analysis of bacterial EF-hand proteins

The EF-hand protein with a helix-loop-helix Ca(2+) binding motif constitutes one of the largest protein families and is involved in numerous biological processes. To facilitate the understanding of the role of Ca(2+) in biological systems using genomic information, we report, herein, our improvement on the pattern search method for the identification of EF-hand and EF-like Ca(2+)-binding proteins. The canonical EF-hand patterns are modified to cater to different flanking structural elements. In addition, on the basis of the conserved sequence of both the N- and C-terminal EF-hands within S100 and S100-like proteins, a new signature profile has been established to allow for the identification of pseudo EF-hand and S100 proteins from genomic information. The new patterns have a positive predictive value of 99% and a sensitivity of 96% for pseudo EF-hands. Furthermore, using the developed patterns, we have identified zero pseudo EF-hand motif and 467 canonical EF-hand Ca(2+) binding motifs with diverse cellular functions in the bacteria genome. The prediction results imply that pseudo EF-hand motifs are phylogenetically younger than canonical EF-hand motifs. Our prediction of Ca(2+) binding motifs provides not only an insight into the role of Ca(2+) and Ca(2+)-binding proteins in bacterial systems, but also a way to explore and define the role of Ca(2+) in other biological systems (calciomics).     

Ca2+-myristoyl switch in the neuronal calcium sensor recoverin requires different functions of Ca2+-binding sites

Recoverin is an EF-hand Ca(2+)-binding protein that is suggested to control the activity of the G-protein-coupled receptor kinase GRK-1 or rhodopsin kinase in a Ca(2+)-dependent manner. It undergoes a Ca(2+)-myristoyl switch when Ca(2+) binds to EF-hand 2 and 3. We investigated the mechanism of this switch by the use of point mutations in EF-hand 2 (E85Q) and 3 (E121Q) that impair their Ca(2+) binding. EF-hand 2 and 3 display different properties and serve different functions. Binding of Ca(2+) to recoverin is a sequential process, wherein EF-hand 3 is occupied first followed by the filling of EF-hand 2. After EF-hand 3 bound Ca(2+), the subsequent filling of EF-hand 2 triggers the exposition of the myristoyl group and in turn binding of recoverin to membranes. In addition, EF-hand 2 controls the mean residence time of recoverin at membranes by decreasing the dissociation rate of recoverin from membranes by 10-fold. We discuss this mechanism as one critical step for inhibition of rhodopsin kinase by recoverin.     

Molecular Cloning of Testican-2

We have screened a human cDNA library using an expressed sequence tag related to the BM-40/secreted protein, acidic and rich in cysteine (SPARC)/osteonectin family of proteins and isolated a novel cDNA. It encodes a protein precursor of 424 amino acids that consists of a signal peptide, a follistatin-like domain, a Ca2+-binding domain, a thyroglobulin-like domain, and a C-terminal region with two putative glycosaminoglycan attachment sites. The protein is homologous to testican-1 and was termed testican-2. Testican-1 is a proteoglycan originally isolated from human seminal plasma that is also expressed in brain. Northern blot hybridization of testican-2 showed a 6.1-kb mRNA expressed mainly in CNS but also found in lung and testis. A widespread expression in multiple neuronal cell types in olfactory bulb, cerebral cortex, thalamus, hippocampus, cerebellum, and medulla was detected by in situ hybridization. A recombinant fragment consisting of the Ca2+-binding EF-hand domain and the thyroglobulin-like domain of testican-2 showed a reversible Ca2+-dependent conformational change in circular dichroism studies. Testican-1 and -2 form a novel Ca2+-binding proteoglycan family built of modular domains with the potential to participate in diverse steps of neurogenesis.     

Centrin is essential for the activity of the ciliary reversal-coupled voltage-gated Ca2+ channels

Voltage-gated Ca(2+) channels play a critical role in controlling Ca(2+) entry in various cells. Ciliary reversal in Paramecium depends on the Ca(2+) influx through voltage-gated Ca(2+) channels on the ciliary membrane. One of the voltage-gated Ca(2+) channel mutants in Paramecium caudatum, cnrC, neither produces Ca(2+) action potentials nor responds to any depolarizing stimuli. Here, we report that the cnrC(+) gene product is P. caudatum centrin (Pccentrin1p), a member of the Ca(2+)-binding EF-hand protein superfamily. The Pccentrin1p gene of cnrC was found to contain a single-base deletion, a mutation that caused the loss of the fourth EF-hand of Pccentrin1p. Moreover, the wild-type Ca(2+) channel function was impaired by Pccentrin1p gene silencing, leading to the loss of current-evoked Ca(2+) action potentials and stimulated ciliary reversal. These results demonstrate that Pccentrin1p is indispensable for the activity of the voltage-gated Ca(2+) channels that control ciliary reversal in Paramecium.     

Characterization of tescalcin, a novel EF-hand protein with a single Ca2+-binding site: metal-binding properties, localization in tissues and cells, and effect on calcineurin

The tescalcin gene is preferentially expressed during mouse testis differentiation. Here, we demonstrate that this gene encodes a 24 kDa Ca(2+)- and Mg(2+)-binding protein with one consensus EF-hand and three additional domains with EF-hand homology. Equilibrium dialysis with (45)Ca(2+) revealed that recombinant tescalcin binds approximately one Ca(2+) ion at physiological concentrations (pCa 4.5). The intrinsic tryptophan fluorescence of tescalcin was significantly reduced by Ca(2+), indicative of a conformational change. The apparent K(d) for Ca(2+) was 0.8 microM. A point mutation in the consensus EF-hand (D123A) abolished (45)Ca(2+) binding and prevented the fluorescence quenching, demonstrating that the consensus EF-hand alone mediates the Ca(2+)-induced conformational change. Tescalcin also binds Mg(2+) (K(d) 73 microM), resulting in a much smaller fluorescence decrease. In the presence of 1 mM Mg(2+), tescalcin's Ca(2+) affinity is shifted to 3.5 microM. These results illustrate that tescalcin should bind Mg(2+) constitutively in a quiescent cell, replacing it with Ca(2+) during stimulation. We also show that tescalcin is most abundant in adult mouse heart, brain, and stomach, as well as in HeLa and HL-60 cells. Immunofluorescence microscopy revealed that tescalcin is present in the cytoplasm and nucleus, with concentration in membrane ruffles and lamellipodia in the presence of serum, where it colocalizes with the small guanosine triphosphatase Rac-1. Tescalcin shares sequence and functional homology with calcineurin-B homologous protein (CHP), and we found that tescalcin, like CHP, can inhibit the phosphatase activity of calcineurin A. Hence, tescalcin is a novel calcineurin B-like protein that binds a single Ca(2+) ion.     

Calcium and lanthanide affinity of the EF-loops from the C-terminal domain of calmodulin

To obtain site-specific information about individual EF-hand motifs, the EF-hand Ca(2+)-binding loops from site III and site IV of calmodulin (CaM) were inserted separately into a non-Ca(2+)-binding cell adhesion protein, domain 1 of CD2 (denoted as CaM-CD2-III-5G-52 and CaM-CD2-IV-5G-52). Structural analyses using various spectroscopic methods have shown that the host protein CD2 retains its native structure after the insertion of the 12-residue loops. The Tb(3+) fluorescence enhancement upon formation of a Tb(3+)-protein complex and the direct competition by La(3+) and Ca(2+) suggest that native Ca(2+)-binding pockets are formed in both engineered proteins. Moreover, as revealed by NMR, both Ca(2+) and La(3+) specifically interact with the residues at the grafted EF-loop. The CaM-CD2-III-5G-52 has stronger affinities to Ca(2+), Tb(3+) and La(3+) than CaM-CD2-IV-5G-52, indicating differential intrinsic metal-binding affinities of the EF-loops.     

Low-affinity Ca(2+)-binding sites versus Zn(2+)-binding sites in histidine-rich Ca(2+)-binding protein of skeletal muscle sarcoplasmic reticulum.

Histidine-rich Ca(2+)-binding protein (HRC) is a 170 kDa protein that can be identified in the isolated sarcoplasmic reticulum from rabbit skeletal muscle by its ability to bind [125I]low-density lipoprotein on blots after SDS-PAGE and that appears to be bound to the junctional membrane through calcium bridges. Molecular cDNA cloning of this protein predicts the existence of a Ca(2+)-binding domain and of a distinct heavy-metal binding domain at the cystein-rich COOH-terminus. Here we demonstrate, using radioactive ligand blot techniques, that HRC protein binds 45Ca at low affinity, as well as being able to bind 65Zn, but at different sites, that are largely inhibitable by prior reductive alkylation of the protein. In contrast to Ca(2+)-binding protein calsequestrin not having detectable 65Zn-binding sites, HRC protein bound selectively to immobilized Zn2+ on IDA-agarose affinity columns. Our results also indicate that rabbit and human 140 kDa HRC protein have common properties.