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.     

Five members of a novel Ca(2+)-binding protein (CABP) subfamily with similarity to calmodulin

Five members of a novel Ca(2+)-binding protein subfamily (CaBP), with 46-58% sequence similarity to calmodulin (CaM), were identified in the vertebrate retina. Important differences between these Ca(2+)-binding proteins and CaM include alterations within their second EF-hand loop that render these motifs inactive in Ca(2+) coordination and the fact that their central alpha-helixes are extended by one alpha-helical turn. CaBP1 and CaBP2 contain a consensus sequence for N-terminal myristoylation, similar to members of the recoverin subfamily and are fatty acid acylated in vitro. The patterns of expression differ for each of the various members. Expression of CaBP5, for example, is restricted to retinal rod and cone bipolar cells. In contrast, CaBP1 has a more widespread pattern of expression. In the brain, CaBP1 is found in the cerebral cortex and hippocampus, and in the retina this protein is found in cone bipolar and amacrine cells. CaBP1 and CaBP2 are expressed as multiple, alternatively spliced variants, and in heterologous expression systems these forms show different patterns of subcellular localization. In reconstitution assays, CaBPs are able to substitute functionally for CaM. These data suggest that these novel CaBPs are an important component of Ca(2+)-mediated cellular signal transduction in the central nervous system where they may augment or substitute for CaM.     

Interaction of HRC (histidine-rich Ca(2+)-binding protein) and triadin in the lumen of sarcoplasmic reticulum

HRC (histidine-rich Ca(2+) binding protein) has been identified from skeletal and cardiac muscle and shown to bind Ca(2+) with high capacity and low affinity. While HRC resides in the lumen of the sarcoplasmic reticulum, the physiological function of HRC is largely unknown. In the present study, we have performed co-immunoprecipitation experiments and show that HRC binds directly to triadin, which is an integral membrane protein of the sarcoplasmic reticulum. Using a fusion protein binding assay, we further identified the histidine-rich acidic repeats of HRC as responsible for the binding of HRC to triadin. These motifs may represent a novel protein-protein interaction domain. The HRC binding domain of triadin was also localized by fusion protein binding assay to the lumenal region containing the KEKE motif that was previously shown to be involved in the binding of triadin to calsequestrin. Notably, the interaction of HRC and triadin is Ca(2+)-sensitive. Our data suggest that HRC may play a role in the regulation of Ca(2+) release from the sarcoplasmic reticulum by interaction with triadin.     

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.     

Identification of a novel interaction between the Ca(2+)-binding protein S100A11 and the Ca(2+)- and phospholipid-binding protein annexin A6

S100A11 is a member of the S100 family of EF-hand Ca²⁺-binding proteins, which is expressed in smooth muscle and other tissues. Ca²⁺ binding to S100A11 induces a conformational change that exposes a hydrophobic surface for interaction with target proteins. Affinity chromatography with immobilized S100A11 was used to isolate a 70-kDa protein from smooth muscle that bound to S100A11 in a Ca²⁺-dependent manner and was identified by mass spectrometry as annexin A6. Direct Ca²⁺-dependent interaction between S100A11 and annexin A6 was confirmed by affinity chromatography of the purified bacterially expressed proteins, by gel overlay of annexin A6 with purified S100A11, by chemical cross-linking, and by coprecipitation of S100A11 with annexin A6 bound to liposomes. The expression of S100A11 and annexin A6 in the same cell type was verified by RT-PCR and immunocytochemistry of isolated vascular smooth muscle cells. The site of binding of S100A11 on annexin A6 was investigated by partial tryptic digestion and deletion mutagenesis. The unique NH₂ terminal head region of annexin A6 was not required for S100A11 binding, but binding sites were identified in both NH₂- and COOH-terminal halves of the molecule. We hypothesize that an agonist-induced increase in cytosolic free [Ca²⁺] leads to formation of a complex of S100A11 and annexin A6, which forms a physical connection between the plasma membrane and the cytoskeleton, or plays a role in the formation of signaling complexes at the level of the sarcolemma. smooth muscle; protein-protein interaction     

cDNA cloning of a neural visinin-like Ca(2+)-binding protein.

A 21,000-dalton Ca(2+)-binding protein (Walsh, M.P., Valentine, K.A., Ngai, P.K., Carruthers, C.A., and Hollengerg, M.D. (1984) Biochem. J. 224, 117-127) was purified from the rat brain and through the use of oligonucleotide probe based on partial amino acid sequence, cDNA clones were obtained from rat brain cDNA library. The complete amino acid sequence deduced from the cDNA contains 191 residues and has a calculated molecular mass of 22,142 daltons. There are three potential Ca(2+)-binding sites like the EF hands in the sequence. It displays striking sequence homology with visinin and recoverin, retina-specific Ca(2+)-binding proteins. Northern blot analysis revealed that the protein is highly and specifically expressed in the brain.     

Ca(2+)-dependent interaction of triadin with histidine-rich Ca(2+)-binding protein carboxyl-terminal region.

A direct binding of HRC (histidine-rich Ca(2+)-binding protein) to triadin, the main transmembrane protein of the junctional sarcoplasmic reticulum (SR) of skeletal muscle, seems well supported. Opinions are still divided, however, concerning the triadin domain involved, either the cytoplasmic or the lumenal domain, and the exact role played by Ca(2+), in the protein-to-protein interaction. Further support for colocalization of HRC with triadin cytoplasmic domain is provided here by experiments of mild tryptic digestion of tightly sealed TC vesicles. Accordingly, we show that HRC is preferentially phosphorylated by endogenous CaM K II, anchored to SR membrane on the cytoplasmic side, and not by lumenally located casein kinase 2. We demonstrate that HRC can be isolated as a complex with triadin, following equilibrium sucrose-density centrifugation in the presence of mM Ca(2+). Here, we characterized the COOH-terminal portion of rabbit HRC, expressed and purified as a fusion protein (HRC(569-852)), with respect to Ca(2+)-binding properties, and to the interaction with triadin on blots, as a function of the concentration of Ca(2+). Our results identify the polyglutamic stretch near the COOH terminus, as the Ca(2+)-binding site responsible, both for the acceleration in mobility of HRC on SDS-PAGE in the presence of millimolar concentrations of Ca(2+), and for the enhancement by high Ca(2+) of the interaction between HRC and triadin cytoplasmic segment. (c)2001 Elsevier Science.     

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.     

Crystal structure of the Ca(2+)-form and Ca(2+)-binding kinetics of metastasis-associated protein, S100A4

S100A4 takes part in control of tumour cell migration and contributes to metastatic spread in in vivo models. In the active dimeric Ca(2+)-bound state it interacts with multiple intracellular targets. Conversely, oligomeric forms of S100A4 are linked with the extracellular function of this protein. We report the 1.5A X-ray crystal structure of Ca(2+)-bound S100A4 and use it to identify the residues involved in target recognition and to derive a model of the oligomeric state. We applied stopped-flow analysis of tyrosine fluorescence to derive kinetics of S100A4 activation by Ca(2+) (k(on)=3.5 microM(-1)s(-1), k(off)=20s(-1)).     

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.     

Ca(2+) signalling in the Golgi apparatus

The Golgi apparatus plays a central role in lipid and protein post-translational modification and sorting. Morphologically the organelle is heterogeneous and it is possible to distinguish stacks of flat cysternae (cis- and medial Golgi), tubular-reticular networks and vesicles (trans-Golgi). These morphological differences parallel a distinct functionality with a selective distribution and complementary roles of the enzymes found in the different compartments.The Golgi apparatus has been also shown to be involved in Ca²⁺ signalling: it is indeed endowed with Ca²⁺ pumps, Ca²⁺ release channels and Ca²⁺ binding proteins and is thought to participate in determining the spatio-temporal complexity of the Ca²⁺ signal within the cell, though this role is still poorly understood.Recently, it has been demonstrated that the organelle is heterogeneous in terms of Ca²⁺ handling and selective reduction of Ca²⁺ concentration, both in vitro and in a genetic human disease, within one of its sub-compartment results in alterations of protein trafficking within the secretory pathway and of the entire Golgi morphology.In this paper we review the available information on the Ca²⁺ toolkit within the Golgi, its heterogeneous distribution in the organelle sub-compartments and discuss the implications of these characteristics for the physiopathology of the Golgi apparatus.     

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.     

Detection of Ca(2+)-binding proteins by electrophoretic migration in the presence of Ca2+ combined with 45Ca2+ overlay of protein blots.

When high affinity Ca(2+)-binding proteins like calmodulin, or proteins with a high Ca(2+)-binding capacity like calsequestrin, underwent sodium dodecyl sulfate-gel electrophoresis in Laemmli systems, their electrophoretic migration rates were much higher in gels containing 1 mM Ca2+ than in gels containing ethylene glycol bis(beta-aminoethyl ether) N,N'-tetraacetic acid (EGTA). Replacement of EGTA by Ca2+ in the gel, combined with the blotting of electrophoretically separated proteins on polyvinylidene difluoride membranes and subsequent 45Ca2+ overlay, proved a very effective means of detecting Ca(2+)-binding proteins. This combined approach is important since artifacts occur in both techniques when used separately. We found that the usual procedure of adding Ca2+ to the sample before electrophoresis without including it in the gel itself (C.B. Klee, T. H. Crouch, and M. H. Krinks, 1979, Proc. Natl. Acad. Sci. USA 76, 6270-6273) permitted the detection of only very high affinity Ca(2+)-binding proteins.     

Intralumenal sarcoplasmic reticulum Ca(2+)-binding proteins

The sarcoplasmic reticulum (SR) controls the level of intracellular Ca2+ in cardiac and skeletal muscle by storing and releasing Ca2+. A set of intralumenal SR Ca(2+)-binding proteins has been identified that may serve important roles in SR Ca2+ storage and mobilization. The most prominent of these SR proteins, calsequestrin, is discretely localized to junctional SR. Other intralumenal proteins are more widely distributed throughout the SR. All of these intralumenal SR Ca(2+)-binding proteins are acidic, stain blue with dye Stains-All, and appear to be substrates for casein kinase II. The biochemistry and cell biology of lumenal SR proteins may conform to a paradigm now emerging from the study of endoplasmic reticulum proteins.     

Calumin, a novel Ca2+-binding transmembrane protein on the endoplasmic reticulum

We have identified a novel endoplasmic reticulum (ER)-resident protein, named "calumin", which is expressed in various tissues. This protein has a molecular mass of approximately 60 kDa and is composed of an ER-luminal domain rich in acidic residues, a single transmembrane segment, and a large cytoplasmic domain. Biochemical experiments demonstrated that the amino-terminal luminal domain is capable of binding Ca2+ with a high capacity and moderate affinity. In embryonic fibroblasts derived from calumin-knockout mice exhibiting embryonic and neonatal lethality, fluorometric Ca2+ imaging detected insufficient Ca2+ contents in intracellular stores and attenuated store-operated Ca2+ entry. Moreover, the mutant fibroblasts were highly sensitive to cell death induced by ER stress. These observations suggest that calumin plays an essential role in ER Ca2+ handling and is also implicated in signaling from the ER, which is closely associated with cell-fate decision.     

Calnuc,an EF-hand Ca(2+)-binding protein,is stored and processed in the Golgi and secreted by the constitutive-like pathway in AtT20 cells

Calnuc is an ubiquitous, EF-hand Ca(2+) binding protein found in the cytoplasm where it binds to Galphai3, in the Golgi lumen where it constitutes a Ca(2+) storage pool, and secreted outside the cell. Here we investigated the pathway of secretion of calnuc in AtT20 cells. We found by pulse-chase experiments that calnuc is synthesized in the endoplasmic reticulum, transported to the Golgi where it remains greater than 12 h and undergoes posttranslational modification (O-glycosylation and sulfation) followed by secretion into the culture medium. We examined if calnuc is secreted by the constitutive or regulated secretory pathway in AtT20 cells. By immunofluorescence and immunogold labeling, endogenous calnuc is found in immature secretion granules (ISG) but not mature regulated secretory granules (RSG), whereas overexpressed calnuc-green fluorescent protein (GFP) is found in both ISG and RSG, where it colocalizes with ACTH. Neither calnuc nor calnuc-GFP are released by the regulated secretory pathway, suggesting that endogenous calnuc and calnuc-GFP are progressively removed from ISG and RSG during granule maturation. We conclude that calnuc is secreted via the constitutive-like pathway and represents a useful endogenous marker for this pathway in AtT20 cells. Together, these observations indicate that calnuc has a unique itinerary as it is retained in the Golgi and is then constitutively secreted extracellularly where it may influence cell behavior via its Ca(2+)-binding properties.     

Annexins--a new family of Ca(2+)-binding proteins

Annexins, the Ca(2+)- and phospholipid-binding proteins, are able to induce Ca(2+)-dependent aggregation of biomembranes. All the representatives of this family contain four or eight tandem repeats, 60-80 amino acids each. All these repeats include a highly conservative 17-member amino acid consensus sequence (an endonexin fold). The central domain comprises all these repeats and contains, in addition, the site(s) with a binding affinity for Ca2+ and phospholipids. Annexins are devoid of the classical "EF-hand" Ca(2+)-binding domain and can therefore be assigned to a new family of Ca(2+)-binding proteins.     

Characterization of a Listeria monocytogenes Ca(2+) pump: a SERCA-type ATPase with only one Ca(2+)-binding site

We have characterized a putative Ca(2+)-ATPase from the pathogenic bacterium Listeria monocytogenes with the locus tag lmo0841. The purified and detergent-solubilized protein, which we have named Listeria monocytogenes Ca(2+)-ATPase 1 (LMCA1), performs a Ca(2+)-dependent ATP hydrolysis and actively transports Ca(2+) after reconstitution in dioleoylphosphatidyl-choline vesicles. Despite a high sequence similarity to the sarcoplasmic reticulum Ca(2+)-ATPase (SERCA1a) and plasma membrane Ca(2+)-ATPase (PMCA), LMCA1 exhibits important biochemical differences such as a low Ca(2+) affinity (K(0.5) ～80 μm) and a high pH optimum (pH ～9). Mutational studies indicate that the unusually high pH optimum can be partially ascribed to the presence of an arginine residue (Arg-795), corresponding in sequence alignments to the Glu-908 position at Ca(2+) binding site I of rabbit SERCA1a, but probably with an exposed position in LMCA1. The arginine is characteristic of a large group of putative bacterial Ca(2+)-ATPases. Moreover, we demonstrate that H(+) is countertransported with a transport stoichiometry of 1 Ca(2+) out and 1 H(+) in per ATP hydrolyzed. The ATPase may serve an important function by removing Ca(2+) from the microorganism in environmental conditions when e.g. stressed by high Ca(2+) and alkaline pH.