The Mammalian Calcium-binding Protein, Nucleobindin (CALNUC), Is a Golgi Resident Protein

We have identified CALNUC, an EF-hand, Ca²⁺-binding protein, as a Golgi resident protein. CALNUC corresponds to a previously identified EF-hand/calcium-binding protein known as nucleobindin. CALNUC interacts with Gα_i3 subunits in the yeast two-hybrid system and in GST-CALNUC pull-down assays. Analysis of deletion mutants demonstrated that the EF-hand and intervening acidic regions are the site of CALNUC's interaction with Gα_i3. CALNUC is found in both cytosolic and membrane fractions. The membrane pool is tightly associated with the luminal surface of Golgi membranes. CALNUC is widely expressed, as it is detected by immunofluorescence in the Golgi region of all tissues and cell lines examined. By immunoelectron microscopy, CALNUC is localized to cis-Golgi cisternae and the cis-Golgi network (CGN). CALNUC is the major Ca²⁺-binding protein detected by ⁴⁵Ca²⁺-binding assay on Golgi fractions. The properties of CALNUC and its high homology to calreticulin suggest that it may play a key role in calcium homeostasis in the CGN and cis-Golgi cisternae.     

The divergent domains of the NEFA and nucleobindin proteins are derived from an EF-hand ancestor

The human protein NEFA (DNA binding, EF-hand, Acidic region) has previously been isolated from a KM3 cell line and immunolocalized on the plasma membrane, in the cytoplasma, and in the culture medium. Sequence analysis of a cDNA clone encoding NEFA identified a hydrophilic domain, two EF-hands, and a leucine zipper at the C- terminus. These characters are shared with nucleobindin (Nuc). In this paper we have further characterized NEFA and probed its evolutionary origins. Circular dichroism (CD) spectra of recombinant NEFA indicated a helical content of 51% and showed that the EF-hands are capable of binding Ca2+. Experiments with recombinant NEFA and synthesized peptides revealed that the leucine zipper cannot form a homodimer. The leucine zipper may allow heterodimer formation of NEFA and an unknown protein. Phylogenetic analyses suggest that this protein is derived from a four-domain EF-hand ancestor with subsequent duplications and fusions. The leucine zipper and putative DNA-binding domains of NEFA have evolved secondarily from existing EF-hand sequences. These analyses provide insights into how complex proteins may originate and trace the precursor of NEFA to the common ancestor of eukaryotes.      

Overexpression of CALNUC (nucleobindin) increases agonist and thapsigargin releasable Ca2+ storage in the Golgi

We previously demonstrated that CALNUC, a Ca2+-binding protein with two EF-hands, is the major Ca2+-binding protein in the Golgi by 45Ca2+ overlay (Lin, P., H. Le-Niculescu, R. Hofmeister, J.M. McCaffery, M. Jin, H. Henneman, T. McQuistan, L. De Vries, and M. Farquhar. 1998. J. Cell Biol. 141:1515-1527). In this study we investigated CALNUC's properties and the Golgi Ca2+ storage pool in vivo. CALNUC was found to be a highly abundant Golgi protein (3.8 microg CALNUC/mg Golgi protein, 2.5 x 10(5) CALNUC molecules/NRK cell) and to have a single high affinity, low capacity Ca2+-binding site (Kd = 6.6 microM, binding capacity = 1.1 micromol Ca2+/micromol CALNUC). 45Ca2+ storage was increased by 2.5- and 3-fold, respectively, in HeLa cells transiently overexpressing CALNUC-GFP and in EcR-CHO cells stably overexpressing CALNUC. Deletion of the first EF-hand alpha helix from CALNUC completely abolished its Ca2+-binding capability. CALNUC was correctly targeted to the Golgi in transfected cells as it colocalized and cosedimented with the Golgi marker, alpha-mannosidase II (Man II). Approximately 70% of the 45Ca2+ taken up by HeLa and CHO cells overexpressing CALNUC was released by treatment with thapsigargin, a sarcoplasmic/endoplasmic reticulum calcium ATPase (SERCA) (Ca2+ pump) blocker. Stimulation of transfected cells with the agonist ATP or IP3 alone (permeabilized cells) also resulted in a significant increase in Ca2+ release from Golgi stores. By immunofluorescence, the IP3 receptor type 1 (IP3R-1) was distributed over the endoplasmic reticulum and codistributed with CALNUC in the Golgi. These results provide direct evidence that CALNUC binds Ca2+ in vivo and together with SERCA and IP3R is involved in establishment of the agonist-mobilizable Golgi Ca2+ store.     

Golgi retention of human protein NEFA is mediated by its N-terminal Leu/Ile-rich region.

The subcellular localization of the human Ca(2+)-binding EF-hand/leucine zipper protein NEFA was studied in HeLa cells by immunofluorescence microscopy. Double immunostaining using mouse anti-NEFA monoclonal antibody 1H8D12 and rabbit anti-ERD2 polyclonal antibody proved that NEFA is localized in the Golgi apparatus. The result was confirmed by the expression of NEFA-green fluorescent protein (GFP) fusion protein in the Golgi in the same cell line. Cycloheximide treatment proved NEFA to be a Golgi-resident protein. Seven NEFA deletion mutants were constructed to ascertain the peptide region relevant for Golgi retention. The expression of each NEFA-GFP variant was detected by fluorescence microscopy and immunoblotting. Only the DeltaN mutant, lacking the N-terminal Leu/Ile-rich region, failed to be retained in the Golgi after cycloheximide treatment. The other six deletion mutants in which either the basic region, the complete EF-hand pair domain, the two EF-hand motifs separately, the leucine zipper and the leucine zipper plus the C-terminal region is deleted, were localized to the Golgi. The peptide sequence within the Leu/Ile-rich region is discussed as a novel Golgi retention motif.     

Cloning and Expression of a cDNA Encoding a Vorticella convallaria Spasmin: an EF-Hand Calcium-Binding Protein

The stalked, ciliated protozoan Vorticella convallaria possesses a highly contractile cytoskeleton consisting of spasmonemes and myonemes. The major component of these contractile organelles is the calcium-binding protein(s) called spasmin. Cloning and characterization of spasmin would help elucidate this contractile system. Therefore, enriched spasmoneme protein preparations from these contractile stalks were used to produce a monoclonal antibody to spasmin. A monoclonal antibody, 1F5, was obtained that immunolocalized specifically to the spasmonemes and the myonemes and recognized a 20-kD calcium-binding protein in spasmoneme protein preparations. A putative spasmin cDNA was obtained from a V. convallaria cDNA library and the derived amino acid sequence of this cDNA revealed an acidic, 20-kD protein with calcium-binding helix-loop-helix domains. The physical properties of the putative spasmin were assessed by characterization of a recombinantly-produced spasmin protein. The recombinant spasmin protein was shown to bind calcium using calcium gel-shift assays and was recognized by the anti-spasmin antibody. Therefore, a V. convallaria spasmin was cloned and shown to be a member of the EF-hand superfamily of calcium-binding proteins.     

The third calmodulin family protein in Tetrahymena. Cloning of the cDNA for Tetrahymena calcium-binding protein of 23 kDa (TCBP-23)

Recently, we proved the existence of the second calmodulin family protein in Tetrahymena (Tetrahymena calcium-binding protein of 25 kDa, TCBP-25) by analyzing its cDNA (Takemasa, T., Ohnishi, K., Kobayashi, T., Takagi, T., Konishi, K., and Watanabe, Y. (1989) J. Biol. Chem. 264, 19293-19301). During the amino acid sequence determination of TCBP-25, we became aware of the fact that another polypeptide carrying calcium-binding domains of EF-hand type existed in addition to Tetrahymena calmodulin and TCBP-25. This third calmodulin family protein from Tetrahymena was confirmed by isolating its cDNA clones. One of the cloned cDNAs contains 763 nucleotides and encodes a protein that is composed of 207 amino acid residues and has a molecular mass of 23,413 daltons. This predicted protein possesses four EF-hand type calcium-binding domains, so we have designated it as Tetrahymena calcium-binding protein of 23 kDa (TCBP-23). TCBP-23 is similar (35% homology) but clearly different from TCBP-25. The TCBP-23 gene is actively transcribed in vivo as a 0.84-kilobase RNA. Thus, it follows that Tetrahymena cells have three different calmodulin family proteins: calmodulin, TCBP-25 and TCBP-23. These proteins are expected to provide important clues for solving the mechanisms of calcium-dependent phenomena, such as ciliary reversal.     

The calcium-binding protein p54/NEFA is a novel luminal resident of medial Golgi cisternae that traffics independently of mannosidase II

A new Golgi resident, p54, has been demonstrated in several eukaryotic species and in multiple organs. Based on Triton X-114 partition, carbonate extraction and trypsin protection assays, p54 behaved as an extrinsic membrane protein, facing the luminal compartment. p54 was purified by two-dimensional electrophoresis and identified by matrix-assisted laser desorption ionization/time-of-flight (MALDI-TOF) mass spectrometry as NEFA, a calcium-binding protein (Barnikol-Watanabe et al., 1994, Biol. Chem. Hoppe Seyler, 375, 497-512). By immunofluorescence, p54/NEFA essentially colocalized with the medial Golgi marker mannosidase II, and did not overlap with the cis-Golgi marker p58, nor with the trans-Golgi network (TGN) marker TGN38. By immuno-electron microscopy, p54/NEFA localized in the medial cisternae and in Golgi-associated vesicles. p54/NEFA remained associated with mannosidase II despite Golgi disruption by nocodazole, caffeine, or, to some extent, potassium depletion (a new procedure to induce Golgi disassembly), but the two markers rapidly dissociated upon brefeldin A treatment and at metaphase, and reassociated upon drug removal and at the end of anaphase. Since p54/NEFA is a peripheral luminal membrane constituent, its distinct trafficking from the transmembrane marker mannosidase II suggests a novel Golgi retention mechanism, by strong association of this soluble protein with another integral transmembrane resident.     

A novel 40-kDa membrane-associated EF-hand calcium-binding protein in Plasmodium falciparum.

A 40-kDa sexual stage radiolabeled surface protein of Plasmodium falciparum, Pfs40, was previously identified as a potential target antigen of transmission blocking immunity by an immunogenetic approach. Synthetic oligonucleotide "guessmers," based on microsequenced tryptic peptides of Pfs40 purified by two-dimensional gel electrophoresis, were used to clone the full length cDNA and genomic DNA encoding Pfs40. The deduced amino acid sequence predicted an integral membrane protein containing five EF-hand calcium-binding domains. The biological activity of one or more of these domains was confirmed by binding of 45Ca to both native and recombinant Pfs40. Antisera to recombinant Pfs40 immunoprecipitated the native radiolabeled 40-kDa surface protein. The predicted noncytosolic membrane-associated localization of Pfs40 is unique within the EF-hand calcium-binding protein superfamily.     

Molecular cloning of hippocalcin, a novel calcium-binding protein of the recoverin family exclusively expressed in hippocampus.

We have isolated a cDNA clone encoding a novel calcium-binding protein of the recoverin family from rat brain cDNA library. This clone (PCB11) has 588 nucleotides in the open reading frame including the termination codon, 174 nucleotides of the 5' leader and 800 nucleotides of the 3' noncoding region. The complete amino acid sequence deduced from the cDNA is composed of 195 residues, has a calculated molecular mass of 22,574 Daltons, and contains three putative calcium-binding domains of the EF-hand structure. The deduced amino acid sequence has a striking sequence homology to those of the retinal recoverin family (recoverin, visinin, P26, 23kD protein, S-modulin) and the brain-derived recoverin family (P23k, 21-kDa CaBP and neurocalcin). Northern blot, in situ hybridization, immunoblot and immunohistochemical analyses revealed that the protein is exclusively expressed in pyramidal layer of the hippocampus. The protein was therefore designated hippocalcin.     

NUCB1, the Drosophila melanogaster homolog of the mammalian EF-hand proteins NEFA and nucleobindin.

Mammalian NEFA and nucleobindin are calcium-binding proteins containing a signal peptide, two EF-hand motifs, acidic and basic regions and a leucine-zipper motif. Although they have been discussed to be involved in autoimmunity, apoptosis and calcium homeostasis in the Golgi apparatus and bone matrix, their exact role remains unknown. Here we report the cloning of their Drosophila homolog, nucb1, as well as the analysis of its expression pattern during embryogenesis and the subcellular localization of the NUCB1 protein. The nucb1 mRNA and the NUCB1 protein were found to be expressed maternally and zygotically, and they accumulate ubiquitously at low levels during all embryonic stages due to a maternal component. From stage 11 onward, high levels of zygotic expression can be detected specifically in the salivary glands and their placodes. In contrast to the known mammalian family members, the NUCB1 protein localizes in a subpattern of cytoplasmic substructures, probably the Golgi apparatus.     

Full sequence of neurocalcin, a novel calcium-binding protein abundant in central nervous system.

We determined the cDNA sequence for neurocalcin, a novel calcium-binding protein in bovine brain. This clone (pCalN) has 582 nucleotides in the open reading frame including the termination codon TGA, 11 nucleotides of the 5' leader and 1251 nucleotides of the 3' noncoding region. The deduced amino acid sequence revealed that neurocalcin is composed of 193 amino acids, has a molecular mass of 22,284 daltons, and contains three putative calcium-binding sites (EF-hand motifs). By Northern blot analysis, 3.8kbp mRNA was detected in brain. The deduced amino acid sequence had a strong homology to visinin (46.5%) and recoverin (51.6%) in retina, suggesting that neurocalcin may play a visinin- or recoverin-like role in brain.     

Characterization of a novel EF-hand homologue, CnidEF, in the sea anemone Anthopleura elegantissima

The superfamily of EF-hand proteins is comprised of a large and diverse group of proteins that contain one or more characteristic EF-hand calcium-binding domains. This study describes and characterizes a novel EF-hand cDNA, CnidEF, from the sea anemone Anthopleura elegantissima (Phylum Cnidaria, Class Anthozoa). CnidEF was found to contain two EF-hand motifs near the C-terminus of the deduced amino acid sequence and two regions near the N-terminus that could represent degenerate EF-hand motifs. CnidEF homologues were also identified from two other sea anemone species. A combination of bioinformatic and molecular phylogenetic analyses was used to compare CnidEF to EF-hand proteins in other organisms. The closest homologues identified from these analyses were a luciferin binding protein (LBP) involved in the bioluminescence of the anthozoan Renilla reniformis, and a sarcoplasmic calcium-binding protein (SARC) involved in fluorescence of the annelid worm Nereis diversicolor. Predicted structure and folding analysis revealed a close association with bioluminescent aequorin (AEQ) proteins from the hydrozoan cnidarian Aequorea aequorea. Neighbor-joining analyses grouped CnidEF within the SARC lineage along with AEQ and other cnidarian bioluminescent proteins rather than in the lineage containing calmodulin (CAM) and troponin-C (TNC).     

Molecular cloning and expression of the cDNA coding for a new member of the S100 protein family from porcine cardiac muscle.

We isolated a new calcium-binding protein from porcine cardiac muscle by calcium-dependent hydrophobic and dye-affinity chromatography. It showed an apparent molecular weight of 11,000 on SDS-PAGE. Amino acid sequence determination revealed that the protein contained two calcium-binding domains of the EF-hand motif. The cDNA gene coding for this protein was cloned from the porcine lung cDNA library. Sequence analysis of the cloned cDNA showed that the protein was composed of 99 amino acid residues and its molecular weight was estimated to be 11,179. Immunological and functional characterization showed that the recombinant S100C protein expressed in Escherichia coli was identical to the natural protein. Homologies to calpactin light chain, S100 alpha and beta protein were 41.1%, 40.9% and 37.5%, respectively. The protein was expressed at high levels in lung and kidney, and low levels in liver and brain. The tissue distribution was apparently different from those of the other S100 protein family. These results indicate that this protein represents a new member of the S100 protein family, and thus we refer to it as S100C protein.     

Enhancement of transport-dependent decarboxylation of phosphatidylserine by S100B protein in permeabilized Chinese hamster ovary cells

Phosphatidylethanolamine synthesis through the phosphatidylserine (PtdSer) decarboxylation pathway requires PtdSer transport from the endoplasmic reticulum or mitochondrial-associated membrane to the mitochondrial inner membrane in mammalian cells. The transport-dependent PtdSer decarboxylation in permeabilized Chinese hamster ovary (CHO) cells was enhanced by cytosolic factors from bovine brain. A cytosolic protein factor exhibiting this enhancing activity was purified, and its amino acid sequence was partially determined. The sequence was identical to part of the amino acid sequence of an EF-hand type calcium-binding protein, S100B. A His(6)-tagged recombinant CHO S100B protein was able to remarkably enhance the transport-dependent PtdSer decarboxylation in permeabilized CHO cells. Under the standard assay conditions for PtdSer decarboxylase, the recombinant S100B protein did not stimulate PtdSer decarboxylase activity and exhibited no PtdSer decarboxylase activity. These results implicated the S100B protein in the transport of PtdSer to the mitochondrial inner membrane.     

Selective Accumulation of the Endoplasmic Reticulum–Golgi Intermediate Compartment Induced by the Antitumor Drug KRN5500

KRN5500 is a semisynthetic spicamycin analogue consisting of a seven-carbon amino sugar linked to a C₁₄ unsaturated fatty acid through glycine and to the amino group of adenine. The drug inhibits cell growth potently and has antitumor activity in in vivo models. The mechanism of the antiproliferative effect of KRN5500 remains to be elucidated. We have found that acute exposure of drug-sensitive HT-29 colon adenocarcinoma cells to the drug results initially in swelling of the Golgi apparatus. Continuous exposure to the drug resulted in the emergence of a resistant population of cells characterized by numerous intracellular vacuoles. These KRN5500-resistant tumor cells exhibited increased staining with the Golgi stain NBD C₆–ceramide and the ER–Golgi fluorescent dye BODIPY–brefeldin A, which, unlike the parental drug-sensitive cells, was dispersed throughout the cytoplasm. Marker enzymes associated with the ER (glucose 6-phosphatase) and cis-Golgi (GalNAc transferase) were elevated >2-fold and nearly 4-fold, respectively, in drug-resistant cell lines while the trans-Golgi marker enzyme, galactosyltransferase, was not. The additional findings that the KRN5500-resistant cells have a >2-fold elevation in ERGIC-53, a cis-Golgi marker protein of the ER–Golgi intermediate compartment (ERGIC), as well as increased 58K, a 58-kDa microtubule-binding protein with formiminotransferase cyclodeaminase activity, and tubulin indicate that the cellular secretory pathway is a primary determinant of sensitivity to KRN5500, as resistance to this agent corresponds with accumulation of several components relatable to ER and cis-Golgi function. Further support for this conclusion is provided by studies which demonstrate that KRN5500 alters the distribution of newly synthesized carcinoembryonic antigen within the secretory pathway, including arrest of this N-glycosylated protein in the Golgi of LS-174T colon carcinoma cells.     

Genomic structure of the sponge,Halichondria okadai calcyphosine gene

Calcyphosine is an EF-hand Ca(2+)-binding protein, which was first isolated from the canine thyroid. It is phosphorylated in a cyclic AMP (cAMP)-dependent manner; then it is thought to be implicated in the cross-signaling between the cAMP and calcium-phosphatidylinositol cascades. Here, we isolated the DNA complementary to RNA (cDNA) of an EF-hand Ca(2+)-binding protein from the sponge, Halichondria okadai and determined its genomic structure. The deduced sequence of the sponge Ca(2+)-binding protein showed significant similarity (about 40% identity) with those of mammal calcyphosines, and the intron positions were well conserved between the sponge and human calcyphosine genes. We considered that the isolated cDNA was that of sponge calcyphosine, and that sponge and mammalian calcyphosines evolved from a common ancestor gene. Recent cDNA projects have revealed that a calcyphosine cDNA is also expressed by human, mouse, and the ascidia. These cDNAs have more than 60% identity with sponge calcyphosine and each other, and all are composed of 208 amino acid residues. On the constructed phylogenetic trees, calcyphosines are essentially divided into two groups, types-I and -II calcyphosines. Type-I calcyphosine may be specific to mammals, and type-II is widely distributed among metazoan species. This suggests that type-II calcyphosine is a rather ancient gene with some essential function.     

Selective accumulation of the endoplasmic reticulum-Golgi intermediate compartment induced by the antitumor drug KRN5500

KRN5500 is a semisynthetic spicamycin analogue consisting of a seven-carbon amino sugar linked to a C(14) unsaturated fatty acid through glycine and to the amino group of adenine. The drug inhibits cell growth potently and has antitumor activity in in vivo models. The mechanism of the antiproliferative effect of KRN5500 remains to be elucidated. We have found that acute exposure of drug-sensitive HT-29 colon adenocarcinoma cells to the drug results initially in swelling of the Golgi apparatus. Continuous exposure to the drug resulted in the emergence of a resistant population of cells characterized by numerous intracellular vacuoles. These KRN5500-resistant tumor cells exhibited increased staining with the Golgi stain NBD C(6)-ceramide and the ER-Golgi fluorescent dye BODIPY-brefeldin A, which, unlike the parental drug-sensitive cells, was dispersed throughout the cytoplasm. Marker enzymes associated with the ER (glucose 6-phosphatase) and cis-Golgi (GalNAc transferase) were elevated >2-fold and nearly 4-fold, respectively, in drug-resistant cell lines while the trans-Golgi marker enzyme, galactosyltransferase, was not. The additional findings that the KRN5500-resistant cells have a >2-fold elevation in ERGIC-53, a cis-Golgi marker protein of the ER-Golgi intermediate compartment (ERGIC), as well as increased 58K, a 58-kDa microtubule-binding protein with formiminotransferase cyclodeaminase activity, and tubulin indicate that the cellular secretory pathway is a primary determinant of sensitivity to KRN5500, as resistance to this agent corresponds with accumulation of several components relatable to ER and cis-Golgi function. Further support for this conclusion is provided by studies which demonstrate that KRN5500 alters the distribution of newly synthesized carcinoembryonic antigen within the secretory pathway, including arrest of this N-glycosylated protein in the Golgi of LS-174T colon carcinoma cells.     

Identification of a novel smooth muscle associated protein,smap2, upregulated during neointima formation in a rat carotid endarterectomy model

Proliferation of aortic smooth muscle cells is an important event in vascular lesion formation. To identify new genes that are involved in neointima formation, we constructed an aortic 3'-directed cDNA library. The novel cDNA of a gene designated smooth muscle associated protein 2 (smap2) was isolated. The full-length cDNA of smap2 is 2914 base pairs long and contains an open reading frame of 1338 base pairs. Dot blot analysis revealed that smap2 was expressed particularly in aorta. The deduced amino acid sequence of smap2 contains two thyroglobulin type-1 domains, two EF-hand calcium-binding domains and putative signal peptide. Furthermore, we demonstrated that smap2 mRNA was upregulated during neointima formation in a rat carotid endarterectomy model. These findings suggest that smap2 might be involved in the progression of atherosclerosis in aorta.     

Isolation of human retinal genes: recoverin cDNA and gene.

A human retina cDNA library enriched for retina-specific clones was prepared by subtraction with a non-retina population of cDNA in combination with polymerase chain reaction (PCR) amplifications. A highly retina-specific cDNA clone (1190 bp) was obtained through this library encoding a 200 amino acid protein with three calcium binding sites and 87% homology to the bovine photoreceptor protein, recoverin, which has been shown to mediate the recovery of the dark current after photoactivation, and 58% homology to the calcium-binding chick cone protein, visinin. Analysis of the gene indicated a 9-10 kb single-copy gene with at least three exons and two introns. The three exons contained the entire coding sequence, and all of the calcium-binding EF-hand regions were in putative exon 1. The recoverin gene was mapped to human chromosome 17 by hybridization to a panel of human-rodent hybrid DNAs.