Molecular analysis of two genes, DD9A and B, which are expressed during the postmolt stage in the decapod crustacean Penaeus japonicus

In decapod crustaceans, deposition of calcium carbonate crystals (calcification) in the exoskeleton takes place during the postmolt phase of the molt cycle. In an attempt to identify proteins which regulate the calcification process, the differential display technique was used to identify genes which were specifically expressed in the integument during the postmolt stage in the penaeid prawn Penaeus japonicus. One of the genes thus identified, named DD9A, was expressed in the epithelial cells of the tail fan. DD9A encoded a putative precursor of a secreted protein of 113 amino acids which exhibited sequence similarities to a group of crustacean and insect cuticular proteins, suggesting that DD9A was a protein component of the exoskeleton. Another gene, DD9B, which was also transcribed specifically during the postmolt period was identified based on its sequence similarity to DD9A. Potential roles of the DD9A protein in the calcification of the exoskeleton will be discussed.     

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.     

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.     

Characterization of a Ca2+ binding and regulatory site in the Ca2+ release channel (ryanodine receptor) of rabbit skeletal muscle sarcoplasmic reticulum.

A region in the skeletal muscle ryanodine receptor between amino acids 4014 and 4765 was expressed as a trpE fusion protein. Overlay studies revealed that this region bound Ca2+ and ruthenium red, an indicator of Ca(2+)-binding sites. Ca2+ binding was mapped to subregion 13b between amino acids 4246 and 4377, encompassing a predicted high affinity Ca(2+)-binding site, and to subregion 13c between amino acids 4364 and 4529, encompassing two predicted high affinity Ca(2+)-binding sites. Ca2+ binding was then mapped to three shorter sequences, 22(13b1), 36(13c1), and 35(13c2), amino acids long, each encompassing one of the three predicted Ca(2+)-binding sites. Site-directed polyclonal antibodies were raised against these three short sequences and purified on antigen affinity columns. The antibody against sequence 13c2, lying between residues 4478 and 4512, specifically recognized both denatured and native forms of the ryanodine receptor, suggesting that at least part of the 35 amino acid sequence containing the Ca(2+)-binding site is surface-exposed. The affinity purified antibody increased the Ca2+ sensitivity of ryanodine receptor channels incorporated into planar lipid bilayers, resulting in increased open probability and opening time without altering channel conductance. The antibody-activated channel was still modulated by Ca2+, Mg2+, ATP, ryanodine, and ruthenium red. These observations suggest that sequence 13c2 may be involved in Ca(2+)-induced Ca2+ release.     

Isolation and sequence of rat testis cDNA for a calcium binding polypeptide similar to the regulatory subunit of calcineurin.

We have cloned and sequenced rat testis cDNAs coding for a calcium binding polypeptide similar to calcineurin beta subunit, the Ca(2+)-binding subunit of the Ca2+/calmodulin stimulated protein phosphatase. Rat testis cDNA library was screened with a monoclonal antibody Va1 raised against bovine brain calcineurin beta subunit. The deduced amino acid sequence is similar to that of human brain calcineurin beta subunit with respect to containing four putative calcium binding sites. However, distinct differences were found: 1) The cloned cDNA had six amino acids polypeptide tail at carboxy-terminal which is absent in human brain calcineurin beta subunit. This amino acids tail makes the carboxy-terminal highly hydrophilic in contrast to the human brain beta subunit which is hydrophobic at carboxy-terminal; 2) eleven amino acids at the N terminal of the cloned cDNA were completely different from the corresponding region of the brain calcineurin beta subunit.     

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.     

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.     

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.     

Calmodulin binding to the Fas death domain. Regulation by Fas activation

Fas (APO-1/CD95) is a cell surface receptor that initiates apoptotic pathways, and its cytoplasmic domain interacts with various molecules suggesting that Fas signaling is complex and regulated by multiple proteins. Calmodulin (CaM) is an intracellular Ca(2+)-binding protein, and it mediates many of the effects of Ca2+. Here, we demonstrate that CaM binds to Fas directly and identify the CaM-binding site on the cytoplasmic death domain (DD) of Fas. Fas binds to CaM-Sepharose and is co-immunoprecipitated with CaM. Other death receptors, such as tumor necrosis factor receptor, DR4, and DR5 do not bind to CaM. The interaction between Fas and CaM is Ca(2+)-dependent. Deletion mapping analysis with various GST-fused Fas cytoplasmic domain fragments revealed that the fragment containing helices 1, 2, and 3 of the Fas DD has the CaM-binding ability. Sequence analysis of this fragment predicted a potential CaM-binding site in helix 2 and connected loops. A valine 254 to asparagine mutation in this region, which is analogous to the identified mutant allele of Fas in lpr mice that have a deficiency in Fas-mediated apoptosis, showed reduced CaM binding. Computer modeling of the interaction between CaM and helix 2 of the Fas DD predicted that amino acids, which are important for Fas-CaM binding, and point mutations of these amino acids caused reduced Fas-CaM binding. The interaction between Fas and CaM is increased approximately 2-fold early upon Fas activation (at 30 min) and is decreased to approximately 50% of control at 2 h. These findings suggest a novel function of CaM in Fas-mediated apoptosis.     

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.     

The sequence of chick alpha-actinin reveals homologies to spectrin and calmodulin

We have sequenced a cDNA, isolated from a chick embryo fibroblast lambda gt11 library, that encodes all 887 amino acids of alpha-actinin. Sequence from 10 different peptides from chick smooth muscle alpha-actinin was found to match that derived from the cDNA. The deduced protein sequence can be divided into three distinct domains: (a) the N-terminal 240 amino acid contains a highly conserved region (compared with Dictyostelium alpha-actinin) which probably represents the actin-binding domain, (b) amino acids 270-740 contain four repeats of a spectrin-like sequence, and (c) the C-terminal sequence contains two EF-hand Ca2+-binding sites. Each of these sites is defective in at least one oxygen-containing Ca2+-chelating amino acid side chain, suggesting that they are nonfunctional. Southern blots suggest that the alpha-actinin cDNA described here hybridizes to only one gene in chicken. Northern blots reveal only one size class of mRNA in fibroblasts and smooth muscle, but no hybridizing species could be detected in skeletal muscle poly(A+) RNA. The results are consistent with the view that smooth and skeletal muscle alpha-actinins are encoded by separate genes, which are considerably divergent.     

The N-terminal domain of 5-lipoxygenase binds calcium and mediates calcium stimulation of enzyme activity

Human 5-lipoxygenase (5-LO) is a key enzyme in the conversion of arachidonic acid into leukotrienes and lipoxins, mediators and modulators of inflammation. In this study, we localized a stimulatory Ca(2+)-binding site to the N-terminal region of the enzyme. Thus, in a (45)Ca(2+) overlay assay, the N-terminal 128 amino acids of recombinant human 5-LO (fused to glutathione S-transferase) bound radioactive calcium to about the same extent as intact 5-LO. The glutathione S-transferase fusion protein of the C-terminal part of 5-LO (amino acids 120-673) showed much weaker binding. A model of a putative 5-LO N-terminal domain was calculated based on the structure of rabbit reticulocyte 15-LO. This model resembles beta-sandwich C2 domains of other Ca(2+)-binding proteins. Comparison of our model with the C2 domain of cytosolic phospholipase A(2) suggested a number of amino acids, located in the loops that connect the beta-strands, as potential Ca(2+) ligands. Indeed, mutations particularly in loop 2 (N43A, D44A, and E46A) led to decreased Ca(2+) binding and a requirement for higher Ca(2+) concentrations to stimulate enzyme activity. Our data indicate that an N-terminal beta-sandwich of 5-LO functions as a C2 domain in the calcium regulation of enzyme activity.     

Structural characterization of a recombinant flagellar calcium-binding protein from Trypanosoma cruzi

Calflagin are flagellar calcium-binding proteins belonging to the EF-hand super family described in several protozoa, including Trypanosoma cruzi. Evidences have shown that Ca(2+) may play an important regulatory role in trypanosomatid flagellar mobility. In these parasites, the response of the cell to variations of Ca(2+) levels is determined by a variety of calcium-modulated proteins. Starting from T. cruzi cDNA lambdagt11 library trypomastigote, a clone encoding a 29-kDa flagellar protein designated recombinant calflagin (rC29) was selected. rC29 is a calcium-acyl switch protein modified by the addition of myristate and palmitate at its amino terminal segment. In this work, unmyristoylated rC29 was expressed in Escherichia coli as an intein fusion protein and purified by affinity chromatography. Circular dichroism (CD) and fluorescence measurements showed conformational changes of rC29 due to Ca(2+) binding. The Ca(2+) binding constants were obtained by tryptophan intrinsic fluorescence spectroscopy. Fluorescence titration exhibited two classes of Ca(2+)-binding sites in the unmyristoylated rC29, which bind calcium with apparent association constant of K(a) of 3.3+/-0.5 (10(6)) and 1.9+/-0.2 (10(4)) M(-1). Experiment using 8-anilinonaphthalene-1-sulfonic acid (ANS) as hydrophobic probe showed that the Ca(2+)-loaded form of rC29 contains exposed hydrophobic surfaces, thus suggesting that rC29 is probably functioning as a calcium sensor.     

The primary structure of NG2, a novel membrane-spanning proteoglycan

The complete primary structure of the core protein of rat NG2, a large, chondroitin sulfate proteoglycan expressed on O2A progenitor cells, has been determined from cDNA clones. These cDNAs hybridize to an mRNA species of 8.9 kbp from rat neural cell lines. The total contiguous cDNA spans 8,071 nucleotides and contains an open reading frame for 2,325 amino acids. The predicted protein is an integral membrane protein with a large extracellular domain (2,224 amino acids), a single transmembrane domain (25 amino acids), and a short cytoplasmic tail (76 amino acids). Based on the deduced amino acid sequence and immunochemical analysis of proteolytic fragments of NG2, the extracellular region can be divided into three domains: an amino terminal cysteine-containing domain which is stabilized by intrachain disulfide bonds, a serine-glycine-containing domain to which chondroitin sulfate chains are attached, and another cysteine-containing domain. Four internal repeats, each consisting of 200 amino acids, are found in the extracellular domain of NG2. These repeats contain a short sequence that resembles the putative Ca(++)-binding region of the cadherins. The sequence of NG2 does not show significant homology with any other known proteins, suggesting that NG2 is a novel species of integral membrane proteoglycan.