Hiroaki Kagawa

Tissue expression of four troponin I genes and their molecular interactions with two troponin C isoforms in Caenorhabditis elegans.

Gene duplication is a major genetic event that can produce multiple protein isoforms. Comparative sequence and functional analysis of related gene products can provide insights into protein family evolution. To characterize the Caenorhabditis elegans troponin I family, we analyzed gene structures, tissue expression patterns and RNAi phenotypes of four troponin I isoforms. Tissue expression patterns were determined using lacZ/gfp/rfp reporter gene assays. The tni‐1, tni‐2/unc‐27 and tni‐3 genes, each encoding a troponin I isoform, are uniquely expressed in body wall, vulval and anal muscles but at different levels; tni‐4 was expressed solely in the pharynx. Expressing tni‐1 and ‐2 gene RNAi caused motility defects similar to unc‐27 (e155) mutant, a tni‐2 null allele. The tni‐3 RNAi expression produced egg laying defects while the tni‐4 RNAi caused arrest at gastrulation. Overlay analyses were used to assay interactions between the troponin I and two troponin C isoforms. The three body wall troponin I isoforms interacted with body wall and pharyngeal troponin C isoforms; TNI‐4 interacted only with pharyngeal troponin C. Our results suggest the body wall genes have evolved following duplication of the pharynx gene and provide important data about gene duplication and functional differentiation of nematode troponin I isoforms.

Molecular dissection, tissue localization and Ca2+ binding of the ryanodine receptor of Caenorhabditis elegans

The ryanodine receptor of Caenorhabditis elegans (CeRyR) which contains 5,071 amino acid residues, is encoded by a single gene, ryr-1/unc-68. The unc-68(kh30) mutation, isolated in an animal showing abnormal response to the anesthetic ketamine, has the substitution Ser1444Asn in CeRyR, predicted to be a phosphorylation site. To elucidate the function of the region of CeRyR, and to determine the localization of CeRyR in this animal, ten region-peptides were produced in Escherichia coli by using expression plasmids and eight antisera were raised against these fusion peptides. One antibody against the region corresponding to the kh30 mutation site enabled detection of CeRyR from mutant animals both in Western analysis and in situ. Specificity of this antiserum was demonstrated using Western analysis, which showed the full size and the partial size bands in wild-type and in the Tc1-induced deletion mutant animals, respectively, but no corresponding bands in unc-68 null mutant animals. CeRyR was detected in I-bands of muscle sarcomeres by double immunostaining. CeRyR was found in the body wall, pharyngeal, vulval, anal and sex muscles of adult worms and also found to be present in embryonic muscle, but not in non-muscle cells. Two EF-hand motifs and the C terminus were demonstrated to be Ca(2+) binding regions. On the basis of these results, we propose a model for the functional domains of CeRyR, which agrees well with the model of mammalian skeletal RyR, which is based on proteolysis and cross-linking analysis. We discuss the usefulness and limitations of the molecular dissection approach, which uses peptides and peptide-specific antibodies to determine the local structure and function of individual domains within a large molecule.

Mapping of conformational B cell epitopes within alpha-helical coiled coil proteins.

An approach to mapping antigenic B cell epitopes within alpha-helical coiled coil proteins has been developed and applied to two proteins: Streptococcal M protein and C. elegans paramyosin protein UNC-15. Overlapping peptides derived from an alpha-helical coiled coil conformational epitope were embedded between helical flanking peptides derived from the completely unrelated GCN4 leucine zipper peptide. The resulting chimeric peptides exhibited helical propensity. Chimeric peptides were tested for antigenicity (recognition by antibody) or immunogenicity (production of appropriate antibody response). A conformational epitope within the Streptococcal M protein recognised by three mAbs spanned 12 residues. Analysis of chimeric peptides based on C. elegans UNC-15 has enabled fine mapping of the minimal B cell epitope recognised by monoclonal antibody NE1-6B2 to seven non-contiguous residues (spanning 15 residues); the footprint of contact residues involved in antibody recognition being restricted to the hydrophilic face of the helix and covering five helical turns. This chimeric peptide epitope when coupled to diphtheria toxoid was highly immunogenic in mice and antisera recognised the conformationally dependent native peptide epitope. This approach has the potential to map conformational epitopes and design minimal epitopes for use as vaccine candidates.

Functional importance of polymerization and localization of calsequestrin in C. elegans

Dual roles of calsequestrin (CSQ-1) being the Ca2+ donor and Ca2+ acceptor make it an excellent Ca2+-buffering protein within the sarcoplasmic reticulum (SR). We have isolated and characterized a calsequestrin (csq-1)-null mutant in Caenorhabditis elegans. To our surprise, this mutant csq-1(jh109) showed no gross defects in muscle development or function but, however, is highly sensitive to perturbation of Ca2+ homeostasis. By taking advantage of the viable null mutant, we investigated the domains of CSQ-1 that are important for polymerization and cellular localization, and required for its correct buffering functions. In transgenic animals rescued with various CSQ-1 constructs, the in vivo patterns of polymerization and localization of several mutated calsequestrins were observed to correlate with the structure-function relationship. Our results suggest that polymerization of CSQ-1 is essential but not sufficient for correct cellular localization and function of CSQ-1. In addition, direct interaction between CSQ-1 and the ryanodine receptor (RyR) was found for the first time, suggesting that the cellular localization of CSQ-1 in C. elegans is indeed modulated by RyR through a physical interaction.

VHA‐8, the E subunit of V‐ATPase, is essential for pH homeostasis and larval development in C. elegans

Vacuolar H+‐ATPase (V‐ATPase) is an ATP‐dependent proton pump, which transports protons across the membrane. It is a multi‐protein complex which is composed of at least 13 subunits. The Caenorhabditis elegans vha‐8 encodes the E subunit of V‐ATPase which is expressed in the hypodermis, intestine and H‐shaped excretory cells. VHA‐8 is necessary for proper intestinal function likely through its role in cellular acidification of intestinal cells. The null mutants of vha‐8 show a larval lethal phenotype indicating that vha‐8 is an essential gene for larval development in C. elegans. Interestingly, characteristics of necrotic cell death were observed in the hypodermis and intestine of the arrested larvae suggesting that pH homeostasis via the E subunit of V‐ATPase is required for the cell survival in C. elegans.

C. ELEGANS MODEL FOR STUDYING TROPOMYOSIN AND TROPONIN REGULATIONS OF MUSCLE CONTRACTION AND ANIMAL BEHAVIOR

There are two muscle tissues in the nematode Caenorhabditis elegans: the pharynx for feeding and the body wall for locomotion. These correspond to cardiac and skeletal muscles in vertebrates, respectively. Study of the muscle genes of C. elegans can be classified into three stages; first, mutant isolation and gene mapping, second, cloning and sequencing of the gene, and third, complete sequences of all genes. Many uncoordinated mutant animals have been isolated (Brenner, 1974; Waterston, 1988; Moerman and Fire, 1997) and the complete amino acid sequence of myosin heavy chain, twitchin, and paramyosin, (invertebrate specific core protein of thick filament), and were the first determined in any animals by analyzing the unc-54, unc-22, and unc-15 mutants, respectively (Karn et al., 1983; Benian et al., 1989; Kagawa et al., 1989). Tropomyosin and troponin components are also present but as with actin and myosin heavy chain in the worm, there are some differences in gene structure and sequence compared to those in other animals (Kagawa et al., 1995; Myers et al., 1996; Moerman and Fire, 1997). Deficiencies of body wall troponin C or tropomyosin in C. elegans cause the Pat (paralyzed arrest at embryonic two-fold stage) phenotype (Williams and Waterston, 1994; Terami et al., 1999) and those of troponin T cause Mup (muscle position abnormal) phenotype (Myers et al., 1996). After determining the complete genome sequences of the nematode (The C. elegans Sequence Consortium, 1998), we can find out how isoforms are related to each other. Only one troponin C gene, pat-10/tnc-1, is expressed in the body wall muscles and the gene defect causes a developmental arrest of the animals (Terami et al., 1999).

Transcription Factors GATA/ELT-2 and Forkhead/HNF-3/PHA-4 Regulate the Tropomyosin Gene Expression in the Pharynx and Intestine of Caenorhabditis elegans

Gene regulation during development is an important biological activity that leads to synthesis of biomolecules at specific locations and specific times. The single tropomyosin gene of Caenorhabditis elegans, tmy-1/lev-11, produces four isoforms of protein: two from the external promoter and two from the internal promoter. We investigated the internal promoter of tropomyosin to identify sequences that regulate expression of tmy-1 in the pharynx and intestine. By promoter deletion of tmy-1 reporters as well as by database analyses, a 100-bp fragment that contained binding sequences for a GATA factor, for a chicken CdxA homolog, and for a forkhead factor was identified. Both the forkhead and CdxA binding sequences contributed to pharyngeal and intestinal expression. In addition, the GATA site also influenced intestinal expression of tmy-1 reporter. We showed that ELT-2 and PHA-4 proteins interact directly with the GATA and forkhead binding sequences, respectively, in gel mobility shift assays. RNA interference knockdown of elt-2 diminished tmy-1::gfp expression in the intestine. In contrast to RNA interference knockdown of pha-4, expression of tmy-1::gfp in pha-4;smg-1 mutants was slightly weaker than that of the wild type. Ectopic expression of PHA-4 and ELT-2 by heat shock was sufficient to elicit widespread expression of tmy-1::lacZ reporter in embryos. We found no indication of a synergistic relation between ELT-2 and PHA-4. Based on our data, PHA-4 and CdxA function as general transcription factors for pharyngeal and intestinal regulation of tmy-1. We present models by which ELT-2, PHA-4, and CdxA orchestrate expression from the internal promoter of tmy-1.

Calcium-bindings of wild type and mutant troponin Cs of Caenorhabditis elegans

Apparent Ca(2+)-binding constant (K(app)) of Caenorhabditis elegans troponin C (CeTnC) was determined by a fluorescence titration method. The K(app) of the N-domain Ca(2+)-binding site of CeTnC was 7.9+/-1.6 x 10(5) M(-1) and that of the C-domain site was 1.2+/-0.6 x 10(6) M(-1), respectively. Mg(2+)-dependence of the K(app) showed that both Ca(2+)-binding sites did not bind competitively Mg(2+). The Ca(2+) dissociation rate constant (k(off)) of CeTnC was determined by the fluorescence stopped-flow method. The k(off) of the N-domain Ca(2+)-binding site of CeTnC was 703+/-208 s(-1) and that of the C-domain site was 286+/-33 s(-1), respectively. From these values we could calculate the Ca(2+)-binding rate constant (k(on)) as to be 5.6+/-2.8 x 10(8) M(-1) s(-1) for the N-domain site and 3.4+/-2.1 x 10(8) M(-1) s(-1) for the C-domain site, respectively. These results mean that all Ca(2+)-binding sites of CeTnC are low affinity, fast dissociating and Ca(2+)-specific sites. Evolutional function of TnC between vertebrate and invertebrate and biological functions of wild type and mutant CeTnCs are discussed.

Genetic analysis of ryanodine receptor function in Caenorhabditis elegans based on unc-68 revertants

The Caenorhabditis elegans ryanodine receptor is encoded by the unc-68 gene, and functions as a Ca2+-induced Ca2+ release channel during muscle contraction. To investigate the factors that suppress calcium release and identify molecules that interact with the ryanodine receptor, we isolated revertants from two unc-68 mutants. Three of the revertants obtained from the null allele unc-68(e540), which displayed normal motility, had intragenic mutations that resulted in failure to splice out intron 21. The other two, kh53 and kh55, had amino acid insertions in the third of the four RyR domains. The brood size and the egg laying rate remain abnormal in these revertants. This suggests the third RyR domain may be required for egg laying and embryogenesis, although we can not determine a molecular mechanism. Five ketamine sensitive revertants recovered from the missense mutant unc-68(kh30) showed altered responses to caffeine, ryanodine, levamisole and ouabain relative to those of the unc-68(kh30) animals. These may carry second-site suppressor mutations, which may define genes for proteins that regulate the Ca2+ concentration in body-wall muscle. One of these mutants, kh52 , shows lower motility and higher sensitivity to drugs, and this mutation was mapped to chromosome X. These observations provide a basis for the study of ryanodine receptor functions in embryogenesis and in calcium-mediated regulation of muscle contraction in C. elegans. This is the first study to show that the conserved RyR domain of the receptor acts in egg laying and embryogenesis.

In vitro polymerization of polyhook protein from Salmonella SJW880

Polyhooks were isolated from Salmonella SJW880, a non-flagellated mutant, and purified by cesium chloride density gradient centrifugation. The polyhooks were disintegrated into protein subunits (monomer) by heat in the absence of salt. The monomer was repolymerized in the presence of moderately high concentrations of sodium citrate at neutral pH. Three types of polymer were produced. One type of polymer, produced at room temperature and at citrate concentrations less than 0.3 M, had no regular shape and no definite thickness. Another type of polymer, produced at room temperature and at citrate concentrations greater than 0.4 M, had a straight shape and a similar thickness to that of polyhook but was easily dissociated into monomer in the absence of salt. A third type of polymer was produced at low temperature, independently of the concentration of citrate, and seemed to be a tubular polymer with a thickness similar to that of polyhook but had no helical curvature. However, this type of polymer was shown to have a structure locally the same as that of polyhook by electron microscopic observation, optical diffraction and circular dichroism measurements.