Soluble calcium-binding proteins (SCBPs) of the earthworm Lumbricus terrestris: possible role as relaxation factors in muscle.

The soluble Ca2+-binding protein (SCBP) from the earthworm Lumbricus terrestris was analyzed with regard to its role as a soluble muscle relaxation factor. The actomyosin ATPase activity was inhibited by the addition of decalcified SCBP as it binds Ca2+ stronger than the regulatory proteins associated with the actomyosin. Competitive 45Ca2+-binding assays with decalcified actomyosin and SCBP showed that 45Ca2+ is first bound to actomyosin and is subsequently taken over by SCBP with increasing incubation time. Ca2+ competition experiments carried out with 45Ca2+ loaded SCBP and fragmented sarcoplasmic reticulum vesicles revealed that 45Ca2+ bound to SCBP can be deprived by the ATP-dependent Ca2+ uptake of the sarcoplasmic reticulum. Furthermore, experiments in a diffusion chamber showed that the addition of SCBP significantly enhances the 45Ca2+ flux in a concentration dependent manner. The amount of the Ca2+ flux increase tends to reach a maximum value of about 70%. With all protein components isolated from the obliquely striated muscle, our in vitro experiments consistently show that SCBP may accelerate muscle relaxation similar as assumed for vertebrate parvalbumin.

Four paralog gelsolin genes are differentially expressed in the earthworm Lumbricus terrestris.

We have identified and characterized four distinct variants of the gelsolin-related protein (EWAM P1-P4) in the earthworm L. terrestris. All of these proteins biochemically qualify as gelsolins since they sever actin filaments in a calcium dependent manner. P1, P2 and P3 are present in the Lumbricus body wall muscle whereas in the gizzard muscle P3 and P4 were found. P1-P4 are encoded by four paralog genes and are differentially expressed in various muscle cell tissues. While the genes for P1 and P2 contain one intron, there was no intron in both P3 and P4 genes. The coding sequences consist of 1104bp (368 amino acids) for P1/P4 and 1101bp (367 amino acids) for P2/P3. Corresponding genes were confirmed by northern blot analysis which revealed three (calculated lengths: 3100, 2300 and 2100 nucleotides) and two (calculated lengths: 2300 and 1700 nucleotides) mRNA transcripts in the body wall and the gizzard, respectively. EWAM mRNA was localized by fluorescence in situ hybridization in the body wall and the gizzard muscle. P1 mRNA was detected in the inner proximal layers of both the circular and longitudinal muscle of the body wall whereas in the gizzard no significant staining was observed for P1. P2-P4 mRNAs were abundant in the outer distal layers of both the circular and the longitudinal muscles of both body wall and gizzard. The differential expression of four paralog gelsolin genes suggests a functional adaptation of different muscle cells with respect to actin filament turnover and modulation of its polymer state.

Gelsolin in Onychophora and Tardigrada with notes on its variability in the Ecdysozoa.

Rearrangements of the filamentous actin network involve a broad range of actin binding proteins. Among these, the gelsolin proteins sever actin filaments, cap their fast growing end and nucleate actin assembly in a calcium-dependent manner. Here, we focus on the gelsolin of the onychophoran Peripatoides novaezealandiae and the eutardigrade Hypsibius dujardini. From the cDNA of P. novaezealandiae we obtained the complete coding sequence with an open reading frame of 2178bp. It encodes a protein of 726 amino acids with a calculated molecular mass of 82,610.9Da and a pI of 5.57. This sequence is comprised of six segments (S1-S6). However, analysis of data from TardiBase reveals that the gelsolin of the eutardigrade Hypsibius dujardini has only three segments (S1-S3). The coding sequence consist of 1119bp for 373 amino acids with a calculated molecular mass of 42,440.95Da and a pI of 6.17. The Peripatoides and Hypsibius gelsolin revealed both conserved binding motifs for G-actin, F-actin and phosphatidylinositol 4,5-bisphosphate (PIP2), along with a full set of type-1 and type-2 Ca2+-binding sites which could result in the binding of eight and four calcium ions, respectively. Both gelsolin proteins lack a C-terminal latch-helix indicating a more rapid activation in the submicromolar Ca2+ range. We suggest that a gelsolin with three segments was present in the last common ancestor of the ecdysozoan clade Panarthropoda (Onychophora, Tardigrada, Arthropoda), primarily because the gelsolin of all non-Ecdysozoa studied so far (except Chordata) reveals this number of segments. Mapping of our molecular data onto a well-established phylogeny revealed that the number of gelsolin segments does not correlate with the phylogenetic lineage but rather with particular functional demands to alter the kinetics of actin polymerization.

Soluble calcium-binding proteins (SCBPs) of the earthworm Lumbricus terrestris: molecular characterization and localization by FISH in muscle and neuronal tissue.

Soluble calcium-binding proteins (SCBPs) of invertebrates probably serve like their vertebrate counterpart-the parvalbumins-as soluble relaxing factors in muscles. Three SCBP isoforms (SCBP1-3) have been isolated and biochemically characterized in the earthworm Lumbricus terrestris (Huch et al. in J Comp Physiol B 158:325-334, 1988). For SCBP2, we found two isoforms named SCBP2a/2b. Both of them together with SCBP3 are present in the body wall muscle. In the gizzard solely, SCBP2b and no SCBP2a or SCBP3 could be detected. The coding sequences of all three isoforms consist of 534 bp for 178 amino acids and contain four EF-hand motifs, of which the second EF-hands are truncated. Recombinant proteins show heat stability and a Ca2+-dependent mobility shift similar to the native proteins, indicating comparable calcium-binding properties. All three isoforms are encoded by three distinct and differentially expressed genes. The genes for SCBP2a, SCBP2b, and SCBP3 are interrupted by only one intron, inserting at nearly the same positions. Northern blot analysis revealed two mRNA transcripts for SCBP2 of approximately 1250 and 1500 kb and one transcript for SCBP3 of approximately 1250 kb. SCBP mRNA was localized by fluorescent in situ hybridization in the body wall and the gizzard. The distribution of the staining intensities resembles that for the myosin ATPase activity and indicates a correlation between the amount of SCBP and speed of muscle contraction. In addition, SCBP mRNA was localized within the nervous tissue, the cerebral and subesophageal ganglia and the ventral nerve cord.