Satoshi Odo

Interspecific Variations in Adhesive Protein Sequences of Mytilus edulis, M. galloprovincialis, and M. trossulus

Variation in the adhesive protein gene sequences of Mytilus edulis, M. galloprovincialis, and M. trossulus collected in Delaware, Kamaishi (Japan), and Alaska, respectively, was analyzed by the polymerase chain reaction (PCR) using two sets of oligonucleotide primers. The first set, Me 13 and Me 14, was designed to amplify the repetitive region. The length of the amplified fragments was highly variable, even among samples of the same species. Another set, Me 15 and Me 16, was designed to amplify a part of the nonrepetitive region. The length of the amplified fragments was uniform in each species and differed interspecifically; 180, 168, and 126 bp for M. edulis, M. trossulus, and M. galloprovincialis, respectively. The amplified sequence of M. trossulus resembled that of M. edulis. Mussels from other sites were also examined by PCR using Me 15 and Me 16. Wild mussels from Tromsö (Norway) and cultured mussels from Brittany (France) were identified as M. edulis. Cultured mussels from the Mediterranean coast of France and wild mussels from Shimizu (Japan) were identified as M. galloprovincialis. Some wild mussels from Hiura (Japan) were identified as a hybrid between M. galloprovincialis and M. trossulus. Thus, the length of this part (variable region) of the sequence is proposed as a diagnostic marker for these three morphologically similar species and their hybrids.

Cement Proteins of the Acorn-Barnacle, Megabalanus rosa

Components of the proteinaceous cement secreted by barnacles have yet to be studied because of their insolubility. We solubilized and characterized the proteins of secondary cement, which is produced when the barnacle is detached from the substratum, in Megabalanus rosa. The cement was fractionated, according to its solubility in aqueous formic acid, into a soluble fraction, SF1 (21%); a fraction soluble after reduction, SF2 (37%); and a fraction insoluble after reduction, IF (42%). Analysis of the SF1 and SF2 by sodium dodecylsulfate-polyacrylamide gel electrophoresis (SDS-PAGE) revealed that they contained three polypeptides (SF1-60 k, -57 k, -47 k) and one polypeptide (SF2-60 k), respectively. The amino acid compositions of these polypeptides were similar and their N-terminal amino acid sequences were identical. These polypeptides had an unusual amino acid composition, rich in Ser, Thr, Ala, and Gly, like the tube cement of a marine polychaete, Phragmatopoma californica. The IF, solubilized in aqueous formic acid after cleavage with cyanogen bromide, was shown by SDS-PAGE to contain eight fragment peptides (CB-peptides). N-terminal amino acid sequences of the CB-peptides were also determined. We conclude that the barnacle cement is composed of at least two types of protein: highly hydroxylated protein in the SF1 and SF2 and insoluble protein in the IF. The SDS-PAGE pattern of CB-peptides from the secondary cement was identical to that of the primary cement produced while the barnacle is attached to a substratum. In addition, immunoblot analysis, using a polyclonal antibody against one of the CB-peptides from the secondary cement, also cross-reacted with a CNBr-fragment peptide of the primary cement. These results indicate that the primary and secondary cements are similar in protein composition.

The Adhesive Protein cDNA of Mytilus galloprovincialis Encodes Decapeptide Repeats but No Hexapeptide Motif

A mussel is attached to hard surfaces by its byssus, which consists of a bundle of threads, each with a fibrous collagenous core coated with adhesive proteins. We constructed a cDNA library from RNA isolated from the foot of the mussel Mytilus galloprovincialis sampled in Japan. The library was probed with a nucleotide sequence corresponding to a part of the decapeptide repeat motif in the major adhesive protein of the closely related species M. edulis, and a clone including the whole coding region of the same adhesive protein of M. galloprovincialis was isolated. The sequences of the signal and nonrepetitive regions of the protein of M. galloprovincialis were homologous to those of M. edulis, despite several substitutions and a deletion of 18 amino acids. The repetitive region included a tetradecapeptide sequence and 62 repeats of the same decapeptide motif as in M. edulis, but hexapeptide sequences present in M. edulis were absent in the protein of M. galloprovincialis. In the decapeptide motif, two tyrosine residues, two lysine residues, and one of the two proline residues were highly conserved, but other residues were frequently substituted. In some residues in the decapeptide motif, specific codon usages were observed, suggesting that the nucleotide sequence itself has a function.

Inhibition of the growth of calcium carbonate crystals by multiple lectins in the coelomic fluid of the acorn barnacle Megabalanus rosa

Abstract The interaction between the multiple lectins (BRA-2, BRA-3) in the coelomic fluid of the acorn barnacle Megabalanus rosa and calcium ions was studied by ultraviolet difference spectroscopy. The association constants for BRA-2 and BRA-3 were 1.4 × 10 4 M −1 and 2.2 × 10 4 M −1 at pH 8.0, respectively. Multiple lectins inhibited the crystal growth of supersaturated calcium carbonate solution at a lectin concentration of > 0.1 mg/30 ml. Destruction of the carbohydrate-recognition domains of the lectins decreased the inhibitory activities on the crystal growth. Incorporation of BRAs into the crystal was demonstrated using FITC-labeled lectins.

Expression Sites of Two Byssal Protein Genes of Mytilus galloprovincialis

Mussels form byssal threads that can attach tenaciously to wet and irregular surfaces. The byssus consists of a fibrous collagenous core, and at least two types of polyphenolic proteins surround it. One of these proteins, designated Mgfp-1, coats the collagenous core; the other, designated Mgfp-2, is the major component of the terminal adhesive plaque of byssal threads. Both proteins contain 3,4-dihydroxyphenylalanine (DOPA) in their primary sequences. In this study, the sites of expression of the genes encoding the polyphenolic proteins were investigated in Mytilus galloprovincialis. By northern blot analysis, we found that the expression of both genes is foot-specific. Northern blot analysis of RNA isolated from the distal end and the remaining proximal portion of the foot indicated that the Mgfp-2 gene is expressed primarily in the distal part, whereas Mgfp-1 expression occurs in both parts. In situ hybridization indicated that the Mgfp-1 gene transcript is localized in the accessory gland along the ventral groove of the foot, and the Mgfp-2 gene transcript is localized in the phenol gland near the foot apex. Thus, it was shown that tissues expressing Mgfp-1 and Mgfp-2 are located around the ventral groove in an arrangement appropriate for byssus formation.

Adhesive protein cDNA sequence of the mussel Mytilus coruscus and its evolutionary implications.

AbstractcDNA encoding the adhesive protein of the musselMytilus coruscus (Mgfpl) was isolated. The coding region encoded 848 amino acids (a.a.) comprising the 20-a.a. signal peptide, the 21-a.a. nonrepetitive linker, and the 805-a.a. repetitive domain. Although the first 204 nucleotides and the 3′-untranslated region of Mgfpl cDNA were homologous to corresponding parts ofM. galloprovincialis adhesive protein (Mgfpl) cDNA, the other parts diverged. The representative repeat motif of the repetitive domain, YKPK(1/P)(S/T)YPP(T/S), was similar but slightly different from the repeat motif of Mgfpl. The codon usage patterns for the same amino acids were different in different positions of the decapeptide motif. Almost identical nucleotide sequences encoding the two to 13 repeats appeared several times in the repetitive region, which suggests that the adhesive protein genes of mussels have evolved through the duplication of these repeat units.

CULTURED MUSSEL FOOT CELLS EXPRESSING BYSSAL PROTEIN GENES

Primary and secondary culture of isolated foot cells of the mussel Mytilus galloprovincialis was carried out with modified Leibovitzs L-15 medium containing 20% fetal calf serum, 20 mg/l kanamycin and 20 mg/l streptomycin, which was adjusted to pH 7.5 and to the osmolarity of 950 mOsM/kg. Foot cells, dissociated by a collagenase treatment, successfully attached, spread and formed a monolayer in 4 to 7 days on a dish coated with collagen type I. This culture method gave reproducible results in repeated experiments. When cells were detached with trypsin from the first culture and subcultured in a flesh medium, they adhered and flattened onto the surface of cultural dishes, and propagated into confluent monolayers within 7 days. The proliferation of cells in the primary and secondary cultures was confirmed by the measurement of DNA synthesis. By RNA blot analysis using probes for the genes encoding three byssal proteins Mgfp-1, -2 and -3, it was shown that all three genes were transcribed in the primary and secondary culture although the Mgfp-1 transcription was weak. These findings suggest that the primary and secondary culture system of mussel foot cells can be used for the in vitro study of expression of at least two byssal protein genes. J. Exp. Zool. 283:131–136, 1999.

Molluscan Adhesive Protein Genes

In the marine intertidal zones, a variety of sessile invertebrates are found, each of which has evolved a unique strategy for adhesion. The most representative sessile invertebrates are mussels of the family Mytilidae (Bivalvia, Mollusca). Of the various spedes in the Mytilidae, most research has been conducted on the blue mussel (common mussel), Mytilus edulis. This species is commonly seen on temperate coasts of the northern and southem hemispheres, but the taxonomies of this and related species are not simple. Many species have been described as belonging to the genus Mytilus but recently they have been combined into just five species: Mytilus edulis, M. galloprovincialis, N. trossulus, M, californianus and N. coruscus. Of these, the closely related species M. edulis, M. galloprovincialis and M. trossulus are called the Mytilus edulis complex (see also Chapter 18). Since they are morphologically very similar and shell shapes are often influenced by environmental conditions, it is quite difficult to identify them by morphological characters alone (Gosling, 1984; Koehn, 1991; Gardner, 1992; Seed, 1992).