Koji Inoue

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.

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.

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.

Conservative Structure of the Plaque Matrix Protein of Mussels in the Genus Mytilus

Abstract: The complementary DNA encoding the byssal plaque matrix protein (fp-2) of the mussel Mytilus coruscus was isolated. The predicted amino acid sequence (474 amino acids) consists of four parts: the signal peptide, the amino-terminal nonrepetitive domain, the central repetitive domain containing 11 repeats of an epidermal growth factor–like motif, and the carboxy-terminal nonrepetitive domain. The amino acid sequence is 82.7%, similar to that of fp-2 of Mytilus galloprovincialis, and the basic structure including number and motif of repeats is highly conservative. Amino acid substitutions are less frequent in ``consensus positions of the central repetitive domain (13.1%), and most of them are changes from irregular amino acids to regular ones. Thus, the structure of fp-2 was found to be conservative between species. It was presumed that the basic structure of fp-2 is unchangeable to maintain the flexible and durable matrix structure and that variation is not required because fp-2 is protected by other surface proteins.