title: Cloning and localization of MCdef, a defensin from Manila clams (Ruditapes philippinarum).
abstract: A defensin-like peptide was previously detected in hemocytes of Manila clams (Ruditapes philippinarum). In the current study, we cloned and characterized this defensin, designated MCdef. Cloning produced a full-length gene sequence of 201 bp predicted to encode a 66-amino-acid precursor protein maturing to a 44-amino-acid residue. Amino acid sequence analysis showed that MCdef is similar to defensins from marine mollusks and ticks. Phylogenetic analysis suggested that MCdef is closely related to defensins from Mytilus galloprovincialis (Mediterranean mussel) and Crassostrea gigas (Pacific cupped oyster). The three-dimensional structure of MCdef was modeled using the solution structure of C. gigas defensin as a template. With the exception of three variable loop areas, the modeled structure of MCdef was identical to that of C. gigas defensin. MCdef antiserum was raised against a synthetic MCdef peptide and verified by Western blotting using recombinant MCdef. RT-PCR analysis demonstrated high levels of MCdef mRNA in hemocytes and adductor, foot, gill, mantle, palp, and siphon tissues of Vibrio tapetis-infected Manila clams, whereas in V. tapetis-uninfected Manila clams, the level of MCdef mRNA was low in adductor, palp, and siphon tissues and even lower in the other tested tissues. Immunohistochemical analysis revealed high MCdef expression was detected in the gill, the mantle, and the digestive tubules of the diverticulum of V. tapetis-infected Manila clams. Minimum inhibitory concentration (MIC) of the purified rMCdef was determined. MCdef showed highest activity against Streptococcus iniae and Staphylococcus aureus.
authors.affiliation: Biochemistry Lab, Department of Medicine, School of Medicine, Jeju National University, Jeju 690-756, Republic of Korea.
authors.forename: Mausumi
authors.initials: M
authors.lastname: Adhya
authors.forename: Hee-Do
authors.initials: HD
authors.lastname: Jeung
authors.forename: Hyun-Sil
authors.initials: HS
authors.lastname: Kang
authors.forename: Kwang-Sik
authors.initials: KS
authors.lastname: Choi
authors.forename: Dong Sun
authors.initials: DS
authors.lastname: Lee
authors.forename: Moonjae
authors.initials: M
authors.lastname: Cho
mesh_terms: D000595:Amino Acid Sequence; D000818:Animals; D000900:Anti-Bacterial Agents / Q000494:pharmacology; D000917:Antibody Formation / Q000276:immunology; D000942:Antigens, Bacterial / Q000276:immunology; D001483:Base Sequence; D049872:Bivalvia / Q000166:cytology / Q000187:drug effects / Q000235:genetics* / Q000821:virology; D003001:Cloning, Molecular; D018076:DNA, Complementary / Q000235:genetics / Q000302:isolation & purification; D023082:Defensins / Q000737:chemistry / Q000235:genetics* / Q000494:pharmacology; D020869:Gene Expression Profiling; D007106:Immune Sera / Q000276:immunology; D007150:Immunohistochemistry; D008969:Molecular Sequence Data; D009928:Organ Specificity / Q000235:genetics; D010679:Philippines; D016133:Polymerase Chain Reaction; D021381:Protein Transport / Q000187:drug effects; D012333:RNA, Messenger / Q000235:genetics / Q000378:metabolism; D015203:Reproducibility of Results; D017422:Sequence Analysis, DNA; D014733:Vibrio / Q000187:drug effects / Q000276:immunology / Q000502:physiology
chemical_list: D000900:Anti-Bacterial Agents; D000942:Antigens, Bacterial; D018076:DNA, Complementary; D023082:Defensins; D007106:Immune Sera; D012333:RNA, Messenger