Cloning, expression and antimicrobial activity of crustinPm1, a major isoform of crustin, from the black tiger shrimp Penaeus monodon.
Crustin antibacterial homologues, containing a whey acidic protein (WAP) domain, have been identified from the haemocyte library of the black tiger shrimp, Penaeus monodon. Sequence analysis of these cDNAs indicates the presence of several isoforms of crustin in P. monodon. CrustinPm1, the most abundant isoform, contains an open reading frame of 435bp encoding a precursor of 145 amino acids that comprises 17 amino acid signal peptides and 128 amino acid mature peptides. The peptides contain a Gly-Pro rich region at the amino-terminus and a single whey acidic protein (WAP) domain at the carboxyl-terminus. In order to characterize the properties and biological activities of this peptide, crustinPm1 was overexpressed in Escherichia coli. The recombinant crustinPm1 has a molecular mass of 14.7kDa with a predicted pI of 8.3. Antimicrobial assays demonstrated that recombinant crustinPm1 exhibited antimicrobial activity against only Gram-positive bacteria with strong inhibition against Staphylococcus aureus and Streptococcus iniae. In addition, the study of inhibition mechanism revealed that the antimicrobial activity of crustinPm1 was a result of bactericidal effect. In situ hybridization with crustinPm1 antisense probes showed strong hybridization signals in a certain haemocyte population of unchallenged shrimp, indicating that crustinPm1 transcript is differentially expressed in different subsets of haemocyte cells.
Supungul
Premruethai
P
Shrimp Molecular Biology and Genomics Laboratory, Department of Biochemistry, Faculty of Science, Chulalongkorn University, Bangkok 10330, Thailand.
Tang
Sureerat
S
Maneeruttanarungroj
Cherdsak
C
Rimphanitchayakit
Vichien
V
Hirono
Ikuo
I
Aoki
Takashi
T
Tassanakajon
Anchalee
A
D000595:Amino Acid Sequence; D000818:Animals; D000890:Anti-Infective Agents / Q000378:metabolism* / Q000494:pharmacology; D023181:Antimicrobial Cationic Peptides / Q000096:biosynthesis* / Q000235:genetics / Q000494:pharmacology; D001483:Base Sequence; D003001:Cloning, Molecular; D018076:DNA, Complementary / Q000378:metabolism; D006090:Gram-Negative Bacteria / Q000187:drug effects; D006094:Gram-Positive Bacteria / Q000187:drug effects; D006434:Hemocytes / Q000378:metabolism; D008969:Molecular Sequence Data; D033561:Penaeidae / Q000378:metabolism*; D010802:Phylogeny; D020033:Protein Isoforms / Q000235:genetics / Q000378:metabolism / Q000494:pharmacology
D000890:Anti-Infective Agents; D023181:Antimicrobial Cationic Peptides; D018076:DNA, Complementary; D020033:Protein Isoforms