Production and characterization of recombinant tachycitin, the Cys-rich chitin-binding protein.
Tachycitin is an invertebrate chitin-binding protein with an amidated C-terminus, and possesses antimicrobial activity against both fungi and bacteria. The (1)H-NMR-based tertiary structure of tachycitin was recently determined [Suetake et al. (2000) J. Biol. Chem., 275, 17929-17932]. In order to examine the structural and functional features of tachycitin more closely, we performed for the first time, gene expression, refolding, (15)N-NMR-based characterizations, and antimicrobial activity measurements of a recombinant tachycitin (rTcn) that does not have the amide group at the C-terminus. The NMR analysis indicated that rTcn possesses the same structural construction as the native tachycitin. The backbone (15)N relaxation measurements showed that the molecular motional correlation time of rTcn increases as its concentration increases, indicating that tachycitins have a tendency to aggregate with each other. rTcn exhibits antimicrobial activity against fungi but not against bacteria. The cell surface of fungi contains chitin as an essential constituent, but that of bacteria does not. These results suggest that not only the chitin-binding region but also the C-terminal amide group of tachycitin plays a significant role in its antimicrobial properties.
Suetake
Tetsuya
T
Division of Biological Sciences, Graduate School of Science, Hokkaido University, Sapporo 060-0810, Japan.
Aizawa
Tomoyasu
T
Koganesawa
Nozomi
N
Osaki
Tsukasa
T
Kobashigawa
Yoshihiro
Y
Demura
Makoto
M
Kawabata
Shun-Ichiro
S
Kawano
Keiichi
K
Tsuda
Sakae
S
Nitta
Katsutoshi
K
D000595:Amino Acid Sequence; D000818:Animals; D000900:Anti-Bacterial Agents / Q000737:chemistry / Q000494:pharmacology; D001483:Base Sequence; D001665:Binding Sites; D001798:Blood Proteins / Q000737:chemistry* / Q000378:metabolism / Q000494:pharmacology; D002352:Carrier Proteins / Q000737:chemistry* / Q000378:metabolism / Q000494:pharmacology; D002686:Chitin / Q000378:metabolism; D003545:Cysteine / Q000737:chemistry; D004247:DNA / Q000235:genetics; D066298:In Vitro Techniques; D008958:Models, Molecular; D019906:Nuclear Magnetic Resonance, Biomolecular; D011487:Protein Conformation; D015202:Protein Engineering; D011994:Recombinant Proteins / Q000737:chemistry / Q000378:metabolism / Q000494:pharmacology
D000900:Anti-Bacterial Agents; D001798:Blood Proteins; D002352:Carrier Proteins; D011994:Recombinant Proteins; C104989:tachycitin protein, Tachypleus tridentatus; D002686:Chitin; D004247:DNA; D003545:Cysteine