Novel cathepsin B and cathepsin B-like cysteine protease of Naegleria fowleri excretory-secretory proteins and their biochemical properties.
Naegleria fowleri causes a lethal primary amoebic meningoencephalitis (PAM) in humans and experimental animals, which leads to death within 7-14 days. Cysteine proteases of parasites play key roles in nutrient uptake, excystment/encystment, host tissue invasion, and immune evasion. In this study, we cloned N. fowleri cathepsin B (nfcpb) and cathepsin B-like (nfcpb-L) genes from our cDNA library of N. fowleri. The full-length sequences of genes were 1,038 and 939 bp (encoded 345 and 313 amino acids), and molecular weights were 38.4 and 34 kDa, respectively. Also, nfcpb and nfcpb-L showed a 56 and 46 % identity to Naegleria gruberi cathepsin B and cathepsin B-like enzyme, respectively. Recombinant NfCPB (rNfCPB) and NfCPB-L (rNfCPB-L) proteins were expressed by the pEX5-NT/TOPO vector that was transformed into Escherichia coli BL21, and they showed 38.4 and 34 kDa bands on sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) and Western blot analysis using their respective antibodies. Proteolytic activity of refolded rNfCPB and rNfCPB-L was maximum at a pH of 4.5, and the most effective substrate was Z-LR-MCA. rNfCPB and rNfCPB-L showed proteolytic activity for several proteins such as IgA, IgG, IgM, collagen, fibronectin, hemoglobin, and albumin. These results suggested that NfCPB and NfCPB-L cysteine protease are important components of the N. fowleri ESP, and they may play important roles in host tissue invasion and immune evasion as pathogens that cause N. fowleri PAM.
Department of Microbiology, Ajou University School of Medicine, Suwon, 443-721, Republic of Korea.
Jinyoung
J
Lee
Jong-Hyun
JH
Kim
Hae-Jin
HJ
Sohn
Hee-Jong
HJ
Yang
Byoung-Kuk
BK
Na
Yong-Joon
YJ
Chwae
Sun
S
Park
Kyongmin
K
Kim
Ho-Joon
HJ
Shin
D000595:Amino Acid Sequence; D001483:Base Sequence; D002401:Cathepsin B / Q000235:genetics / Q000378:metabolism*; D003001:Cloning, Molecular; D057056:Cysteine Proteases / Q000235:genetics / Q000378:metabolism*; D004795:Enzyme Stability; D015723:Gene Library; D008969:Molecular Sequence Data; D016848:Naegleria fowleri / Q000201:enzymology* / Q000235:genetics; D059748:Proteolysis; D016053:RNA, Protozoan / Q000235:genetics; D011994:Recombinant Proteins / Q000235:genetics / Q000378:metabolism; D013379:Substrate Specificity
D016053:RNA, Protozoan; D011994:Recombinant Proteins; D057056:Cysteine Proteases; D002401:Cathepsin B