Enhanced anti-rotavirus action of human cystatin C by site-specific glycosylation in yeast.
The cDNA encoding human cystatin C (HCC) was subjected to site-specific substitution of alanine for serine at the position 37, to obtain the Asn(35)-Lys(36)-Ser(37) sequence that is a signal for asparagine-linked (N-linked) glycosylation of protein in eukaryotes, and was transformed into Pichia pastoris X33. As a result, 1.2 mg/L oligomannosyl HCC with a carbohydrate chain of Man(10)GlcNAc(2) was produced by the Pichia transformant. The oligomannosyl HCC was more stable at the low ionic strength condition of 50 mM potassium phosphate buffer, pH 7.0, than the wild-type. In addition, the oligomannosylation substantially improved the molecular stability of cystatin against an aspartic proteinase, cathepsin D, in which the susceptibility decreased to less than 50% of nonglycosylated one. The anti-rotavirus activity of HCC was substantially enhanced by the site-directed glycosylation using the yeast expression system. A MA-104 cell line was used as a host cell for human rotavirus type-2 Wa strain in this study, to which both the wild-type and oligomannosyl HCCs did not show cytotoxicity at a concentration of 100 mug/mL. More than 80% viability of the host cell infected with 1.0 x 10(5) PFU/mL of rotavirus was conserved under the condition coexisting with 75 mug/mL of the oligomannosyl HCC, which was 15.2% higher than that of wild-type HCC. Thus, the in vitro anti-rotavirus assay indicated that the supplement of a proper amount of the oligomannosyl HCC could be used as an anti-rotavirus agent.
Nakamura
Soichiro
S
Department of Life and Environmental Science, Shimane University, Shimane, Japan. soichiro@edu.shimane-u.ac.jp
Hata
Junya
J
Kawamukai
Makoto
M
Matsuda
Hideyuki
H
Ogawa
Masahiro
M
Nakamura
Kosuke
K
Jing
Hao
H
Kitts
David D
DD
Nakai
Shuryo
S
D019943:Amino Acid Substitution / Q000235:genetics; D000998:Antiviral Agents / Q000378:metabolism* / Q000494:pharmacology*; D001665:Binding Sites / Q000187:drug effects / Q000235:genetics; D002470:Cell Survival / Q000187:drug effects / Q000235:genetics; D055316:Cystatin C; D015891:Cystatins / Q000235:genetics / Q000378:metabolism* / Q000494:pharmacology*; D006031:Glycosylation / Q000187:drug effects; D006801:Humans; D016297:Mutagenesis, Site-Directed; D010843:Pichia / Q000235:genetics / Q000378:metabolism*; D012401:Rotavirus / Q000187:drug effects* / Q000378:metabolism; D014161:Transduction, Genetic
D000998:Antiviral Agents; C525030:CST3 protein, human; D055316:Cystatin C; D015891:Cystatins