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Structural insights into the unique inhibitory mechanism of the silkworm protease inhibitor serpin18.

Guo PC, Dong Z, Zhao P, Zhang Y, He H, Tan X, Zhang W, Xia Q - Sci Rep (2015)

Bottom Line: Notably, this inhibitiory reaction results from the formation of an intermediate complex, which then follows for the digestion of protease and inhibitor into small fragments.This activity differs from previously reported modes of inhibition for serpins.Our findings have thus provided novel structural insights into the unique inhibitory mechanism of serpin18.

View Article: PubMed Central - PubMed

Affiliation: State Key Laboratory of Silkworm Genome Biology, Southwest University, 216, Tiansheng Road, Beibei, Chongqing 400716, People's Republic of China.

ABSTRACT
Serpins generally serve as inhibitors that utilize a mobile reactive center loop (RCL) as bait to trap protease targets. Here, we present the crystal structure of serpin18 from Bombyx mori at 1.65 Å resolution, which has a very short and stable RCL. Activity analysis showed that the inhibitory target of serpin18 is a cysteine protease rather than a serine protease. Notably, this inhibitiory reaction results from the formation of an intermediate complex, which then follows for the digestion of protease and inhibitor into small fragments. This activity differs from previously reported modes of inhibition for serpins. Our findings have thus provided novel structural insights into the unique inhibitory mechanism of serpin18. Furthermore, one physiological target of serpin18, fibroinase, was identified, which enables us to better define the potential role for serpin18 in regulating fibroinase activity during B. mori development.

No MeSH data available.


Related in: MedlinePlus

The inhibitory activity of serpin18.(A) The bar graph diagrams of residual activities of proteases incubated with serpin18 at molar ratios (inhibitor: protease) of 5. (B) Titration of papain with the wild-type serpin18 and variants, monitored by the loss of papain activity. (C) SDS-PAGE of the reaction between serpin18 and papain were analysed by Coomassie brilliant blue staining (CBBS). Lanes 1 to 3: 0.5 mg/ml, 1.0 mg/ml, 1.5 mg/ml serpin18 without papain; M, protein marker; Lane 4, 0.5 mg/ml papain; Lane 5–7, samples corresponding to lanes 1 to 3, respectively, with 0.5 mg/ml papain. Western blot analysis of the reaction between serpin18 and papain with antibodies against (D) anti-serpin and (E) anti-papain under the denaturing conditions. Lane 1, 0.5 mg/ml papain; Lanes 2 to 4: 0.5 mg/ml, 1.0 mg/ml, 1.5 mg/ml serpin18 with 0.5 mg/ml papain. The gels were run under the same experimental conditions.
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f3: The inhibitory activity of serpin18.(A) The bar graph diagrams of residual activities of proteases incubated with serpin18 at molar ratios (inhibitor: protease) of 5. (B) Titration of papain with the wild-type serpin18 and variants, monitored by the loss of papain activity. (C) SDS-PAGE of the reaction between serpin18 and papain were analysed by Coomassie brilliant blue staining (CBBS). Lanes 1 to 3: 0.5 mg/ml, 1.0 mg/ml, 1.5 mg/ml serpin18 without papain; M, protein marker; Lane 4, 0.5 mg/ml papain; Lane 5–7, samples corresponding to lanes 1 to 3, respectively, with 0.5 mg/ml papain. Western blot analysis of the reaction between serpin18 and papain with antibodies against (D) anti-serpin and (E) anti-papain under the denaturing conditions. Lane 1, 0.5 mg/ml papain; Lanes 2 to 4: 0.5 mg/ml, 1.0 mg/ml, 1.5 mg/ml serpin18 with 0.5 mg/ml papain. The gels were run under the same experimental conditions.

Mentions: To evaluate the inhibitory activity of serpin18, the candidate proteases trypsin, chymotrypsin, elastase, subtilisin, protease K, and papain were each incubated with the inhibitor. The residual proteolytic activity of trypsin, chymotrypsin, elastase, subtilisin, and protease K were each reduced by 4%–10%, implying that serpin18 has almost no inhibitory activity towards these serine proteases (Fig. 3A). Interestingly, serpin18 inhibited the proteolytic activity of papain (a cysteine protease) by 90% in a dose-dependent fashion (Fig. 3A,B). Moreover, the papain-like mammalian cysteine protease, cathepsin L, was also inactivated by serpin18 (Fig. 3A). These results demonstrated that serpin18 might act as a cysteine protease inhibitor, instead of as a serine protease inhibitor.


Structural insights into the unique inhibitory mechanism of the silkworm protease inhibitor serpin18.

Guo PC, Dong Z, Zhao P, Zhang Y, He H, Tan X, Zhang W, Xia Q - Sci Rep (2015)

The inhibitory activity of serpin18.(A) The bar graph diagrams of residual activities of proteases incubated with serpin18 at molar ratios (inhibitor: protease) of 5. (B) Titration of papain with the wild-type serpin18 and variants, monitored by the loss of papain activity. (C) SDS-PAGE of the reaction between serpin18 and papain were analysed by Coomassie brilliant blue staining (CBBS). Lanes 1 to 3: 0.5 mg/ml, 1.0 mg/ml, 1.5 mg/ml serpin18 without papain; M, protein marker; Lane 4, 0.5 mg/ml papain; Lane 5–7, samples corresponding to lanes 1 to 3, respectively, with 0.5 mg/ml papain. Western blot analysis of the reaction between serpin18 and papain with antibodies against (D) anti-serpin and (E) anti-papain under the denaturing conditions. Lane 1, 0.5 mg/ml papain; Lanes 2 to 4: 0.5 mg/ml, 1.0 mg/ml, 1.5 mg/ml serpin18 with 0.5 mg/ml papain. The gels were run under the same experimental conditions.
© Copyright Policy - open-access
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC4493575&req=5

f3: The inhibitory activity of serpin18.(A) The bar graph diagrams of residual activities of proteases incubated with serpin18 at molar ratios (inhibitor: protease) of 5. (B) Titration of papain with the wild-type serpin18 and variants, monitored by the loss of papain activity. (C) SDS-PAGE of the reaction between serpin18 and papain were analysed by Coomassie brilliant blue staining (CBBS). Lanes 1 to 3: 0.5 mg/ml, 1.0 mg/ml, 1.5 mg/ml serpin18 without papain; M, protein marker; Lane 4, 0.5 mg/ml papain; Lane 5–7, samples corresponding to lanes 1 to 3, respectively, with 0.5 mg/ml papain. Western blot analysis of the reaction between serpin18 and papain with antibodies against (D) anti-serpin and (E) anti-papain under the denaturing conditions. Lane 1, 0.5 mg/ml papain; Lanes 2 to 4: 0.5 mg/ml, 1.0 mg/ml, 1.5 mg/ml serpin18 with 0.5 mg/ml papain. The gels were run under the same experimental conditions.
Mentions: To evaluate the inhibitory activity of serpin18, the candidate proteases trypsin, chymotrypsin, elastase, subtilisin, protease K, and papain were each incubated with the inhibitor. The residual proteolytic activity of trypsin, chymotrypsin, elastase, subtilisin, and protease K were each reduced by 4%–10%, implying that serpin18 has almost no inhibitory activity towards these serine proteases (Fig. 3A). Interestingly, serpin18 inhibited the proteolytic activity of papain (a cysteine protease) by 90% in a dose-dependent fashion (Fig. 3A,B). Moreover, the papain-like mammalian cysteine protease, cathepsin L, was also inactivated by serpin18 (Fig. 3A). These results demonstrated that serpin18 might act as a cysteine protease inhibitor, instead of as a serine protease inhibitor.

Bottom Line: Notably, this inhibitiory reaction results from the formation of an intermediate complex, which then follows for the digestion of protease and inhibitor into small fragments.This activity differs from previously reported modes of inhibition for serpins.Our findings have thus provided novel structural insights into the unique inhibitory mechanism of serpin18.

View Article: PubMed Central - PubMed

Affiliation: State Key Laboratory of Silkworm Genome Biology, Southwest University, 216, Tiansheng Road, Beibei, Chongqing 400716, People's Republic of China.

ABSTRACT
Serpins generally serve as inhibitors that utilize a mobile reactive center loop (RCL) as bait to trap protease targets. Here, we present the crystal structure of serpin18 from Bombyx mori at 1.65 Å resolution, which has a very short and stable RCL. Activity analysis showed that the inhibitory target of serpin18 is a cysteine protease rather than a serine protease. Notably, this inhibitiory reaction results from the formation of an intermediate complex, which then follows for the digestion of protease and inhibitor into small fragments. This activity differs from previously reported modes of inhibition for serpins. Our findings have thus provided novel structural insights into the unique inhibitory mechanism of serpin18. Furthermore, one physiological target of serpin18, fibroinase, was identified, which enables us to better define the potential role for serpin18 in regulating fibroinase activity during B. mori development.

No MeSH data available.


Related in: MedlinePlus