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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

Inhibitory activity of serpin18 towards fibroinase.(A) The bar graph diagrams of residual activities of fibroinase incubated with serpin18 at molar ratios of 5. (B) The reaction between serpin18 and fibroinase were analyzed by SDS-PAGE method followed by Coomassie-staining and Lanes 1 to 4: 0.5 mg/ml, 1.0 mg/ml, 1.5 mg/ml, 2.0 mg/ml serpin18 without fibroinase; Lane 5, protein marker; Lane 6, 0.25 mg/ml fibroinase; Lanes 7 to 10: samples corresponding to lanes 1 to 4, respectively, with 0.25 mg/ml fibroinase. (C) Western blot analysis the reaction between serpin18 and fibroinase using the antibody of fibroinase. Lane 1: 0.25 mg/ml fibroinase; Lanes 2 to 5: 0.5 mg/ml, 1.0 mg/ml, 1.5 mg/ml, 2.0 mg/ml serpin18 with 0.25 mg/ml fibroinase. (D) Western blot analysis for fibroinase in the developmental stages of the fifth instar and the wandering periods in the A-MSG. The gels were run under the same experimental conditions and the full length blots and gels are presented in Supplementary Figure S7.
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f5: Inhibitory activity of serpin18 towards fibroinase.(A) The bar graph diagrams of residual activities of fibroinase incubated with serpin18 at molar ratios of 5. (B) The reaction between serpin18 and fibroinase were analyzed by SDS-PAGE method followed by Coomassie-staining and Lanes 1 to 4: 0.5 mg/ml, 1.0 mg/ml, 1.5 mg/ml, 2.0 mg/ml serpin18 without fibroinase; Lane 5, protein marker; Lane 6, 0.25 mg/ml fibroinase; Lanes 7 to 10: samples corresponding to lanes 1 to 4, respectively, with 0.25 mg/ml fibroinase. (C) Western blot analysis the reaction between serpin18 and fibroinase using the antibody of fibroinase. Lane 1: 0.25 mg/ml fibroinase; Lanes 2 to 5: 0.5 mg/ml, 1.0 mg/ml, 1.5 mg/ml, 2.0 mg/ml serpin18 with 0.25 mg/ml fibroinase. (D) Western blot analysis for fibroinase in the developmental stages of the fifth instar and the wandering periods in the A-MSG. The gels were run under the same experimental conditions and the full length blots and gels are presented in Supplementary Figure S7.

Mentions: To determine whether serpin18 could inactivate fibroinase, we purified the naturally occurring fibroinase protein from silk gland lysates as previously described2728 and tested the inhibiton of fibroinase by serpin18. Serpin18 showed significant inhibitory activity against fibroinase, with its proteolytic activity showing a 73% reduction (Fig. 5A). Similar to the inhibition of papain by serpin18, the reduction in fibroinase was not a consequence of the formation of a stable serpin-fibroinase complex, but instead the digestion of both protease and serpin was observed (Fig. 5B,C).These results suggested that fibroinase is one of the physiological targets of serpin18. Notably, fibroinase from B. mori silk glands is a cathepsin L-like cysteine protease that catalyzes the hydrolysis of liquid silk proteins (including fibroin and sericin)2829. Thus, we conclude that serpin18 plays a physiological role in regulating the activity of fibroinase and protecting silk proteins from degradation during B. mori development.


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)

Inhibitory activity of serpin18 towards fibroinase.(A) The bar graph diagrams of residual activities of fibroinase incubated with serpin18 at molar ratios of 5. (B) The reaction between serpin18 and fibroinase were analyzed by SDS-PAGE method followed by Coomassie-staining and Lanes 1 to 4: 0.5 mg/ml, 1.0 mg/ml, 1.5 mg/ml, 2.0 mg/ml serpin18 without fibroinase; Lane 5, protein marker; Lane 6, 0.25 mg/ml fibroinase; Lanes 7 to 10: samples corresponding to lanes 1 to 4, respectively, with 0.25 mg/ml fibroinase. (C) Western blot analysis the reaction between serpin18 and fibroinase using the antibody of fibroinase. Lane 1: 0.25 mg/ml fibroinase; Lanes 2 to 5: 0.5 mg/ml, 1.0 mg/ml, 1.5 mg/ml, 2.0 mg/ml serpin18 with 0.25 mg/ml fibroinase. (D) Western blot analysis for fibroinase in the developmental stages of the fifth instar and the wandering periods in the A-MSG. The gels were run under the same experimental conditions and the full length blots and gels are presented in Supplementary Figure S7.
© Copyright Policy - open-access
Related In: Results  -  Collection

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getmorefigures.php?uid=PMC4493575&req=5

f5: Inhibitory activity of serpin18 towards fibroinase.(A) The bar graph diagrams of residual activities of fibroinase incubated with serpin18 at molar ratios of 5. (B) The reaction between serpin18 and fibroinase were analyzed by SDS-PAGE method followed by Coomassie-staining and Lanes 1 to 4: 0.5 mg/ml, 1.0 mg/ml, 1.5 mg/ml, 2.0 mg/ml serpin18 without fibroinase; Lane 5, protein marker; Lane 6, 0.25 mg/ml fibroinase; Lanes 7 to 10: samples corresponding to lanes 1 to 4, respectively, with 0.25 mg/ml fibroinase. (C) Western blot analysis the reaction between serpin18 and fibroinase using the antibody of fibroinase. Lane 1: 0.25 mg/ml fibroinase; Lanes 2 to 5: 0.5 mg/ml, 1.0 mg/ml, 1.5 mg/ml, 2.0 mg/ml serpin18 with 0.25 mg/ml fibroinase. (D) Western blot analysis for fibroinase in the developmental stages of the fifth instar and the wandering periods in the A-MSG. The gels were run under the same experimental conditions and the full length blots and gels are presented in Supplementary Figure S7.
Mentions: To determine whether serpin18 could inactivate fibroinase, we purified the naturally occurring fibroinase protein from silk gland lysates as previously described2728 and tested the inhibiton of fibroinase by serpin18. Serpin18 showed significant inhibitory activity against fibroinase, with its proteolytic activity showing a 73% reduction (Fig. 5A). Similar to the inhibition of papain by serpin18, the reduction in fibroinase was not a consequence of the formation of a stable serpin-fibroinase complex, but instead the digestion of both protease and serpin was observed (Fig. 5B,C).These results suggested that fibroinase is one of the physiological targets of serpin18. Notably, fibroinase from B. mori silk glands is a cathepsin L-like cysteine protease that catalyzes the hydrolysis of liquid silk proteins (including fibroin and sericin)2829. Thus, we conclude that serpin18 plays a physiological role in regulating the activity of fibroinase and protecting silk proteins from degradation during B. mori development.

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