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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

Expression patterns and localization of serpins in the silk gland.(A) Expression patterns of serpin16, serpin18 and serpin22 genes in the day1, 3, 5, 7 of the fifth instar (V-1d, V-3d, V-5d, V-7d), and the hour 12 after wandering (W-1d). (B) Expression patterns of serpin were from female or male larvae in the different segments of the silk gland in the day 5 of the fifth instar. Western blot analysis for serpin18 and its paralogs in the (C) developmental stages of the fifth instar and (D) different segments of the silk gland. The silkworm tubulin was used as internal control. The gels were run under the same experimental conditions and the full length blots and gels are presented in Supplementary Figure S6. (E) Immunofluorescence analysis of serpin18 and its paralogs in the A-MSG on fifth day of the fifth instar. Slides were incubated with anti-serpin16 antibody followed by the secondary antibody labeled with FITC (greeen) and counterstained with DAPI (blue). Control experiments were also performed using pre-immune serum. Bar, 100 μm.
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f4: Expression patterns and localization of serpins in the silk gland.(A) Expression patterns of serpin16, serpin18 and serpin22 genes in the day1, 3, 5, 7 of the fifth instar (V-1d, V-3d, V-5d, V-7d), and the hour 12 after wandering (W-1d). (B) Expression patterns of serpin were from female or male larvae in the different segments of the silk gland in the day 5 of the fifth instar. Western blot analysis for serpin18 and its paralogs in the (C) developmental stages of the fifth instar and (D) different segments of the silk gland. The silkworm tubulin was used as internal control. The gels were run under the same experimental conditions and the full length blots and gels are presented in Supplementary Figure S6. (E) Immunofluorescence analysis of serpin18 and its paralogs in the A-MSG on fifth day of the fifth instar. Slides were incubated with anti-serpin16 antibody followed by the secondary antibody labeled with FITC (greeen) and counterstained with DAPI (blue). Control experiments were also performed using pre-immune serum. Bar, 100 μm.

Mentions: To elucidate the physiological roles of serpins in the silk gland, semi-quantitative RT–PCR was used to analyze the temporal–spatial expression profiles of serpin18 and its paralogs (serpin16 and serpin22). These serpins showed similar expression patterns during the silkworm development: expression levels increased from the first to the fifth day, decreased on the seventh day, and disappeared by the wandering stage (Fig. 4A). We then further divided the silk gland into five morphologically and functionally distinct compartments (anterior silk gland, ASG; anterior/middle/posterior regions of the middle silk gland, A/M/P-MSG; and posterior silk gland, PSG; Fig. S4), and then investigated the expression patterns in each region on the fifth day of the fifth instar. We found that all three serpins were expressed exclusively in the MSG, with the high expression levels in the A-MSG and the low levels in the M-MSG (Fig. 4B).


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)

Expression patterns and localization of serpins in the silk gland.(A) Expression patterns of serpin16, serpin18 and serpin22 genes in the day1, 3, 5, 7 of the fifth instar (V-1d, V-3d, V-5d, V-7d), and the hour 12 after wandering (W-1d). (B) Expression patterns of serpin were from female or male larvae in the different segments of the silk gland in the day 5 of the fifth instar. Western blot analysis for serpin18 and its paralogs in the (C) developmental stages of the fifth instar and (D) different segments of the silk gland. The silkworm tubulin was used as internal control. The gels were run under the same experimental conditions and the full length blots and gels are presented in Supplementary Figure S6. (E) Immunofluorescence analysis of serpin18 and its paralogs in the A-MSG on fifth day of the fifth instar. Slides were incubated with anti-serpin16 antibody followed by the secondary antibody labeled with FITC (greeen) and counterstained with DAPI (blue). Control experiments were also performed using pre-immune serum. Bar, 100 μm.
© Copyright Policy - open-access
Related In: Results  -  Collection

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

f4: Expression patterns and localization of serpins in the silk gland.(A) Expression patterns of serpin16, serpin18 and serpin22 genes in the day1, 3, 5, 7 of the fifth instar (V-1d, V-3d, V-5d, V-7d), and the hour 12 after wandering (W-1d). (B) Expression patterns of serpin were from female or male larvae in the different segments of the silk gland in the day 5 of the fifth instar. Western blot analysis for serpin18 and its paralogs in the (C) developmental stages of the fifth instar and (D) different segments of the silk gland. The silkworm tubulin was used as internal control. The gels were run under the same experimental conditions and the full length blots and gels are presented in Supplementary Figure S6. (E) Immunofluorescence analysis of serpin18 and its paralogs in the A-MSG on fifth day of the fifth instar. Slides were incubated with anti-serpin16 antibody followed by the secondary antibody labeled with FITC (greeen) and counterstained with DAPI (blue). Control experiments were also performed using pre-immune serum. Bar, 100 μm.
Mentions: To elucidate the physiological roles of serpins in the silk gland, semi-quantitative RT–PCR was used to analyze the temporal–spatial expression profiles of serpin18 and its paralogs (serpin16 and serpin22). These serpins showed similar expression patterns during the silkworm development: expression levels increased from the first to the fifth day, decreased on the seventh day, and disappeared by the wandering stage (Fig. 4A). We then further divided the silk gland into five morphologically and functionally distinct compartments (anterior silk gland, ASG; anterior/middle/posterior regions of the middle silk gland, A/M/P-MSG; and posterior silk gland, PSG; Fig. S4), and then investigated the expression patterns in each region on the fifth day of the fifth instar. We found that all three serpins were expressed exclusively in the MSG, with the high expression levels in the A-MSG and the low levels in the M-MSG (Fig. 4B).

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