Structure-function Relationship Of Serpin-type Trypsin Inhibitor In Silkworm, Bombyx Mori

Protease inhibitor is responsible for inhibiting the decomposition by harmful protease, which is widely distributed in animals, plants, fungi, bacteria and viruses in vivo. The most widely studied protease inhibitors are serine protease inhibitors, the main regulator of the protease hydrolysis cascade, regulating animal, insect coagulation, inflammation, elastin repair, tissue remoldeling and apoptosis processes; in plants, serine protease inhibitors can also be a defense against insects and pathogens, excessive regulation of adversity proteolytic enzyme destruction, as well as an increase in insect resistance and so play an important role. In accordance with the mechanism of serine proteases can be divided into three categories:canonical inhibitors, non-canonical inhibitors and serpins. Canonical inhibitors use simple "lock and key" molecules that bind to and block access to the protease active site, by contrast serpins are covalently bound to a solid substrate through the RCL formed complex, as a result the inhibitory conformation is irreversible.In this study, we used silkworm as the main object of study. Previous investigations have shown that serine protease inhibitors are precise regulation of proteolytic involved in silkworm growth, development, molting and metamorphosis, such as growth and development, and is closely related to the response against invading pathogens process. However, previous studies on serpin-type trypsin inhibitor mainly in the fat body, silk glands, digestive fluids and cocoon shell, yet body wall is less studied. In addition, previous studies concentrated on the biochemistry and genetic ructions, but very few details about how structures related to functions were revealed. In the innar-immune defense process of the silkworm, pathogenic microorganisms mainly secret proteolytic penetrate that dissolve the body wall, so this paper made a detailed insight on trypsin inhibitor origined from silkworm body wall.After many experiments, we established a purification method with ammonium sulfate precipitation and Trypsin inhibitor Sepharose 4B affinity chromatography as a major step, and partially seperated the trypsin inhibitors purified from P50 strains total protein in the body wall. Gelatin-Native PAGE technology was also apllied in this study, which can be intuitivlye qualified the ability of inhibitory activity by observing gel bands, and the separation gel with trypsin inhibitor showed activity of more than 12 bands. Comparing with the trypsin activity bands of hemolymph presents four differences. I thermal stability studies, the silkworm body wall TIs activity bands began to decrease and fade above 60 ℃, indicating that the thermal stability is not high; Only two bands with molecular weight of 18-25 kDa performed a extremely high thermal stability, even after treatment at 100 ℃, can still maintain a certain activity. In pH stability test, observed the body wall TIs remained stable in weakly acidic （pH 3） or alkaline （pH 10） environment.After the partially purified, TIs from body wall went through SDS-PAGE and Gelatin-Native PAGE electrophoresis, we selected the clear protein bands on SDS-PAGE separating gel for MALDI-TOF-MASS spectrometry analysis, which was also have the greatest activity of glues on the Gelatin-Native PAGE gel. Then we collected a large number of peptide mass fingerprint data （PMF）, analysised by searching protein corresponding information in UniProt database of Mascot. Finally, determined three corresponding proteins, which are silkworm antitrypsin isoform 1 （SW-AT-1）, serine proteinase inhibitor-like protein and a peptidase which could inhibit cysteine protease, and also obtained theoretical molecular weight 43428.4 Da,43345.43 Da and 77611.43 Da, respectively; isoelectric points are 5.41,5.19 and 6.37, respectively. Their UniProt aecession numbers are:C7ASM9, Q1HPQ5 and D2KMR2. Bioinformatic analysis showed silkworm antitrypsin isoform 1 （SW-AT-1） belongs to serpin family; Q1HPQ5 is an inactive precursor zymogens that are cleaved during limited proteolysis to generate their active forms. The next step in the research is intended to explore the relationship between structure and their function, so we chose to SW-AT-1 and serine proteinase-like protein gene was cloned, and through prokaryotic vector for recombinant expression in vitro. The Expression of recombinant proteins were used to structural and functional studies.The accquired full length of amino acid sequence was submitted to NCBI Protein BLAST, and we found the whole mRNA sequence coding SW-AT-1 and serine proteinase-like protein, GenBank accession number, respectively:FJ613793 and NM₀01046997.2. For SW-AT sequence bioinformatics analysis showed that SW-AT gene has 10 exons and 9 introns,9 exons constitute four isomers, namely SW-AT-1, SW-AT-2, SW-AT-3 and SW-AT-4, however, SW-AT-1 gene have been reported that has the highest expression in the body wall, and mainly expressed in the body wall, and the difference between the base of the four isomers’genes was located at the mRNA sequence 1200-121 Obp, which also the end of the coding region of the mature peptide, so we chose SW-AT-1 as a template for cloning research. By PCR amplification and sequencing of the fragment detected only SW-AT-1 gene, we did not detect other three isomers. CDS region of the SW-AT-1 gene contains 1310 nucleotides, encoding 392 amino acids, and experiments designed primers removed the signal peptide coding region. Protein structure analysis also showed SW-AT-1 contains the typical serpin superfamily domains. Experiment by bacteria PCR, restriction and DNA sequencing validation verified recombinant expression vector pET-28a-SW-AT-1 successfully constructed. The recombinant expression vector transferred into E. coli strain C43 in order to get prokaryotic expressioned protein, the result showed the presence of rSW-AT-1 abundant in supernatant of disrupted cells, however serine proteinase-like protein was formed as inactive inclusion bodies precipitated.Collected recombinant SW-AT-1 protein expressed in E.coli strain C43 and purified using Ni-NTA affinity chromatography, and large amounts of purified protein were eluted. In the determination of rSW-AT-1 protein inhibitory activity, we found that the thermal stability is not high, the denaturation temperature between 47-50 ℃, identical with the gelatin-Native PAGE electrophoresis. The inhibitory activity was relatively high between pH 4-pH 10, and activities is stable only in a weakly alkaline or acid environment. While in chymotrypsin inhibitory activity assay, SW-AT-1 exhibited the same characters. Followed by determination of SW-AT-1 inhibitor constant analysis showed a high affinity with two proteases, and Ki values were 1.73×10-10 M and 0.86x10-10 M, respectively. The Ki values indicate the high affinity of rSW-AT-1 with trypsin as well as chymotrypsin. The Dixon plot performed competitive inhibition effect on the dynamics of the relationship between enzymes.Protein three-dimensional structure predictions were initiated by using SWISS MODEL （http://swissmodel.expasy.org）. The program was run using Manduca sexta Serpin-protease 1K complex （1SEK） as the template （Protein Data Bank code 1SEK, GenBank ID:AAA29334）. The semi refined model was then sent to the SWISS MODEL server for final refinement. The evaluation was carried out on PSVS server （http://psvs-1₄-dev.nesg.org/）, with Procheck （http://www.ebi.ac.uk/thornton-srv/software/PROCHECK/） score:87.2% most favoured regions,12.2% additionally allowed regions,0.6% generously allowed regions, and none disallowed regions. Images were generated in the modeling package Pymol vl.4. Then, in the NCBI conserved domain search service, and again determined the reliability of structural simulation, three-dimensional simulations show that the structure of RCL region amino acid sites identified G327, A328, E329 and A330 RCL located N-terminal, F352, N353, A354, N355, K356 and P357 located on the C-terminal sides of RCL. Corresponding to the 10-bit fixed-point,10 mutants G327A, A328G, E329G, A330G, F352A, N353D, A354G, N355S, K356G and P357G was to construct. Take the same steps prokaryotic expression and purification, the purified protein of wild-type and mutants were carried out on circular dichroism chromatographic analysis. The secondary and tertiary structure of rSW-AT-1 was analyzed by circular dichroism （CD） under different temperatures and pH. As shown in results, the percentages of secondary structural elements of rSW-AT-1 showed high contents of a-helices （33.7%） and unordered structures （31.4%）, and low content of β-sheets （16.6%） and turns （16.8%）. Under thermal treatments between 25 ℃ to 65 ℃, the secondary structure remained comparatively stable. In contrast, the tertiary structure, as shown by the 272-nm absorbance in near-UV CD, was greatly changed under different temperatures. Similar with the changes in tertiary structure, the inhibitory activity was dropped rapidly above 45℃, and merely lost above 50℃. pH had clearly severe impact on secondary structures. However, the changes of inhibitory activity still fitted well with the changes of tertiary structure. These results indicated that the tertiary structure, other than secondary elements, was the determinant to the inhibitory ability of SW-AT-1. In this study, mutation of E329 impaired SW-AT-1 activity. Since the tertiary structure of E329G was almost disrupted, we proposed that mutation of E329 might lead to the insertion of the RCL into beta-sheet, where it could not interact with the target protease. Surprisingly, we found that the carboxy-terminal side of RCL also affected the inhibitory activity. Mutations at N355 and K356 with small residues such as serine or glycine promoted the inhibitory activity. It could be proposed that in these mutants, RCL might be expelled from beta-sheet and the P1 residue flips to an exposed protease-accessible conformation. However, such conformational rearrangements have just little effects on the structure of the molecule.Taken together, we characterized SW-AT-1 as both trypsin and chymotrypsin inhibitor, and such inhibitory activities were mainly attributed to the proper conformation of RCL in the structure. Both of the N- and C-terminal sides of RCL have effects on the activity, and G327 and E329 play an important role in the proper folding of RCL.