Identification And Functional Characterization Of Protease Inhibitors In The Silk Gland Of Silkworm, Bombyx Mori

The silkworm, Bombyx mori, is a lepidopteran insect, which was first domesticated by human5000years ago, since it could spin cocoon. At the beginning of last century, B. mori became a kind of model organism, and made great contributions to the development of biological basic science. At present, as the lepidopteran model insect, the silkworm has become a major research object for all kinds of biological subjects, such as genetics, physiology, molecular biology, cell biology and genomics.Silkworm is an important economic insect, since it can produce and secrete silk proteins efficiently. Researches on the mechanism of silk secretion make silkworm become a model organism of silk-produce insects. The silk gland is the only spinning organ, and could be divided into three compartments according to the morphology and function, including anterior silk gland, middle silk gland, and posterior silk gland. Fibroins are produced by the posterior silk gland, and wrapped by the sericins when flow through the middle silk gland, and finally secreted to the exterior by the narrow anterior silk gland. Under actions of multiple factors, the fibroin conformation changes dramatically, and forms water-insoluble fibre. As a natural polymer material, the silkworm silk now is mainly applied in textile industry, but it has an increasingly wide utilization in other fileds, such as biomedical, commetic, and health care. Silkworm could produce silk at the normal atmospheric temperature and pressure. It is related with the complex processing of silk proteins in the silk gland. Many researches have revealed some changes of factors in the process of silk formation, for instance, the changes of ion categories and contents in the silk gland, and the change of fibroin conformation in the silk gland, but these are still not enough to reveal the whole formation process of silk fibre. Since the silkworm genome database was open for the whole world from2004, silkworm researches have entered into the big data times. To aim at important characteristic of silkworm, such as development, immunity, and silk synthesis, researchers performed analysis and systematization in many aspects, such as genomics, transcriptomics, proteomics, and metabolomics, with the hope of finding some complex biological networks and discovering novel important signaling pathways.The formation process of silk fibre is the transition process of silk protein conformation. Except fibroins and sericins, other proteins in the silk gland lumen must play important roles in this process, but no one has researched on what proteins are in the silk gland lumen and what their fuctions are. Therefore, we extracted proteins from the silk gland lumen, and performed high-revolution proteomics analysis by shotgun liquid chromatography-tandem mass spectrometry (LC-MS/MS), and then did systematic functional annotation and quantitative analysis. In order to reveal the fate of proteins in the silkg land, we also identified the protein components of silk fibres. The proteome results of silk gland luminal content and silk fibres indicated that protease inhibitors are very abundant. Therefore, we further analyzed the structure and classification of protease inhibitors in the silk gland and silk fibre, and determined their activities, and then isolated and purified several abundant protease inhibitors. We mainly researched the functions of Serpin-type serine protease inhibitors. Because that our Serpin16antibody could well recognize three high-homology proteins, including Serpinl6, Serpin18and Serpin22. We systematically investigated the variatations of the three Serpins in different developmental stages and different compartments, determined their inhibitory effection on various proteases, and verified their interactions with a protease which is also secreted by the silk gland.The main results are as follows:1. Shotgun proteomics analyses of silk gland luminal contents and silk fibres(1) Identification and quantification of proteins in the silk gland lumen: We collected the silk gland luminal content from five compartments (anterior silk gland (ASG), anterior part of middle silk gland (A-MSG), middle part of middle silk gland (M-MSG), posterior part of middle silk gland (P-MSG), and posterior silk gland (PSG)) in two stages (day5of the fifth instar (V5) and day1of the wandering (W1)), and identified1271proteins by LC-MS/MS. By systematically analyzing the gene ontology annotation, all the proteins were classified into11functional categories. After label-free quantification by using intensity based on absolute quantification (iBAQ) methods, we found that extracellular proteins have fewer protein numbers, but have much higher abundances than those of intracellular proteins. Fibroins and sericins constitute the most abundant compositions. From V5to W1, all the3fibroins and sericinl flow from the P-MSG and M-MSG to the A-MSG and ASG; both sericin2and sericin3are distributed in the lumen of A-MSG and ASG, but sericin2is down-regulated while sericin3is up-regulated; other abundant proteins include metabolism-related proteins, protease inhibiotors and proteins of unknown functions. From V5to W1, most of metabolism-related enzymes are significantly down-regulated in the lumen of A-MSG and ASG, suggesting the variation of biochemical reactions in the lumen of these two compartments.(2) Identification and quantification of proteins in the silk fibres:We analyzed seven silk fibre samples by LC-MS/MS, and identified500proteins. By systematically analyzing the gene ontology annotation, all the proteins were classified into8functional categories. By calculating the abundance proportion, we found that fibroins&sericins, proteins of unknown functions, and protease inhibitors constitute three major protein classifications. Fibroins and sericins account for46.2%-74.7%molar abundance in seven silk fibres. Due to the fact that fibroins and sericins have much higher molecular weight (100kDa-400kDa) than normal proteins, their relative mass proportion will be much higher than the relative molecular abudance proportion, accounting for84.6%-96.7%of the total silk proteins.2. Structure and activity analyses of protease inhibitors in the silk gland lumen and silk fibre(1) Structure analyses of protease inhibitors in the silk gland lumen and silk fibre:51protease inhibitors were detected in the silk gland lumen and silk fibre, and could be classified into13families according to their domains, including Serpin, TIL, Kunitz_BPTI, Kazal, amfpi, WAP, Pacifastin, PBP, A2M, Inhibitor_I29, Inhibitor_I68, Cystatin and proteasome inhibitor. The first9families belong to serine protease inhibitors,6families of which belong to the canonical protease inhibitors, including TIL, Kunitz_BPTI, Kazal, amfpi, WAP, Pacifastin, because that they have similar structures and inhibitory mechanisms. Canonical protease inhibitors are rich in cysteine residues, which could form multiple pairs of intradisulfide bonds, giving these inhibitors very stable structures. Canonical protease inhibitors always contain multiple tandem domains, making their inhibitory efficiency doubled and redoubled..(2) Inhibitory activities of protease inhibitors in the silk gland lumen and silk fibre: By prediction of the active sites of serine protease inhibitors, we found that silk gland could secrete multiple protease inhibitors, such as trypsin inhibitors, chymotrypsin inhibitors, elastase inhibitors and subtilisin inhibitors. The protease inhibitors that contain tandem inhibitor domains have multiple active sites, and thus have abilities to inhibit various proteases. We used5commercial proteases as the target enzymes to determine the activities and specificities of protease inhibitors in the cocoon silk. We found abundant protease K inhibitors and a small amount of trypsin inhibitors in the cocoon silk, and detected no or little chymotrypsin inhibitors, elastase inhibitors and cysteine inhibitors in the cocoon silk. The microorganisms such as bacteria and fungi could secrete subtilisins (eg. protease K) to digest proteins, and thus take in nutrients and invade other organisms. Therefore, abundant protease K inhibitors in the cocoon silk may play roles in defending against microorganisms.(3) Purification of protease inhibitors from cocoon silk: We prepared a trypsin affinity column and used it to isolate inhibitors from cocoon silk proteins, and finally got two tryspin inhibitors (TIs) with molecular weight of15kDa and13kDa, respectively, and thus named them as TI15and TI13. We found that TI15is the serine protease inhibitor BmSPI51by MALDI-TOF/TOF MS, which belongs to the Kunitz family. However, we can’t identify the TI13by mass spectrometry. We also prepared a protease K affinity column and used it to isolate inhibitors from cocoon silk proteins. A protease K inhibitor with around30kDa was purified and named as KI30. Although no reliable mass spectrometry result was got, the western blot result showed that the antibody of BmSPI39can cross-react with KI30, suggesting that KI30belongs to the TIL family and may be BmSPI39or a highly homologous protein of BmSPI39. Our label-free quantification results have shown that BmSPI51and BmSPI39are two most abundant protease inhibitors in the cocoon silk, implying that KI30is probably the BmSPI39. Both the BmSPI51and BmSPI39belong to the canonical protease inhibitors, and they were considered to play roles in proventing cocoon silk from being degraded in previous studies.3. Expression pattern analysis, purification and activity assay of Serpin-type serine protease inhibitors secreted by the silk gland(1) Specificity detection of the Serpin16antibody:Serpin16is the most abundant Serpin protein in the silk gland. In this study, we prepared rabbit polyclonal antibody against Serpin16protein, and identified its specificity by combinations of two-dimensional electrophoresis, western blot and MALDI TOF MS. After separation of silk gland proteins from day3of the fifth instar by two-dimensional electrophoresis, we performed western blot analysis and found that Serpin16antibody can react with two protein spots, named as spot1and spot2. MALDI TOF MS was used to identify the two protein spots, and found that spot1contains the unique peptides of Serpin16, and spot2contains the unique peptides of Serpin18and Serpin22, suggesting that Serpin16antibody can react with these three proteins, including Serpin16, Serpin18and Serpin22It is due to that their amino acids sequences are highly homologous. Therefore, we can use this antibody to investigate the expression stage and location of Serpin16, Serpin18and Serpin22.(2) Synthesis and secretion of Serpin16, Serpin18and Serpin22:The semi-quantitative RT-PCR results showed that silkworm Serpinl6, Serpinl8and Serpin22are expressed in the anterior part of middle silk gland before day7of the fifth instar. We also got similar results in the protein level by western blot. It showed that Serpin16, Serpin18and Serpin22proteins exist in the anterior part of middle silk gland and anterior silk gland. Serpin16increases from day1of the fifth instar to day5of the fifth instar, and then decreases, while Serpin18and Serpin22increase from early stage of the fifth instar to day7of the fifth instar, and then decreases. Both the western blot and immunofluorescent results suggested that Serpin16, Serpin18and Serpin22mainly exist in the lumen of silk gland. In conclusion, Serpin16, Serpin18and Serpin22are synthesized by the cells of the anterior part of middle silk gland from the early stage to the middle stage, and secreted into the silk gland lumen, and then decrease before the spinning stage.(3) Purification and activity assay of Serpin16, Serpin18and Serpin22:By comprehensive utilization of the anion-exchange chromatography and gel filtration, we purified Serpinl6, Serpin18and Serpin22from the lumen of anterior part of middle silk gland. The activity assay results indicated that Serpinl6, Serpinl8and Serpin22show significant inhibitory activities against subtilising A, protease K and papain, but weak inhibitory activities against trypsin, chymotrypsin, and elastase. The determination of protease acitivity on luminal proteins in the anterior silk gland suggested that protease activity is low on day5of the fifth instar, but increase significantly on day7of the fifth instar. Serpinl6, Serpin18and Serpin22showed significant inhibitory activities against proteases in the lumen of anterior part of middle silk gland on day7of the fifth instar larvae. In conclusion, on day7of the fifth instar before spinning, proteases show increasing acitivities in the lumen of anterior part of middle silk gland, but their inhibitors Serpin16, Serpin18and Serpin22decrease significantly. We speculate that Serpinl6, Serpin18and Serpin22may play roles in restricting their target proteases to special place and stage.4. Interaction between Serpin-type serine protease inhibitors and a protease secreted by the silk gland(1) Expression pattern of BmSP82:The label-free quantification result indicated that BmSP82is more abundant than other proteases in the lumen of the silk gland, and mainly exists in the anterior part of the middle silk gland. BmSP82is a potential target protease of Serpinl6, Serpin18and Serpin22. Therefore, we used semi-quantitative RT-PCR to investigate the expression pattern of BmSP82, and found that BmSP82is expressed only in the malpighian tubule and silk gland. After dividing the silk gland into5compartments, we found that BmSP82has a high expression in the anterior part of middle silk gland, but a low expression in other compartments of silk gland, which is similar with that of Serpinl6, Serpinl8and Serpin22. The temporal expression pattern indicated that BmSP82is highly expressed in the whole fifth instar, has the highest expression from day5of the fifth instar to day7of the fifth instar, which is different from that of Serpinl6, Serpinl8and Serpin22.(2) Interaction between BmSP82and Serpinl6, Serpin18and Serpin22:We constructed the recombinant expression vector of BmSP82. After transforming it into Pichia pastoris, we screened multiple inserts of BmSP82. After induction by methanol, we finally got BmSP82protein secreted by the yeast cells. Serine protease BmSP82is composed of the N-terminal CUB domian and the C-terminal catalytic domain. We used trypsin to digest the BmSP82, making the CUB domian and catalytic domain apart from each other, which finally caused mature BmSP82from pro-protease. We incubated the mature BmSP82with Serpin16, Serpin18and Serpin22, and found that they form complexes with much higher molecular weight by western blot.