Study On The Function Of Hemolymph Protein Bmhp20 In Silkworm, Bombyx Mori

Insects developed a powerful immune system in order to combat with the pathogens for their survival and reproduction in the special environment. Insects only have innate immune system, in which the humoral immunity and cell immunity are triggered by recognition of the conserved pathogen-associated molecular patterns (PAMPs) by pattern recognition receptors (PRRs). The humoral immunity refers to antimicrobial peptide (AMP) production, melanization, and clotting. The cell immunity includes the phagocytosis, coagulation, and encapsulation. Pathogen-recognition protein plays an important part in the insect innate immune system. Peptidoglycan (PGN)recognition proteins recognize the PGN on the surface of pathogens to elicit the production of anti-bacterial peptide.β-glucan recognition proteins also elicit similar physiological effect in insects. In addition to these two well-studied pattern-recognition proteins, there are also some other recognition proteins in insects responsible for immune response to the environment.Chymotrypsin inhibitor b1 (CIb1), which was purified from the silkworm larval hemolymph, is a Kunitz-type inhibitor. Recent studies showed that CIb1 participated in the immune response in Bombyx mori and had lipopolysaccharide (LPS)-binding activity. We speculated that CIb1 might mediate the pathogens-elicited signal transduction in insects.The present studies aimed to explore the function of CIb1 in B. mori. We purified a CIb1-binding protein(named as Bmhp20) and cloned the gene encoding Bmhp20 based on the N-terminal peptide sequencing. Then recombinant Bmhp20 protein was purified for functional analysis. The main results are as follows:1. The purification of silkworm CIb1 and its interaction with hemolymph proteins CIb1, a basic chymotrypsin inhibitor with a small molecular mass, was able to inhibit chymotrypsin activity in vitro. CIb1 purification strategy was designed by tracking chymotrypsin inhibitor activity. First, ammonium sulfate precipitation was used to remove some contaminous proteins. The pellet was further purified through gel filtration and ion exchange resin based on its molecular mass and isoelectric point. The purified CIb1 fraction was confirmed to contain a single protein by mass-spectrometry and chymotrypsin inhibitor activity staining. Biotin-labeled CIb1 was used to incubate with silkworm hemolymph. Far-Western results domestrated that CIb1 bound to silkworm hemolymph proteins when silkworm hemolymph was treated with lipopolysaccharide.2. Recombinant CIb1 protein expression, purification and its interaction with hemolymph proteinsThe gene encoding mature CIb1 was cloned into the pDEST17 for protein expression. The plasmid construct was transformed into four E. coli host strains:Rosetta, Origami B (DE3), BL21(DE3), JM109(DE3). CIb1 was a chymotrypsin inhibitor with 3 disulfide-bonds, thus the expressed recombinant protein correctly folded was used for further study. Chymotrypsin inhibitor activity assay suggested rCIb1 expressed in origami B(DE3) had the chymotrypsin inhibitor activity. rCIb1 was mainly present in the inclusion body. The rCIb1 in the inclusion body was dissolved by addition of urea, subjected to the Sephacryl S-200 purification and allowed for renaturation at the same time. Chymotrypsin inhibitor activity assay showed that the purified rCIb1 was active. And our previous studies showed CIb1 bound to silkworm hemolymph proteins post induced with lipopolysaccharide. To isolate the protein bound to CIb1, an in vitro binding assay was performed using recombinant CIb1-coupled Ni-NTA and larval hemolymph from LPS-immunized larvae (L24) of silkworm. The Ni-NTA column was washed with washing buffer to remove non-specific proteins and eluted with high concentration imidazole. The eluted high molecular mass proteins could be blotted with rCIb1 antibody, suggesting the eluted proteins may be a complex including rCIb1 and silkworm hemolymph proteins. And the complex was very stable in the present of SDS. We used electro-elution to isolate the protein from the eluted fraction in the pull-down assay. The amino-terminal sequence of the isolated protein "HGSSDVDGSGEVEAVAGTLK" was sequenced by automated Edman degradation.3. Gene cloning and expression analysis of CIb1-binding protein Bmhp20The N-terminal sequences were subjected to the silkworm database for a tBLASTn search and a putative gene BGIBMGA002175 showed similarity to the N-terminal sequences. The gene was cloned using primers designed according to its EST sequence and sequenced. And we found that the cloned sequence of the gene was identical to that of N-terminal sequences of CIb1-binding protein, suggesting the cloned gene was the CIb1-binding protein. Then, the 5'of the gene was cloned and the whole gene was obtained. The whole gene was 740 bp, with an 645 bp open reading frame encoding a 215-amino-acid peptide. The encoded protein with 18-amino-acid signal peptide at the N-terminus had a predicted molecular mass of 21.5 kDa and a predicted isoelectric point of 5.5. The protein was named as Bmhp20 (accession number: GU015849) according to its molecular mass and expression in hemolymph. Bmhp20 had three exons and two introns. And the sequences encoding the mature protein were located on the last exon. No proteins were found to show significant similarity to Bmhp20 through NCBI blast. Amino-acid composition analysis of the protein showed that Bmhp20 was composed of several amino acids:glycine (26.9%), serine (13.2%), phenylalanine (12.2%), aspartate acid (10.7%), and histidine (9.1%). ELM database analysis found that Bmhp20 was rich in glycosaminoglycan binding sites (DGSGE, DNSGF, NSGF, HSGF, DSGF, and DSAL).Silkworm microarray analysis using the fifth instar day 3 larvae domonstrated that Bmhp20 expression showed no difference between the male and female and was found in four tissues:head, cuticle, fat body and ovary. The expression pattern of Bmhp20 was consistent with that of RT-PCR analysis. RT-PCR analysis on the expression of Bmhp20 during development showed that Bmhp20 was highly expressed in all the larval stages, on the first day of wandering stage, and then decreased. Bmhp20 showed a high expression on day 4 and 5 of pupal.Previous research demonstrated that the CIb1 played a role in silkworm innate immunity response. Since Bmhp20 interacts with CIb1, we speculated that Bmhp20 might be involved in the immunity response too. So, real-time RT-PCR was carried out to measure the transcriptional regulation of Bmhp20 in the fat body challenged by B. bombyseptieus, B. bassiana, and E. coli. Compared to the control, Bmhp20 expression was increased rapidly after injection of E. coli for 1 hour and then decreased. Bmhp20 expression was increased rapidly when treated with B. bassiana, decreased after injection for 3-6 hours and then increased again 12 hours post injection. B. bombyseptieus exerted the most powerful effect on Bmhp20 expression compared to the other two pathogens and showed the highest expression on the 24 hours after injection. The strong response of Bmhp20 to B. bombyseptieus implied that Bmhp20 might played an important role in the immune reponse of silkworm to its main bacterial septic pathogens (B. bombyseptieus).4. Recombinant Bmhp20 expression, purification and antibody preparationDNA sequences encoding the mature Bmhp20 were inserted into the expression vector pET28a and then the construct was transformed into Rosetta E. coli host cell to express His-Bmhp20 protein. Analysis on the induced E. coli cells showed that Bmhp20 protein was expressed as soluble protein, which would facilitate the subsequenct functional analysis of Bmhp20 protein. The expressed Bmhp20 had a hexa-histidine tag and was subjected to Ni-NTA resin for affinity purification. The purified fraction was further purified through the Superdex 75 gel column. Since Bmhp20 had many histidines, the his-tag was removed to avoid the experimental background in the subsequent experiments. The purified His-Bmhp20 was digested with thrombin and the remanent thrombin was then removed via streptavidin agarose. The Bmhp20 without the his-tag was collected through the Superdex 75 gel column. Bmhp20 polyclonal antibody was prepared through Bmhp20 immunization of adult male rabbit. The serum was collected when the valence reached 1:100,000 and stored in-30℃for use later.5. Functional analysis of Bmhp201) The protein was produced and then secreted into silkworm hemolymphAmino acid sequence analysis of Bmhp20 indicated many glycosaminoglycan binding site located on Bmhp20 which may be involved in immune recognition of B. mori. According to the results of immune blotting of each tissue in three-day fifth instar silkworm larvae, Bmhp20 signals only appeared in the hemolymph rather than other tissues. And Bmhp20 was the protein identified to interact with Clbl in the silkworm plasma. All of these facts demonstrated that Bmhp20 existed and played a role in silkworm hemolymph.2) Bmhp20 did not have the activity of casein hydrolysisBecause Bmhp20 could bind to Chymotrypsin inhibitor Clbl, the first thing we considered was whether Bmhp20 acted as a hydrolase to regulate the physiological responses in vivo and Clbl was the one to control the extent of this process. In vitro, the assay of Bmhp20 activity showed Bmhp20 lacked the activity of casein hydrolysis. Therefore, we speculated that Bmhp20 played other roles to participate in immune response of silkworm rather than as enzymes.3) The amount of Bmhp20 protein in hemolymph decreased after E. coli induction After challenged by E. coli, B. bombyseptieus and B. bassiana, the silkworm hemolymph was collected via centrifugation and used to detect the expression of Bmhp20. Western blotting results indicated that protein level of Bmph20 changed unsignificantly in silkworm plasma when challenged by B. bombyseptieus or B. bassiana. However, infection of E. coli caused the Bmhp20 mRNA amount decreased in circulation. It's probably because the silkworm hemolymph was treated with centrifugation. Since CIb1 could bind to E.coli, we speculated CIb1 and Bmhp20 bound to each other after LPS stimulation. Under this circumstance, we assume that the interaction between Clbl and Bmhp20 would lead to the decrease of Bmhp20 in circulation.4) Bmhp20 was able to bind to polysaccharide, Gram-positive bacteria and fungi in vivo.In vitro binding assay showed that Bmph20 bound to chitin, cellulose and curdlan. Meanwhile, Bmhp20 also bound to Gram-positive bacteria (S. aureus, B. bombyseptieus) and fungi (B. bassiana, P. pastoris). All of these suggested Bmhp20 played a role in pattern recognition in the silkworm immune response.5) Bmhp20 did not participate in the phagocytosis.Bmhp20 was incubated with FITC labeled S. aureus and B. bassiana respectively, and then we observed whether Bmhp20 would enhance silkworm hemocyte phagocytosis or not. The results showed that Bmph20 did not enhance phagocytosis. After injection of FITC labeled S. aureus and B. bassiana into silkworm, we collected hemocytes 1 hour after injection and observed hemocytes engulfed the labeled bacteria, however, Bmhp20 did not involve in this immune response. In vitro and vivo experiments proved that Bmhp20 didn't participate in the phagocytosis.6) Bmhp20 was involved in the inhibition of silkworm melanizationSilkworm larval plasma (SLP) reagent is a kind of detection reagent. Addition of glucan or peptidoglycan into SLP could cause melanization. The melanization inhibition experiment adopted with SLP reagent indicated that addition of Bmhp20 in advance can inhibit the melanization significantly, in contrast to addition of glucan or peptidoglycan which directly stimulated melanization. The results demonstrated that Bmhp20 played a role in the inhibition of the PO activity in silkworm.