Melanization in insect hemolymph is triggered by the recognition of pathogen-associated molecular patterns via pattern recognition receptors. The signal transduction leads to the activation of the prophenoloxidase and hence the generation of melanin. The proPO activation process must be tightly controlled to minimize the host damage caused by reactive intermediates during melanin synthesis. The full-length cDNA sequence of a 20 kDa hemolymph protein from Bombyx (Bmhp20) was determined. Bmhp20 gene was expressed in larval fat body, integument, trachea, and ovary and was induced by the challenge of B. bombyseptieus. Binding of recombinant Bmhp20 to microbial cell wall components as well as gram-positive bacteria and fungi was confirmed. Phenoloxidase activity assay indicated that recombinant Bmhp20 blocked the proPO activation in hemolymph that was triggered by peptidoglycan or beta-1, 3-glucan. Our data suggest that Bmhp20 plays bifunctional roles in silkworm humoral responses: to participate in pattern recognition and to block the activation of proPO.
Humoral responses of immunity system in insect such as melanization and induced production of plasma proteins are essential for the host to fight against the pathogens [1,2]. For its large body and easily obtained hemolymph, silkworm is one of the earliest insects used to study humoral responses [3,4].
Pathogen-associated molecular patterns (PAMPs) are conserved structures present on the surfaces of microbes including peptidoglycan (PGN), beta-1, 3-glucan, lipoteichoic acid and lipopolysaccharide [
The availability of silkworm genome makes identification of large number of PRR genes. PGRP genes in silkworm are clustered into two groups located on chromosome 1 (PGRP1-5) and 16 (PGRP 8-11) [
The present research is focused on the characterization of this silkworm hemolymph protein. Its full-length cDNA sequence was obtained. We found that the deduced amino acid sequences of Bmhp20 are rich in putative glycosaminoglycan attachment sites. The expression of Bmhp20 gene was enhanced by B. bombyseptieus. In vitro binding assays revealed that recombinant Bmhp20 bound to gram-positive bacteria and fungi. The result of phenoloxidase activity assay indicated that the recombinant Bmhp20 blocked the proPO activation in hemolymph that was activated by PGN or β-1, 3-glucan.
Silkworm strain p50 is maintained at the State Key Laboratory of Silkworm Genome Biology, Southwest University, China. The larvae are reared on mulberry leaves at 25˚C. The curdlan (Sigma, USA), cellulose (Sigma, USA), and chitin (NEB, USA) were purchased and separately suspended in 100 µl PBS (pH 7.4, 200 mM NaCl).
The amino acid sequence data from Edman degradation analysis were blasted against the protein database at SilkDB (http://silkworm.swu.edu.cn/silkdb/). The identified protein sequence and its corresponding nucleic acid sequences (predicted gene BGIBMGA002175) were used as references for primer design.
Total RNA was extracted from the fat body of the fifth instar day 3 larvae using Trizol reagent (Invitrogen, China). RNA samples were treated with DNase I (DNAfree, Ambion) at 37˚C for 1 h to remove the DNA contamination. The concentration of RNA was measured in a spectrophotometer (Amersham Biosciences, Sweden). Three micrograms of total RNAs were used to synthesize first-strand cDNA in 25 µl of reverse transcription PCR (RT-PCR) system with M-MLV Reverse Transcriptase (Promega, USA).
Two primers (forward 5’-ATGGCACTGAAAATTACTT-3’; reverse: 5’-GGAGCTCTAAGCTGTTAAA-3’) were synthesized (Genscript, China) for PCR amplification using the fat body cDNA. PCRs were performed as follows: 94˚C for 3 min, 35 cycles at 94˚C for 40 s, 55˚C for 40 s, 72˚C for 40 s, followed by incubation at 72˚C for 10 min. PCR products were cloned into pMD19-T simple vector (TaKaRa, China). According to the manual of 5’ RACE system Version 2.0 (Invitrogen, China), the partial sequenced sequence were used to design the primers GSP1 (5’-GTGTCCAAATCCAGAATCGTG-3’), GSP2 (5’- CGAACCCAGAGTCACGACCGAA-3’), and nested primer (5’-AACTGCCGAAGCCGCTATGA-3’). The first-strand gene specific cDNA was reverse-transcribed from fat body RNA by GSP1 using SuperScript™ II (Invitrogen, China). The specific cDNA was tailed with poly C. Tailed cDNA was used as template to reamplify the target gene using Abridged Anchor Primer 1 (5’-GGCCACGCGTCGACTACGGGGGGGGGG-3’)
and GSP2. The product was used for the third round PCR amplification using Abridged Universal Amplification Primer 2 (5’-GGCCACGCGTCGACTAGTAC-3’) and nested primer. The amplified nucleic acid fragment was cloned into pMD19-T simple vector (TaKaRa, China) and sequenced.
Gene translation and the prediction of molecular weight and theoretical pI were performed with ExPASy (http://www.expasy.ch/). Gene structure was drawn by SIM4 (http://pbil.univ-lyon1.fr/members/duret/cours/inserm210604/exercise4/sim4.html). Sequence homologous was analyzed on NCBI. Signal peptide prediction was carried out using the program SignalP 3.0 (http://www.cbs.dtu.dk/services/SignalP/). Functional sites in protein were predicted using ELM database (http://elm.eu.org/). TESS (http://www.cbil.upenn.edu/cgi-bin/tess/tess) was used to predict the Upstream regulatory sequences.
To investigate the expression of Bmhp20 mRNA in tissues, the RNAs from ten different tissues (including fat body, integument, ovary, trachea, head, testis, silk gland, midgut, hemocyte, and malpighian tubule) of the fifth instar day 3 larvae were extracted. For developmental expression profile of Bmhp20, RNAs were prepared using whole larval body from the 1st to the 4th instar larvae and the fat body excised from the fifth instar larva day 1 to adult. RNAs isolation and digestion were carried out as mentioned above. The amplification of the BmActin3 served as an internal control. The Bmhp20 primers (forward: 5’-TTTGGAAATCGCAACCCT-3’, reverse: 5’-GATACCGCCCAATGAAGTG-3’) and BmActin3 primers (forward: 5’-AACACCCCGTCCTGCTCACTG-3’, reverse:
5’-GGGCGAGACGTGTGATTTCCT-3’) were used for RT-PCR. PCRs were performed for one cycle at 94˚C for 3 min, 25 cycles at 94˚C for 40 s; 55˚C for 40 s; 72˚C for 40 s; followed by incubation at 72˚C for 10 min.
The cDNA fragment encoding the mature Bmhp20 was amplified using the fat body cDNA by the following primers: forward: 5’-CTAgctagcTTCCCTCATGGCAGTAGCG-3’, reverse: 5’-CCgagctcGTGTTGACATGCCTAGATCAGTGT-3’, and then cloned into pMD19-T simple vector (TaKaRa, China) for sequencing. The recombinant plasmid of pET 28a-Bmhp20 was constructed. One Rosetta colony containing pET28a-Bmhp20 was incubated at 37˚C in 400 ml of LB medium supplemented with 25 µg/ml kanamycin until the OD600 reached 0.4 - 0.8. A final concentration of 1 mM IPTG was added into the culture and then induced for 3 h at 37˚C. Cells were harvested by centrifugation. All the following purification procedures were conducted at 4˚C unless otherwise noted. Cells were washed for 3 times with 20 mM PBS buffer (pH 7.4, 500 mM NaCl), and then suspended in 20 ml of same PBS buffer. Cells were disrupted by sonication on ice and separated by centrifugation for 30 min at 16,000× g. The supernatant was loaded onto a Ni-NTA affinity agarose column (Invitrogen, China). The fractions eluted with 20 mM PBS (pH 7.4, 500 mM NaCl) containing 150 mM imidazole were collected. A gel filtration chromatography was subsequently performed using a Superdex 75 column (AKTA purify). The column was washed with 50 mM PBS (pH 7.4, 200 mM NaCl) at a lined linear velocity of 1ml/min. The fractions containing HisBmhp20 were combined and the proteins were digested by thrombin (Novagen, USA). After the cleavage reaction, biotinylated thrombin was removed with Streptavidin Agarose, and then concentrated by ultrafilter (Amicon Ultra-15 Millipore, USA). The protein concentration was determined by the Bradford protein assay and the bovine serum albumin (BSA) was used as a standard. The cleaved Bmhp20 served as antigen for the production of polyclonal rabbit antiserum. A total of 500 µg of cleaved Bmhp20 was equally mixed with complete Freund’s adjuvant (Sigma, USA). The resultant emulsified mixture was injected into rabbit. Subsequent booster injections in the rabbit were carried out for three times (250 µg per time, once a week). Seven days after the last injection, the rabbit was bled and the blood sample was collected. The antiserum was purified by MAbTrap™ Kit (GE, USA).
The tissues of the fifth instar day 3 larvae were excised and dissolved in 2% SDS solution. The proteins concentration was determined with BCA kit (Beyotime, China). Two micrograms of each sample was loaded on 15% SDS-PAGE gel (2 µg/each well). After electrophoresis, proteins were transferred to a PVDF membrane (0.45 µm Roche, Switzerland). Membranes were blocked with 5% nonfat milk dissolved in Tris-buffered saline Tween-20 (TBST), and then incubated with a 10,000-fold dilution of rabbit-Bmhp20 serum. Immunoreactive bands were detected with anti-rabbit IgG peroxidase (HRP)-conjugated secondary antibodies (1:10,000, Sigma, USA). The signals were visualized using the ECL Plus Western blotting detection reagent (GE, USA).
The fifth instar day 3 larvae were injected with the formaldehyde-fixed E. coli (108 cells per larvae), B. bombyseptieu (107 cells per larvae), B. bassiana (107 cells per larvae), respectively. Injection of 10 µl of 0.85% NaCl was used as a negative control. Fat body from challenged larvae was obtained 1, 3, 6, 12, 24 h after injection. The RNAs were isolated and first-strand cDNAs were synthesized as mentioned above. For real-time PCR, primers of Bmhp20 (forward: 5’-AGTAGCACAAGCCTTCCCTCATG-3’; reverse: 5’-ACTGCCGAAGCCGCTATGAC-3’) and primers of sw22934 (forward: 5’-TTCGTACTGGCTCTTCTCGT-3’; reverse: 5’-CAAAGTTGATAGCAATTCCCT - 3’) were designed. The primer annealing temperatures was 60˚C and the size of product was 139 bp. Primer specificity was visualized by 2% agarose gel. The PCR efficiency was within 95% - 110%. The real-time PCRs were performed using the SYBR Premix Ex Taq kit (TaKaRa, China). Each reaction in 25 µl reaction cocktail including 12.5 µl of Ex Taq, 70 ng cDNA, 10 mM Bmhp20 forward and reverse primers. The PCR reaction was performed on an ABI Prism 7000 Sequence Detection System (Applied Biosystems) using the following program: initial denaturation at 95˚C for 10 s, and 40 cycles of 95˚C for 5 s, 60˚C for 31 s. The primers of sw22934 (transcription initiation factor 2 gene) was used as internal standardizetion.
In vitro binding assays were executed according to published protocol [
Silkworm larva plasma (SLP) reagent (Wako, Japan) was used to measure the phenoloxidase (PO) activity. Two types of polysaccharide, PGN (53243 Sigma, USA) extracted from M. luteus and laminarin (L9634 Sigma, USA), a kind of β-1,3-glucan extracted from L. digitata, were used as inducers. The assays were performed as described in manufacturer’s instructions. In brief, 20 µl recombinant Bmhp20 (1.5 µg/µl), 20 µl PGN (0.2 ng/µl) or 20 µl laminarin (0.2 ng/µl), and 10 µl double distilled water (ddH2O) were mixed together. After incubated at 27˚C for 5 min, 50 µl of SLP reagent was added. Then the mixture was incubated for another 30 min at 27˚C. PO activity was monitored at absorbance of 450 nm. The assay was repeated three times.
The sequence of 20 residues “HGSSDVDGSGEVEAVAGTLK” was used to search the silkDB (http://silkworm.swu.edu.cn/silkdb/) with TBLASTN program. A protein (BGIBMGA002175) with the same deduced amino acid sequence was found. However, the 5’cDNA sequence of this gene is absent. We then performed 5’RACE to clone its full length cDNA. As a result, a 740-bp cDNA sequence was obtained, as shown in
Comparison of the cDNA and its genome sequences revealed that Bmhp20 had three exons separated by two introns (
The deduced amino acid sequences of Bmhp20 were shown in
RT-PCR was carried out using RNAs extracted from fat body, integument, ovary, trachea, head, testis, silk gland, midgut, hemocyte, and malpighian tubule of the fifth instar day 3 larvae. As shown in
Real-time RT-PCR was carried out to measure the transcriptional regulation of Bmhp20 in the fat body challenged by B. bombyseptieus, Beauveria bassiana, and Escherichia coli. As illustrated in
His-Bmhp20 was expressed in prokaryotic expression system and purified by a two-step chromatography. Histag at the N terminal of his-Bmhp20 was removed with biotinylated thrombin. As shown in
The distribution of Bmhp20 in different tissues was examined by Western blotting. Same amount of proteins were separated by SDS-PAGE and stained by silver as a loading control (
As mentioned above, the analysis of the amino acid sequence of Bmhp20 revealed that it has abundant putative glycosaminoglycan attachment sites (
suggesting little binding of Bmhp20 to this gram-negative bacterium.
PGN or β-glucan activates the PO cascade of silkworm larval plasma. The produced melanin can be easily detected with a substrate 3, 4-dihydroxyphenylalanine [
the SLP reagent pre-incubated with his-Bmhp20, the PO activities were no longer increased, indicating that Bmhp20 acts as a proPO activation blocker. Previous studies of proPO-activating proteinases in silkworm revealed at least two enzymes, i.e. an esterase hydrolyzing N-αbenzoyl-L-arginine ethyl ester (BAEEase), a prophenoloxidase activating enzyme (PPAE) were involved in the activation pathway [25,26]. BAEEase activity was shown to be inactivated by silkworm antitrypsin, whereas the activation of PPAE was unaffected by protease inhibitors [
To our knowledge, proteins with recognition activities have not yet been reported to inhibit the proPO-activating system of insect. It is concluded that for the first time, we identified a silkworm hemolymph protein participating in pattern recognition as well as blocking proPO activation.
NH conceived of the study and developed the study design. BL and XQ carried out the analysis and BL drafted the manuscript. QH did the PO activity assay. LJ performed the binding assay. ZX participated in the study design. NH contributed to the critical revision of the manuscript. All authors read and approved the final manuscript.
This work was supported by research grants from the National Basic Research Program of China (Grant No. 2012CB114600), Natural Science Foundation Project of CQ CSTC (Grant No. cstc2011jjjq0010), and 111 project (B07045). The nucleotide sequence reported in this paper has been submitted to GenebankTM with an accession number GU015849.
PAMPs, Pathogen-associated molecular patterns; PRRs, pattern recognition receptors; proPO, prophenoloxidase; PGRP, peptidoglycan recognition protein; PO, phenoloxidase; PGN, peptidoglycan; IPTG, isopropyl β-D-thiogalactoside; SLP, silkworm larva plasma; BAEEase, N- α-benzoyl-L-arginine ethyl ester; PPAE, prophenoloxidase activating enzyme.