Manduca sexta Serpin-7, a Putative Regulator of Hemolymph Prophenoloxidase Activation.
ABSTRACT Serpins regulate various physiological reactions in humans and insects, including certain immune responses, primarily through inhibition of serine proteases. Six serpins have previously been identified and characterized in the tobacco hornworm Manduca sexta. In this study, we obtained a full-length cDNA sequence of another Manduca serpin, named serpin-7. The open reading frame of serpin-7 encodes a polypeptide of 400 amino acid residues with a predicted signal peptide of the first 15 residues. Multiple protein sequence alignment of the reactive center loop region of the M. sexta serpins indicated that serpin-7 contains Arg-Ile at the position of the predicted scissile bond cleaved by protease in the serpin inhibition mechanism. The same residues occur in the scissile bond of the reactive center loop in M sexta serpin-4 and serpin-5, which are protease inhibitors that can block prophenoloxidase activation in plasma. Serpin-7 transcript was detected in hemocytes and fat body, and its expression increased in fat body after injection of larvae with Micrococcus luteus. Recombinant serpin-7 added to larval plasma inhibited spontaneous melanization and decreased prophenoloxidase activation stimulated by bacteria. Serpin-7 inhibited prophenoloxidase-activating protease-3 (PAP3), forming a stable serpin-protease complex. Considering that serpin-3 and serpin-6 are also efficient inhibitors of PAP3, it appears that multiple serpins present in plasma may have redundant or overlapping functions. We conclude that serpin-7 has serine protease inhibitory activity and is likely involved in regulation of proPO activation or other protease-mediated aspects of innate immunity in M. sexta.
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- "leureptin-1mRNAwashighlyinducedinhemocytesandlesssoin fatbody.AmongthefivemiRNAstargetingleureptin-1,mse-let-7a, -2b*and-31*levelsdecreasedinbothtissues.TheincreasesinIg domain-containinghemicentin-1and-2mRNAlevelsinfatbody wereaccompaniedbyabundancedecreasesoftheirpotentialreg- ulatorymiRNAs(mse-miR-2763forhemicentin-1andmse-miR- 970forhemicentin-2)aftertheimmunechallenge.Insummary, transcriptsofonePRRmayberegulatedbyoneormoremiRNAs; mse-miR-2763and-31*couldcontrolmultiplePRRsbytargeting theirtranscripts. 4.2.2.Extracellularsignaltransductionandmelanization WefoundmiRNAsmayregulatethemRNAsof13HPs,2PPO- activatingproteinases(PAPs),3serineproteinasehomologs (SPHs),and5serpinsthatmediateandmodulatetheextracel- lularsignaltransductionforimmuneresponses(Table4).They mayalsocontributetofinetuningofthegeneexpressionfor melanization,involvingPheandTyrhydroxylases,Punch,dopa decarboxylase,andPPOs.AfterexaminationofthemiRNAand mRNAlevels,weidentifiedtheputativeregulatorypairsifthey displayedoppositetrendsofchangeafterthebacterialinjection ineitherfatbodyorhemocytes(Table5).HP6activatesproHP8 andHP8activatespro-SpätzletoinducetheTollpathway(An etal.,2010).Mse-miR-31*seemstoregulatelevelsofbothHP6 andHP8transcripts,whichiscrucialtotheTollpathwayacti- vation.HP14,HP21,HP6,PAPsandSPHsformaPPOactivation systemtogenerateactivecompoundsandmelanintokilland sequestratepathogens.HP21cleavesproPAP2/3andmse-miR-9a potentiallyregulatesHP21andPAP2mRNAlevels.Mse-miR-2763 mayaffectHP14andPAP3transcriptabundances.The30-UTRof HP22containsputativerecognitionsitesofbothstrandsofthe mse-miR-9bduplex.Serpinsinhibitcognateserineproteinasesto modulatetheextracellularsignalingpathways.Serpin-3,-4,-6, and-7inhibitthree(HP8,PAP1,PAP3),three(HP1,6,21),three (HP8,PAP1,PAP3)andone(PAP3)proteinases,respectively (Christenetal.,2012;Jiang,2008;Suwanchaichindaetal.,2013). Notably,mse-miR-11,-263a,and-308maydown-regulatethe transcriptsofserpin-4&6,serpin-3&4,andserpin-4&7, respectively.Whilemse-miR-12mayregulatetheexpressionofa serpin-proteinasepair(i.e.serpin-3andPAP3),mRNAlevelsof serpinsandtargetserineproteinasesseemtobemodulatedby differentmiRNAsinmostcases. "
ABSTRACT: The tobacco hornworm Manduca sexta has served as a model for insect biochemical and physiological research for decades. However, knowledge of the posttranscriptional regulation of gene expression by microRNAs is still rudimentary in this species. Our previous study (Zhang et al., 2012) identified 163 conserved and 13 novel microRNAs in M. sexta, most of which were present at low levels in pupae. To identify additional M. sexta microRNAs and more importantly to examine their possible roles in the expression regulation of immunity-related genes, we constructed four small RNA libraries using fat body and hemocytes from naïve or bacteria-injected larvae and obtained 32.9 million reads of 18-31 nucleotides by Illumina sequencing. Mse-miR-929 and mse-miR-1b (antisense microRNA of mse-miR-1) were predicted in the previous study and now found to be conserved microRNAs in the tissue samples. We also found four novel microRNAs, two of which result from a gene cluster. Mse-miR-281-star, mse-miR-965-star, mse-miR-31-star, and mse-miR-9b-star were present at higher levels than their respective mature strands. Abundance changes of microRNAs were observed after the immune challenge. Based on the quantitative data of mRNA levels in control and induced fat body and hemocytes as well as the results of microRNA target site prediction, we suggest that certain microRNAs and microRNA*s regulate gene expression for pattern recognition, prophenoloxidase activation, cellular responses, antimicrobial peptide synthesis, and conserved intracellular signal transduction (Toll, IMD, JAK-STAT, MAPK-JNK-p38, and small interfering RNA pathways). In summary, this study has enriched our knowledge on M. sexta microRNAs and how some of them may participate in the expression regulation of immunity-related genes.Insect biochemistry and molecular biology 04/2014; 47. DOI:10.1016/j.ibmb.2014.01.008 · 3.42 Impact Factor
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ABSTRACT: Insects possess an innate immune system that responds to invading microorganisms. In this study, a subtractive cDNA library was constructed to screen for immune response-related genes in the fat bodies of Antheraea pernyi (Lepidoptera: Saturniidae) pupa challenged with Escherichia coli. Four hundred putative EST clones were identified by suppression subtractive hybridization (SSH), including 50 immune response-related genes, three cytoskeleton genes, eight cell cycle and apoptosis genes, five respiration and energy metabolism genes, five transport genes, 40 metabolism genes, ten stress response genes, four transcription and translation regulation genes and 77 unknown genes. To verify the reliability of the SSH data, the transcription of a set of randomly selected immune response-related genes were confirmed by semi-quantitative reverse transcription-PCR (RT-PCR) and real-time quantitative reverse transcription-PCR (qRT-PCR). These identified immune response-related genes provide insight into understanding the innate immunity in A. pernyi.Journal of Invertebrate Pathology 09/2013; 114(3). DOI:10.1016/j.jip.2013.09.004 · 2.60 Impact Factor
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ABSTRACT: As important arthropod immune responses, prophenoloxidase (proPO) activation and Toll pathway initiation are mediated by serine proteinase cascades and regulated by serpins. Herein, a serine protease inhibitor (Lvserpin), encoding for 415 amino acids with calculated molecular weight of 46,639 Da and isoelectric point of 7.03 was characterized from the Pacific white shrimp Litopenaeus vannamei. Multiple sequence alignment revealed that Lvserpin shared the highest similarity with Penaeus monodon serpin6 (87%). Quantitative real-time PCR (qRT-PCR) results showed that the transcripts of Lvserpin were detected in all the examined tissues and most highly expressed in gill. The expression profiles of Lvserpin were greatly fluctuated upon infection of Vibrio anguillarum, Micrococcus lysoleikticus or White Spot Syndrome Virus (WSSV). Double stranded RNA-mediated suppression of Lvserpin resulted in a significant increase in the transcripts of two clip-domain serine proteinases (PPAE and PPAF), prophenoloxidase (proPO), anti-lipopolysaccharide factor (ALF), Crustin and penaeidin3 (Pens3) and also increased the high cumulative mortality post V. anguillarum injection. Besides, the recombinant Lvserpin protein (rLvserpin) was purified and exhibited inhibitory activity against trypsin. Also the rLvserpin showed inhibition on prophenoloxidase activation and bacterial growth. Hence, we proposed that the Lvserpin played important role in the shrimp innate immunity.Developmental and comparative immunology 11/2013; 43(1). DOI:10.1016/j.dci.2013.10.012 · 3.71 Impact Factor