Seminars in cell & developmental biology, Jan 4, 2016
Serpins are the largest known family of serine proteinase inhibitors and perform a variety of phy... more Serpins are the largest known family of serine proteinase inhibitors and perform a variety of physiological functions in arthropods. Herein, we review the field of serpins in arthropod biology, providing an overview of current knowledge and topics of interest. Serpins regulate insect innate immunity via inhibition of serine proteinase cascades that initiate immune responses such as melanization and antimicrobial peptide production. In addition, several serpins with anti-pathogen activity are expressed as acute-phase serpins in insects upon infection. Parasitoid wasps can downregulate host serpin expression to modulate the host immune system. In addition, examples of serpin activity in development and reproduction in Drosophila have also been discovered. Serpins also function in host-pathogen interactions beyond immunity as constituents of venom in parasitoid wasps and saliva of blood-feeding ticks and mosquitoes. These serpins have distinct effects on immunosuppression and anticoagu...
BioOne Complete (complete.BioOne.org) is a full-text database of 200 subscribed and open-access t... more BioOne Complete (complete.BioOne.org) is a full-text database of 200 subscribed and open-access titles in the biological, ecological, and environmental sciences published by nonprofit societies, associations, museums, institutions, and presses.
Insect Biochemistry and Molecular Biology, Nov 1, 2018
Members of the serpin superfamily of proteins occur in animals, plants, bacteria, archaea and som... more Members of the serpin superfamily of proteins occur in animals, plants, bacteria, archaea and some viruses. They adopt a variety of physiological functions, including regulation of immune system, modulation of apoptosis, hormone transport and acting as storage proteins. Most members of the serpin family are inhibitors of serine proteinases. In this study, we searched the genome of Manduca sexta and identified 32 serpin genes. We analyzed the structure of these genes and the sequences of their encoded proteins. Three M. sexta genes (serpin-1, serpin-15, and serpin-28) have mutually exclusive alternatively spliced exons encoding the carboxyl terminal reactive center loop of the protein, which is the site of interaction with target proteases. We discovered that MsSerpin-1 has 14 splicing isoforms, including two undiscovered in previous studies. Twentyeight of the 32 M. sexta serpins include a putative secretion signal peptide and are predicted to be extracellular proteins. Phylogenetic analysis of serpins in M. sexta and Bombyx mori indicates that 17 are orthologous pairs, perhaps carrying out essential physiological functions. Analysis of the reactive center loop and hinge regions of the protein sequences indicates that 16 of the serpin genes encode proteins that may lack proteinase inhibitor activity. Our annotation and analysis of these serpin genes and their transcript profiles should lead to future advances in experimental study of their functions in insect biochemistry.
Serpins are serine protease inhibitors that are widely distributed in metazoans, but they have no... more Serpins are serine protease inhibitors that are widely distributed in metazoans, but they have not been characterized previously in Eimeria spp. A serpin from Eimeria acervulina was cloned, expressed, and characterized. Random screening of an E. acervulina sporozoite cDNA library identified a single clone (D14) whose coding region shared high similarity to consensus structure of serpins. Clone D14 contained an entire open reading frame consisting of 1,245 nucleotides that encode a peptide of 413 amino acids, with a predicted molecular weight of 45.5 kDa and containing a signal peptide 28 residues. By Western blot analysis, polyclonal antiserum to the recombinant serpin (rbSp) recognized a major 55-kDa protein band in unsporulated oocysts and in oocysts sporulated up to 24 hr (fully sporulated). The anti-rbSp detected bands of 55 and 48 kDa in sporozoites (SZ) and merozoites (MZ), respectively. Analysis of MZ secretion products revealed a single protein of 48 kDa that may correspond to secreted serpin. By immunostaining, the serpin was located in granules distributed throughout both the SZ and MZ, but granules seemed to be concentrated in the parasites' anterior. Analysis of the structure predicts that the E. acervulina serpin should be an active inhibitor. However, rbSp was without inhibitory activity against common serine proteases. By Western blot analysis, the endogenous serpin in MZ extracts did not form the expected high-molecular-weight complex when coincubated with either trypsin or subtilisin. The results demonstrate that E. acervulina contains a serpin gene and expresses a protein with structural properties similar to an active serine protease inhibitor. Although the function of the E. acervulina serpin remains unknown, the results further suggest that serpin is secreted by the parasite where it may be involved in cell invasion and other basic developmental processes.
CLIP proteases are non-digestive serine proteases present in hemolymph of insects and other arthr... more CLIP proteases are non-digestive serine proteases present in hemolymph of insects and other arthropods. They are composed of one or more amino-terminal clip domains followed by a linker sequence and a carboxyl-terminal S1A family serine protease domain. The genes for CLIP proteases have evolved as four clades (CLIPA, CLIPB, CLIPC, CLIPD), each present as multigene families in insect genomes. CLIP proteases in hemolymph function in innate immune responses. These include proteolytic activation of the cytokine Spätzle, to form an active Toll ligand leading to synthesis of antimicrobial peptides, and specific activation of prophenoloxidase, required for the melanization response. CLIP proteases act in cascade pathways. In the immune pathways that have been characterized, microbial surface molecules stimulate activation of an initiating modular serine protease, which then activates a CLIPC, which in turn activates a CLIPB. The active CLIPB then cleaves and activates an effector molecule (proSpätzle or prophenoloxidase). CLIPA proteins are pseudoproteases, lacking proteolytic activity, but some can function as regulators of the activity of other CLIP proteases and form high molecular weight immune complexes. A few three dimensional structures for CLIP proteases are now available for structure-function analysis of these immune factors, revealing structural features that may act in specific activation or in formation of immune complexes. The functions of most CLIP proteases are unknown, even in well studied insect species. It is very likely that additional proteins activated by CLIP proteases and acting in immunity remain to be discovered.
The body form of holometabolous insects dramatically transforms from larval to adult stages durin... more The body form of holometabolous insects dramatically transforms from larval to adult stages during metamorphosis that occurs in the pupal stage. The larval disorganization and then new adult tissues are built up at this time. In motoneuron, larval neuronal cells degenerate, and new adult neurons are remodeled. Finally, adult neurons reconnect to new adult muscles. However, the factors that control metamorphosis have not yet been fully elucidated. Here, we show that an antioxidant enzyme, Tribolium castaneum superoxide dismutase 6 (TcSOD6), is secreted into the haemolymph and is required for proper movable legs during metamorphosis. TcSOD6 has a unique domain architecture and is mainly expressed in the pupal stage. The depletion of TcSOD6 expression in the pupa inhibits normal axon development and results in adults that display dysfunctional leg motions, suggesting that SOD6 expression is required for the development of properly movable legs. Therefore, we speculate that TcSOD6 might...
Members of the serpin superfamily of proteins occur in animals, plants, bacteria, archaea and som... more Members of the serpin superfamily of proteins occur in animals, plants, bacteria, archaea and some viruses. They adopt a variety of physiological functions, including regulation of immune system, modulation of apoptosis, hormone transport and acting as storage proteins. Most members of the serpin family are inhibitors of serine proteinases. In this study, we searched the genome of Manduca sexta and identified 32 serpin genes. We analyzed the structure of these genes and the sequences of their encoded proteins. Three M. sexta genes (serpin-1, serpin-15, and serpin-28) have mutually exclusive alternatively spliced exons encoding the carboxyl terminal reactive center loop of the protein, which is the site of interaction with target proteases. We discovered that MsSerpin-1 has 14 splicing isoforms, including two undiscovered in previous studies. Twentyeight of the 32 M. sexta serpins include a putative secretion signal peptide and are predicted to be extracellular proteins. Phylogenetic analysis of serpins in M. sexta and Bombyx mori indicates that 17 are orthologous pairs, perhaps carrying out essential physiological functions. Analysis of the reactive center loop and hinge regions of the protein sequences indicates that 16 of the serpin genes encode proteins that may lack proteinase inhibitor activity. Our annotation and analysis of these serpin genes and their transcript profiles should lead to future advances in experimental study of their functions in insect biochemistry.
CLIP proteases are non-digestive serine proteases present in hemolymph of insects and other arthr... more CLIP proteases are non-digestive serine proteases present in hemolymph of insects and other arthropods. They are composed of one or more amino-terminal clip domains followed by a linker sequence and a carboxyl-terminal S1A family serine protease domain. The genes for CLIP proteases have evolved as four clades (CLIPA, CLIPB, CLIPC, CLIPD), each present as multigene families in insect genomes. CLIP proteases in hemolymph function in innate immune responses. These include proteolytic activation of the cytokine Spätzle, to form an active Toll ligand leading to synthesis of antimicrobial peptides, and specific activation of prophenoloxidase, required for the melanization response. CLIP proteases act in cascade pathways. In the immune pathways that have been characterized, microbial surface molecules stimulate activation of an initiating modular serine protease, which then activates a CLIPC, which in turn activates a CLIPB. The active CLIPB then cleaves and activates an effector molecule (proSpätzle or prophenoloxidase). CLIPA proteins are pseudoproteases, lacking proteolytic activity, but some can function as regulators of the activity of other CLIP proteases and form high molecular weight immune complexes. A few three dimensional structures for CLIP proteases are now available for structure-function analysis of these immune factors, revealing structural features that may act in specific activation or in formation of immune complexes. The functions of most CLIP proteases are unknown, even in well studied insect species. It is very likely that additional proteins activated by CLIP proteases and acting in immunity remain to be discovered.
Insect biochemistry and molecular biology, Jan 17, 2015
Members of the multicopper oxidase (MCO) family of enzymes can be classified by their substrate s... more Members of the multicopper oxidase (MCO) family of enzymes can be classified by their substrate specificity; for example, ferroxidases oxidize ferrous iron, ascorbate oxidases oxidize ascorbate, and laccases oxidize aromatic substrates such as diphenols. Our previous work on an insect multicopper oxidase, MCO1, suggested that it may function as a ferroxidase. This hypothesis was based on three lines of evidence: RNAi-mediated knock down of Drosophila melanogaster MCO1 (DmMCO1) affects iron homeostasis, DmMCO1 has ferroxidase activity, and DmMCO1 has predicted iron binding residues. In our current study, we expanded our focus to include MCO1 from Anopheles gambiae, Tribolium castaneum, and Manduca sexta. We verified that MCO1 orthologs have similar expression profiles, and that the MCO1 protein is located on the basal surface of cells where it is positioned to oxidize substrates in the hemolymph. In addition, we determined that RNAi-mediated knock down of MCO1 in A. gambiae affects i...
ÐCuticle sclerotization or tanning is a vital process that occurs during each stage of insect dev... more ÐCuticle sclerotization or tanning is a vital process that occurs during each stage of insect development to harden and stabilize the newly secreted exoskeleton. The structural polymers protein and chitin make up the bulk of the cuticle, and chemical interactions between these biopolymers with quinonoid tanning agents are largely responsible for the physical properties of the mature exoskeleton. The oxidative conjugation of catechols with cuticular proteins plays an important role in this metabolism. The main hypothesis for cuticle sclerotization involves the formation of adducts and cross-links between nucleophilic imidazole nitrogens of histidyl residues in the proteins and electrophilic ring or side-chain carbons of ortho-quinones and para-quinone methides derived from the catechols, N-acetyldopamine, N-betaalanyldopamine, and 3,4-dihydroxyphenylethanol. C±N and C±O linkages between these quinone tanning agents and proteins in cuticles from a variety of insects from several orders have been elucidated. cDNAs for both the tyrosinase and laccase types of phenoloxidases that catalyze the cross-linking reactions have been isolated and sequenced. The sequences of laccase cDNAs from two insect species were more similar to fungal laccases than to those from plants. These results provide insights into how insects use structural proteins, catechols, and oxidative enzymes to form catechol±amino acid adducts during sclerotization. Published by Elsevier Science Ltd.
Proceedings of the National Academy of Sciences, 1998
Activation of pro-phenol oxidase (proPO) in insects and crustaceans is important in defense again... more Activation of pro-phenol oxidase (proPO) in insects and crustaceans is important in defense against wounding and infection. The proPO zymogen is activated by a specific proteolytic cleavage. PO oxidizes phenolic compounds to produce quinones, which may help to kill pathogens and can also be used for synthesis of melanin to seal wounds and encapsulate parasites. We have isolated from the tobacco hornworm, Manduca sexta , a serine proteinase that activates proPO, and have cloned its cDNA. The isolated proPO activating proteinase (PAP) hydrolyzed artificial substrates but required other protein factors for proPO activation, suggesting that proPO-activating enzyme may exist as a protein complex, one component of which is PAP. PAP (44 kDa) is composed of two disulfide-linked polypeptide chains (31 kDa and 13 kDa). A cDNA for PAP was isolated from a hemocyte library, by using a PCR-generated probe based on the amino-terminal amino acid sequence of the 31-kDa catalytic domain. PAP belongs ...
Extracellular serine proteinase cascades stimulate prophenoloxidase (proPO) activation and antimi... more Extracellular serine proteinase cascades stimulate prophenoloxidase (proPO) activation and antimicrobial peptide production in insect innate immune responses. Serpins in plasma regulate such cascades by selective inhibition of proteinases, in reactions which result in the formation of covalent serpin-proteinase complexes. We carried out experiments to identify plasma proteinases that are inhibited by Manduca sexta serpin-3, an immune-inducible serpin known to regulate proPO activation. Immunoaffinity chromatography, using antiserum to serpin-3, yielded serpin-3 complexes with proteinases identified by immunoblot analysis as prophenoloxidase-activating proteinase (PAP)-1, PAP-2, PAP-3, and hemolymph proteinase 8 (HP8). HP8 can cleave and activate the Toll ligand, Spätzle, leading to synthesis of antimicrobial peptides. Analysis by mass spectrometry of tryptic peptides derived from the serpin-3 complexes confirmed the presence of PAP-1, PAP-3, and HP8. Purified recombinant serpin-3 and active HP8 formed an SDS-stable complex in vitro. Identification of serpin-3-proteinase complexes in plasma provides insight into proteinase targets of serpin-3 and extends the understanding of serpin/proteinase function in the immune response of M. sexta.
Leucine-rich repeat containing proteins are involved in immune response in many capacities. In in... more Leucine-rich repeat containing proteins are involved in immune response in many capacities. In insects, these include Toll-like receptors and the Anopheles gambiae proteins APL1 and LRIM1. Here we describe the identification and characterization of leureptin, a novel extracellular protein with 13 leucine-rich repeats from hemolymph of the insect Manduca sexta. After injection of bacteria, leureptin mRNA level increased in fat body, but protein levels in plasma decreased, an indication that leureptin is consumed during the immune response. Leureptin bound to bacterial lipopolysaccharide (LPS). Microscopy using leureptin antiserum showed that leureptin associates with hemocytes after injection of bacteria, an indication that leureptin is involved in hemocyte responses to bacterial infection. Sequence database searches suggest similar proteins are present in other Lepidopteran species.
Extracellular serine proteinase pathways control immune and homeostatic processes in insects. Our... more Extracellular serine proteinase pathways control immune and homeostatic processes in insects. Our current knowledge of their components is limited-prophenoloxidase-activating proteinases (PAPs) are among the few hemolymph proteinases (HPs) with known functions. To identify components of proteinase systems in the hemolymph of Manduca sexta, we amplified cDNAs from larval fat body or hemocytes using degenerate primers coding for two conserved regions in S1 family serine proteinases. PCR yielded fragments encoding seven known (HP1-HP4, PAP-1, PAP-2 and PAP-3) and 18 unknown (HP5-HP22) serine proteinases. We screened cDNA libraries and isolated clones for 17 of the newly discovered HPs (HP5-HP22 except for HP11) and prepared antibodies to 14 recombinant proteins (HP6, HP8-HP10, HP12, HP14-HP19, HP21 and HP22). Fourteen of the HPs contain regulatory clip domain(s) at their amino-terminus-HP1, HP2, HP6, HP8, HP13, HP17, HP18, HP21, HP22 and PAP-1 have one, whereas HP12, HP15, PAP-2 and PAP-3 have two clip domains. Multiple sequence alignment of catalytic domains in these and other arthropod serine proteinases provided useful clues for future functional analysis. Northern blot and reverse transcription PCR (RT-PCR) analyses showed increases in HP2, HP7, HP9, HP10, HP12-HP22 mRNA levels at 24 h after a bacterial challenge, and immunoblot analysis confirmed elevated concentrations of HP12, HP14-HP19, HP21 and HP22 proteins in plasma in response to injected bacteria. Hemocytes express HP13 and HP18; fat body produces HP12, HP20-HP22; both tissues synthesize the other HPs. These results collectively indicate the existence of a complex serine proteinase network in M. sexta hemolymph, predicted to mediate rapid defense responses upon wounding and/or microbial infection.
The innate immune system of insects include the Toll pathway, which is mediated by an extracellul... more The innate immune system of insects include the Toll pathway, which is mediated by an extracellular serine proteinase cascade. In the tobacco hornworm, Manduca sexta, hemolymph proteinase 8 (HP8) promotes the synthesis of antimicrobial proteins by cleaving proSpätzle, the putative ligand of M. sexta Toll. HP8 has been observed to form a complex in hemolymph with M. sexta serpin-1, which has multiple alternative splicing isoforms. To investigate the regulation of HP8 and its processing of proSpätzle, we characterized the interaction of recombinant HP8 with serpin-1 isoform J (serpin-1J). Recombinant serpin-1J formed an SDS-stable complex with HP8 in vitro. The association rate constant of serpin-1J and HP8 was 1.3×10 4 M-1 s-1 , with a stoichiometry of inhibition of 5.4. Serpin-1J inhibited the cleavage of proSpätzle by HP8. Injection of serpin-1J into M. sexta larvae resulted in decreased bacteria-induced antimicrobial activity in hemolymph and reduced expression of cecropin, attacin and hemolin mRNA in fat body. Altogether, these results suggest that serpin-1J functions to inhibit HP8 and thereby modulates the concentration of active Spätzle to regulate the Toll pathway response in M. sexta.
Proceedings of the National Academy of Sciences of the United States of America, Sep 8, 2020
Significance Phenoloxidase-catalyzed melanization and Spätzle-triggered Toll signaling are critic... more Significance Phenoloxidase-catalyzed melanization and Spätzle-triggered Toll signaling are critical innate immune responses of insects that are triggered by specific proteolysis. It has been unclear whether separate protease cascades or an integrated network of serine proteases coordinates these responses. Here we present evidence that hemolymph protease HP5 acts in pathways eliciting activation of both phenoloxidase and Spätzle-1 in Manduca sexta . HP5 has a unique specificity by cleaving proHP6 at His 112 , resulting in HP6 activation. CLIP proteases related to HP6 from other insect species, including Drosophila Persephone, share this activation site feature with proHP6, and therefore, HP5 orthologs in other species are candidates for key roles in regulating and integrating protease cascade pathways in innate immune responses.
Recognition of nonself is the first step in mounting immune responses. In the innate immune syste... more Recognition of nonself is the first step in mounting immune responses. In the innate immune systems of both vertebrates and arthropods, such recognition, termed pattern recognition, is mediated by a group of proteins, known as pattern recognition proteins or receptors. Different pattern recognition proteins recognize and bind to molecules (molecular patterns) present on the surface of microorganisms but absent from animals. These molecular patterns include microbial cell wall components such as bacterial lipopolysaccharide, lipoteichoic acid and peptidoglycan, and fungal β-1,3-glucans. Binding of pattern recognition proteins to these molecular patterns triggers responses such as phagocytosis, nodule formation, encapsulation, activation of proteinase cascades, and synthesis of antimicrobial peptides. In this article, we describe four classes of pattern recognition proteins, hemolin, peptidoglycan recognition protein, β-1,3-glucan recognition proteins, and immulectins (C-type lectins) involved in immune responses of the tobacco hornworm, Manduca sexta.
Proceedings of the National Academy of Sciences, 2006
Serpins are central to the modulation of various innate immune responses in insects and are suspe... more Serpins are central to the modulation of various innate immune responses in insects and are suspected to influence the outcome of malaria parasite infection in mosquito vectors. Three Anopheles gambiae serpins (SRPN1, -2, and -3) were tested for their ability to inhibit the prophenoloxidase cascade, a key regulatory process in the melanization response. Recombinant SRPN1 and -2 can bind and inhibit a heterologous phenoloxidase-activating protease and inhibit phenoloxidase activation in vitro . Using a reverse genetics approach, we studied the effect of SRPN2 on melanization in An. gambiae adult females in vivo . Depletion of SRPN2 from the mosquito hemolymph increases melanin deposition on foreign surfaces such as negatively charged Sephadex beads. As reported, the knockdown of SRPN2 adversely affects the ability of the rodent malaria parasite Plasmodium berghei to invade the midgut epithelium and develop into oocysts. Importantly, we tested whether the absence of SRPN2 from the hem...
Serpins regulate various physiological reactions in humans and insects, including certain immune ... more 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.
Serpins (serine protease inhibitors) regulate some innate immune responses of insects by inhibiti... more Serpins (serine protease inhibitors) regulate some innate immune responses of insects by inhibiting endogenous proteases. In this study, we characterized the serpin (SRPN) gene family in the mosquito Anopheles gambiae, the major malaria vector in Sub-Saharan Africa. We identified 18 A. gambiae SRPN genes, all on chromosomes 2 and 3, through searches of genomic DNA and EST databases. In addition to SRPN10, previously documented to exhibit alternative splicing, we found three splicing isoforms of SRPN4. We completed sequencing of cDNAs for the A. gambiae serpins to obtain complete coding sequence information and to verify or improve gene predictions. The predicted SRPN9 and 15 in the initial genome annotation were determined to be a single gene (SRPN9). Sixteen of the serpins contained putative secretion signal sequences. Multiple sequence alignments showing conserved residues important in structural conformation, including the consensus pattern within the hinge region, indicated that most of the A. gambiae serpins may be inhibitory. Phylogenetic analyses confirmed that SRPN1, 2, 3, 8, 9 and 10 formed phylogenetic clusters with known inhibitory serpins from Drosophila melanogaster and Manduca sexta. Many of the A. gambiae serpins were expressed during all life stages. However, SRPN7, 8, 12, and 19 were expressed at very low levels in the adult stage. SRPN13 was expressed mostly in eggs and young larvae, whereas SRPN5 and 14 were expressed mostly in adults. Such differences in expression pattern suggest that the serpins are involved in multiple physiological processes. Determining the biological functions of the mosquito serpins will require future work to identify the proteases they inhibit in vivo.
Seminars in cell & developmental biology, Jan 4, 2016
Serpins are the largest known family of serine proteinase inhibitors and perform a variety of phy... more Serpins are the largest known family of serine proteinase inhibitors and perform a variety of physiological functions in arthropods. Herein, we review the field of serpins in arthropod biology, providing an overview of current knowledge and topics of interest. Serpins regulate insect innate immunity via inhibition of serine proteinase cascades that initiate immune responses such as melanization and antimicrobial peptide production. In addition, several serpins with anti-pathogen activity are expressed as acute-phase serpins in insects upon infection. Parasitoid wasps can downregulate host serpin expression to modulate the host immune system. In addition, examples of serpin activity in development and reproduction in Drosophila have also been discovered. Serpins also function in host-pathogen interactions beyond immunity as constituents of venom in parasitoid wasps and saliva of blood-feeding ticks and mosquitoes. These serpins have distinct effects on immunosuppression and anticoagu...
BioOne Complete (complete.BioOne.org) is a full-text database of 200 subscribed and open-access t... more BioOne Complete (complete.BioOne.org) is a full-text database of 200 subscribed and open-access titles in the biological, ecological, and environmental sciences published by nonprofit societies, associations, museums, institutions, and presses.
Insect Biochemistry and Molecular Biology, Nov 1, 2018
Members of the serpin superfamily of proteins occur in animals, plants, bacteria, archaea and som... more Members of the serpin superfamily of proteins occur in animals, plants, bacteria, archaea and some viruses. They adopt a variety of physiological functions, including regulation of immune system, modulation of apoptosis, hormone transport and acting as storage proteins. Most members of the serpin family are inhibitors of serine proteinases. In this study, we searched the genome of Manduca sexta and identified 32 serpin genes. We analyzed the structure of these genes and the sequences of their encoded proteins. Three M. sexta genes (serpin-1, serpin-15, and serpin-28) have mutually exclusive alternatively spliced exons encoding the carboxyl terminal reactive center loop of the protein, which is the site of interaction with target proteases. We discovered that MsSerpin-1 has 14 splicing isoforms, including two undiscovered in previous studies. Twentyeight of the 32 M. sexta serpins include a putative secretion signal peptide and are predicted to be extracellular proteins. Phylogenetic analysis of serpins in M. sexta and Bombyx mori indicates that 17 are orthologous pairs, perhaps carrying out essential physiological functions. Analysis of the reactive center loop and hinge regions of the protein sequences indicates that 16 of the serpin genes encode proteins that may lack proteinase inhibitor activity. Our annotation and analysis of these serpin genes and their transcript profiles should lead to future advances in experimental study of their functions in insect biochemistry.
Serpins are serine protease inhibitors that are widely distributed in metazoans, but they have no... more Serpins are serine protease inhibitors that are widely distributed in metazoans, but they have not been characterized previously in Eimeria spp. A serpin from Eimeria acervulina was cloned, expressed, and characterized. Random screening of an E. acervulina sporozoite cDNA library identified a single clone (D14) whose coding region shared high similarity to consensus structure of serpins. Clone D14 contained an entire open reading frame consisting of 1,245 nucleotides that encode a peptide of 413 amino acids, with a predicted molecular weight of 45.5 kDa and containing a signal peptide 28 residues. By Western blot analysis, polyclonal antiserum to the recombinant serpin (rbSp) recognized a major 55-kDa protein band in unsporulated oocysts and in oocysts sporulated up to 24 hr (fully sporulated). The anti-rbSp detected bands of 55 and 48 kDa in sporozoites (SZ) and merozoites (MZ), respectively. Analysis of MZ secretion products revealed a single protein of 48 kDa that may correspond to secreted serpin. By immunostaining, the serpin was located in granules distributed throughout both the SZ and MZ, but granules seemed to be concentrated in the parasites' anterior. Analysis of the structure predicts that the E. acervulina serpin should be an active inhibitor. However, rbSp was without inhibitory activity against common serine proteases. By Western blot analysis, the endogenous serpin in MZ extracts did not form the expected high-molecular-weight complex when coincubated with either trypsin or subtilisin. The results demonstrate that E. acervulina contains a serpin gene and expresses a protein with structural properties similar to an active serine protease inhibitor. Although the function of the E. acervulina serpin remains unknown, the results further suggest that serpin is secreted by the parasite where it may be involved in cell invasion and other basic developmental processes.
CLIP proteases are non-digestive serine proteases present in hemolymph of insects and other arthr... more CLIP proteases are non-digestive serine proteases present in hemolymph of insects and other arthropods. They are composed of one or more amino-terminal clip domains followed by a linker sequence and a carboxyl-terminal S1A family serine protease domain. The genes for CLIP proteases have evolved as four clades (CLIPA, CLIPB, CLIPC, CLIPD), each present as multigene families in insect genomes. CLIP proteases in hemolymph function in innate immune responses. These include proteolytic activation of the cytokine Spätzle, to form an active Toll ligand leading to synthesis of antimicrobial peptides, and specific activation of prophenoloxidase, required for the melanization response. CLIP proteases act in cascade pathways. In the immune pathways that have been characterized, microbial surface molecules stimulate activation of an initiating modular serine protease, which then activates a CLIPC, which in turn activates a CLIPB. The active CLIPB then cleaves and activates an effector molecule (proSpätzle or prophenoloxidase). CLIPA proteins are pseudoproteases, lacking proteolytic activity, but some can function as regulators of the activity of other CLIP proteases and form high molecular weight immune complexes. A few three dimensional structures for CLIP proteases are now available for structure-function analysis of these immune factors, revealing structural features that may act in specific activation or in formation of immune complexes. The functions of most CLIP proteases are unknown, even in well studied insect species. It is very likely that additional proteins activated by CLIP proteases and acting in immunity remain to be discovered.
The body form of holometabolous insects dramatically transforms from larval to adult stages durin... more The body form of holometabolous insects dramatically transforms from larval to adult stages during metamorphosis that occurs in the pupal stage. The larval disorganization and then new adult tissues are built up at this time. In motoneuron, larval neuronal cells degenerate, and new adult neurons are remodeled. Finally, adult neurons reconnect to new adult muscles. However, the factors that control metamorphosis have not yet been fully elucidated. Here, we show that an antioxidant enzyme, Tribolium castaneum superoxide dismutase 6 (TcSOD6), is secreted into the haemolymph and is required for proper movable legs during metamorphosis. TcSOD6 has a unique domain architecture and is mainly expressed in the pupal stage. The depletion of TcSOD6 expression in the pupa inhibits normal axon development and results in adults that display dysfunctional leg motions, suggesting that SOD6 expression is required for the development of properly movable legs. Therefore, we speculate that TcSOD6 might...
Members of the serpin superfamily of proteins occur in animals, plants, bacteria, archaea and som... more Members of the serpin superfamily of proteins occur in animals, plants, bacteria, archaea and some viruses. They adopt a variety of physiological functions, including regulation of immune system, modulation of apoptosis, hormone transport and acting as storage proteins. Most members of the serpin family are inhibitors of serine proteinases. In this study, we searched the genome of Manduca sexta and identified 32 serpin genes. We analyzed the structure of these genes and the sequences of their encoded proteins. Three M. sexta genes (serpin-1, serpin-15, and serpin-28) have mutually exclusive alternatively spliced exons encoding the carboxyl terminal reactive center loop of the protein, which is the site of interaction with target proteases. We discovered that MsSerpin-1 has 14 splicing isoforms, including two undiscovered in previous studies. Twentyeight of the 32 M. sexta serpins include a putative secretion signal peptide and are predicted to be extracellular proteins. Phylogenetic analysis of serpins in M. sexta and Bombyx mori indicates that 17 are orthologous pairs, perhaps carrying out essential physiological functions. Analysis of the reactive center loop and hinge regions of the protein sequences indicates that 16 of the serpin genes encode proteins that may lack proteinase inhibitor activity. Our annotation and analysis of these serpin genes and their transcript profiles should lead to future advances in experimental study of their functions in insect biochemistry.
CLIP proteases are non-digestive serine proteases present in hemolymph of insects and other arthr... more CLIP proteases are non-digestive serine proteases present in hemolymph of insects and other arthropods. They are composed of one or more amino-terminal clip domains followed by a linker sequence and a carboxyl-terminal S1A family serine protease domain. The genes for CLIP proteases have evolved as four clades (CLIPA, CLIPB, CLIPC, CLIPD), each present as multigene families in insect genomes. CLIP proteases in hemolymph function in innate immune responses. These include proteolytic activation of the cytokine Spätzle, to form an active Toll ligand leading to synthesis of antimicrobial peptides, and specific activation of prophenoloxidase, required for the melanization response. CLIP proteases act in cascade pathways. In the immune pathways that have been characterized, microbial surface molecules stimulate activation of an initiating modular serine protease, which then activates a CLIPC, which in turn activates a CLIPB. The active CLIPB then cleaves and activates an effector molecule (proSpätzle or prophenoloxidase). CLIPA proteins are pseudoproteases, lacking proteolytic activity, but some can function as regulators of the activity of other CLIP proteases and form high molecular weight immune complexes. A few three dimensional structures for CLIP proteases are now available for structure-function analysis of these immune factors, revealing structural features that may act in specific activation or in formation of immune complexes. The functions of most CLIP proteases are unknown, even in well studied insect species. It is very likely that additional proteins activated by CLIP proteases and acting in immunity remain to be discovered.
Insect biochemistry and molecular biology, Jan 17, 2015
Members of the multicopper oxidase (MCO) family of enzymes can be classified by their substrate s... more Members of the multicopper oxidase (MCO) family of enzymes can be classified by their substrate specificity; for example, ferroxidases oxidize ferrous iron, ascorbate oxidases oxidize ascorbate, and laccases oxidize aromatic substrates such as diphenols. Our previous work on an insect multicopper oxidase, MCO1, suggested that it may function as a ferroxidase. This hypothesis was based on three lines of evidence: RNAi-mediated knock down of Drosophila melanogaster MCO1 (DmMCO1) affects iron homeostasis, DmMCO1 has ferroxidase activity, and DmMCO1 has predicted iron binding residues. In our current study, we expanded our focus to include MCO1 from Anopheles gambiae, Tribolium castaneum, and Manduca sexta. We verified that MCO1 orthologs have similar expression profiles, and that the MCO1 protein is located on the basal surface of cells where it is positioned to oxidize substrates in the hemolymph. In addition, we determined that RNAi-mediated knock down of MCO1 in A. gambiae affects i...
ÐCuticle sclerotization or tanning is a vital process that occurs during each stage of insect dev... more ÐCuticle sclerotization or tanning is a vital process that occurs during each stage of insect development to harden and stabilize the newly secreted exoskeleton. The structural polymers protein and chitin make up the bulk of the cuticle, and chemical interactions between these biopolymers with quinonoid tanning agents are largely responsible for the physical properties of the mature exoskeleton. The oxidative conjugation of catechols with cuticular proteins plays an important role in this metabolism. The main hypothesis for cuticle sclerotization involves the formation of adducts and cross-links between nucleophilic imidazole nitrogens of histidyl residues in the proteins and electrophilic ring or side-chain carbons of ortho-quinones and para-quinone methides derived from the catechols, N-acetyldopamine, N-betaalanyldopamine, and 3,4-dihydroxyphenylethanol. C±N and C±O linkages between these quinone tanning agents and proteins in cuticles from a variety of insects from several orders have been elucidated. cDNAs for both the tyrosinase and laccase types of phenoloxidases that catalyze the cross-linking reactions have been isolated and sequenced. The sequences of laccase cDNAs from two insect species were more similar to fungal laccases than to those from plants. These results provide insights into how insects use structural proteins, catechols, and oxidative enzymes to form catechol±amino acid adducts during sclerotization. Published by Elsevier Science Ltd.
Proceedings of the National Academy of Sciences, 1998
Activation of pro-phenol oxidase (proPO) in insects and crustaceans is important in defense again... more Activation of pro-phenol oxidase (proPO) in insects and crustaceans is important in defense against wounding and infection. The proPO zymogen is activated by a specific proteolytic cleavage. PO oxidizes phenolic compounds to produce quinones, which may help to kill pathogens and can also be used for synthesis of melanin to seal wounds and encapsulate parasites. We have isolated from the tobacco hornworm, Manduca sexta , a serine proteinase that activates proPO, and have cloned its cDNA. The isolated proPO activating proteinase (PAP) hydrolyzed artificial substrates but required other protein factors for proPO activation, suggesting that proPO-activating enzyme may exist as a protein complex, one component of which is PAP. PAP (44 kDa) is composed of two disulfide-linked polypeptide chains (31 kDa and 13 kDa). A cDNA for PAP was isolated from a hemocyte library, by using a PCR-generated probe based on the amino-terminal amino acid sequence of the 31-kDa catalytic domain. PAP belongs ...
Extracellular serine proteinase cascades stimulate prophenoloxidase (proPO) activation and antimi... more Extracellular serine proteinase cascades stimulate prophenoloxidase (proPO) activation and antimicrobial peptide production in insect innate immune responses. Serpins in plasma regulate such cascades by selective inhibition of proteinases, in reactions which result in the formation of covalent serpin-proteinase complexes. We carried out experiments to identify plasma proteinases that are inhibited by Manduca sexta serpin-3, an immune-inducible serpin known to regulate proPO activation. Immunoaffinity chromatography, using antiserum to serpin-3, yielded serpin-3 complexes with proteinases identified by immunoblot analysis as prophenoloxidase-activating proteinase (PAP)-1, PAP-2, PAP-3, and hemolymph proteinase 8 (HP8). HP8 can cleave and activate the Toll ligand, Spätzle, leading to synthesis of antimicrobial peptides. Analysis by mass spectrometry of tryptic peptides derived from the serpin-3 complexes confirmed the presence of PAP-1, PAP-3, and HP8. Purified recombinant serpin-3 and active HP8 formed an SDS-stable complex in vitro. Identification of serpin-3-proteinase complexes in plasma provides insight into proteinase targets of serpin-3 and extends the understanding of serpin/proteinase function in the immune response of M. sexta.
Leucine-rich repeat containing proteins are involved in immune response in many capacities. In in... more Leucine-rich repeat containing proteins are involved in immune response in many capacities. In insects, these include Toll-like receptors and the Anopheles gambiae proteins APL1 and LRIM1. Here we describe the identification and characterization of leureptin, a novel extracellular protein with 13 leucine-rich repeats from hemolymph of the insect Manduca sexta. After injection of bacteria, leureptin mRNA level increased in fat body, but protein levels in plasma decreased, an indication that leureptin is consumed during the immune response. Leureptin bound to bacterial lipopolysaccharide (LPS). Microscopy using leureptin antiserum showed that leureptin associates with hemocytes after injection of bacteria, an indication that leureptin is involved in hemocyte responses to bacterial infection. Sequence database searches suggest similar proteins are present in other Lepidopteran species.
Extracellular serine proteinase pathways control immune and homeostatic processes in insects. Our... more Extracellular serine proteinase pathways control immune and homeostatic processes in insects. Our current knowledge of their components is limited-prophenoloxidase-activating proteinases (PAPs) are among the few hemolymph proteinases (HPs) with known functions. To identify components of proteinase systems in the hemolymph of Manduca sexta, we amplified cDNAs from larval fat body or hemocytes using degenerate primers coding for two conserved regions in S1 family serine proteinases. PCR yielded fragments encoding seven known (HP1-HP4, PAP-1, PAP-2 and PAP-3) and 18 unknown (HP5-HP22) serine proteinases. We screened cDNA libraries and isolated clones for 17 of the newly discovered HPs (HP5-HP22 except for HP11) and prepared antibodies to 14 recombinant proteins (HP6, HP8-HP10, HP12, HP14-HP19, HP21 and HP22). Fourteen of the HPs contain regulatory clip domain(s) at their amino-terminus-HP1, HP2, HP6, HP8, HP13, HP17, HP18, HP21, HP22 and PAP-1 have one, whereas HP12, HP15, PAP-2 and PAP-3 have two clip domains. Multiple sequence alignment of catalytic domains in these and other arthropod serine proteinases provided useful clues for future functional analysis. Northern blot and reverse transcription PCR (RT-PCR) analyses showed increases in HP2, HP7, HP9, HP10, HP12-HP22 mRNA levels at 24 h after a bacterial challenge, and immunoblot analysis confirmed elevated concentrations of HP12, HP14-HP19, HP21 and HP22 proteins in plasma in response to injected bacteria. Hemocytes express HP13 and HP18; fat body produces HP12, HP20-HP22; both tissues synthesize the other HPs. These results collectively indicate the existence of a complex serine proteinase network in M. sexta hemolymph, predicted to mediate rapid defense responses upon wounding and/or microbial infection.
The innate immune system of insects include the Toll pathway, which is mediated by an extracellul... more The innate immune system of insects include the Toll pathway, which is mediated by an extracellular serine proteinase cascade. In the tobacco hornworm, Manduca sexta, hemolymph proteinase 8 (HP8) promotes the synthesis of antimicrobial proteins by cleaving proSpätzle, the putative ligand of M. sexta Toll. HP8 has been observed to form a complex in hemolymph with M. sexta serpin-1, which has multiple alternative splicing isoforms. To investigate the regulation of HP8 and its processing of proSpätzle, we characterized the interaction of recombinant HP8 with serpin-1 isoform J (serpin-1J). Recombinant serpin-1J formed an SDS-stable complex with HP8 in vitro. The association rate constant of serpin-1J and HP8 was 1.3×10 4 M-1 s-1 , with a stoichiometry of inhibition of 5.4. Serpin-1J inhibited the cleavage of proSpätzle by HP8. Injection of serpin-1J into M. sexta larvae resulted in decreased bacteria-induced antimicrobial activity in hemolymph and reduced expression of cecropin, attacin and hemolin mRNA in fat body. Altogether, these results suggest that serpin-1J functions to inhibit HP8 and thereby modulates the concentration of active Spätzle to regulate the Toll pathway response in M. sexta.
Proceedings of the National Academy of Sciences of the United States of America, Sep 8, 2020
Significance Phenoloxidase-catalyzed melanization and Spätzle-triggered Toll signaling are critic... more Significance Phenoloxidase-catalyzed melanization and Spätzle-triggered Toll signaling are critical innate immune responses of insects that are triggered by specific proteolysis. It has been unclear whether separate protease cascades or an integrated network of serine proteases coordinates these responses. Here we present evidence that hemolymph protease HP5 acts in pathways eliciting activation of both phenoloxidase and Spätzle-1 in Manduca sexta . HP5 has a unique specificity by cleaving proHP6 at His 112 , resulting in HP6 activation. CLIP proteases related to HP6 from other insect species, including Drosophila Persephone, share this activation site feature with proHP6, and therefore, HP5 orthologs in other species are candidates for key roles in regulating and integrating protease cascade pathways in innate immune responses.
Recognition of nonself is the first step in mounting immune responses. In the innate immune syste... more Recognition of nonself is the first step in mounting immune responses. In the innate immune systems of both vertebrates and arthropods, such recognition, termed pattern recognition, is mediated by a group of proteins, known as pattern recognition proteins or receptors. Different pattern recognition proteins recognize and bind to molecules (molecular patterns) present on the surface of microorganisms but absent from animals. These molecular patterns include microbial cell wall components such as bacterial lipopolysaccharide, lipoteichoic acid and peptidoglycan, and fungal β-1,3-glucans. Binding of pattern recognition proteins to these molecular patterns triggers responses such as phagocytosis, nodule formation, encapsulation, activation of proteinase cascades, and synthesis of antimicrobial peptides. In this article, we describe four classes of pattern recognition proteins, hemolin, peptidoglycan recognition protein, β-1,3-glucan recognition proteins, and immulectins (C-type lectins) involved in immune responses of the tobacco hornworm, Manduca sexta.
Proceedings of the National Academy of Sciences, 2006
Serpins are central to the modulation of various innate immune responses in insects and are suspe... more Serpins are central to the modulation of various innate immune responses in insects and are suspected to influence the outcome of malaria parasite infection in mosquito vectors. Three Anopheles gambiae serpins (SRPN1, -2, and -3) were tested for their ability to inhibit the prophenoloxidase cascade, a key regulatory process in the melanization response. Recombinant SRPN1 and -2 can bind and inhibit a heterologous phenoloxidase-activating protease and inhibit phenoloxidase activation in vitro . Using a reverse genetics approach, we studied the effect of SRPN2 on melanization in An. gambiae adult females in vivo . Depletion of SRPN2 from the mosquito hemolymph increases melanin deposition on foreign surfaces such as negatively charged Sephadex beads. As reported, the knockdown of SRPN2 adversely affects the ability of the rodent malaria parasite Plasmodium berghei to invade the midgut epithelium and develop into oocysts. Importantly, we tested whether the absence of SRPN2 from the hem...
Serpins regulate various physiological reactions in humans and insects, including certain immune ... more 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.
Serpins (serine protease inhibitors) regulate some innate immune responses of insects by inhibiti... more Serpins (serine protease inhibitors) regulate some innate immune responses of insects by inhibiting endogenous proteases. In this study, we characterized the serpin (SRPN) gene family in the mosquito Anopheles gambiae, the major malaria vector in Sub-Saharan Africa. We identified 18 A. gambiae SRPN genes, all on chromosomes 2 and 3, through searches of genomic DNA and EST databases. In addition to SRPN10, previously documented to exhibit alternative splicing, we found three splicing isoforms of SRPN4. We completed sequencing of cDNAs for the A. gambiae serpins to obtain complete coding sequence information and to verify or improve gene predictions. The predicted SRPN9 and 15 in the initial genome annotation were determined to be a single gene (SRPN9). Sixteen of the serpins contained putative secretion signal sequences. Multiple sequence alignments showing conserved residues important in structural conformation, including the consensus pattern within the hinge region, indicated that most of the A. gambiae serpins may be inhibitory. Phylogenetic analyses confirmed that SRPN1, 2, 3, 8, 9 and 10 formed phylogenetic clusters with known inhibitory serpins from Drosophila melanogaster and Manduca sexta. Many of the A. gambiae serpins were expressed during all life stages. However, SRPN7, 8, 12, and 19 were expressed at very low levels in the adult stage. SRPN13 was expressed mostly in eggs and young larvae, whereas SRPN5 and 14 were expressed mostly in adults. Such differences in expression pattern suggest that the serpins are involved in multiple physiological processes. Determining the biological functions of the mosquito serpins will require future work to identify the proteases they inhibit in vivo.
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Papers by Michael Kanost