Prophenoloxidase-activating proteinase-3 (PAP-3) from Manduca sexta hemolymph: a clip-domain serine proteinase regulated by serpin-1J and serine proteinase homologs

Jiang, Haobo; Wang, Yan; Yu, Xiao‐Qiang; Zhu, Yifei; Kanost, Michael R.

doi:10.1016/s0965-1748(03)00123-1

Cited by 202 publications

(253 citation statements)

References 27 publications

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“…Only a handful of these hemolymph proteinases have been studied functionally. ProPAP1, proPAP2, and proPAP3 can cleave and activate proPO (39,(45)(46)(47) in a reaction that requires the noncatalytic SPH1 and SPH2 (26,39,45,47). One cascade pathway for proPO activation in M. sexta has been established.…”

Section: Discussionmentioning

confidence: 99%

Functions of Manduca sexta Hemolymph Proteinases HP6 and HP8 in Two Innate Immune Pathways

Ishibashi

Ragan

et al. 2009

Journal of Biological Chemistry

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Serine proteinases in insect plasma have been implicated in two types of immune responses; that is, activation of prophenoloxidase (proPO) and activation of cytokine-like proteins. We have identified more than 20 serine proteinases in hemolymph of the tobacco hornworm, Manduca sexta, but functions are known for only a few of them. We report here functions of two additional M. sexta proteinases, hemolymph proteinases 6 and 8 (HP6 and HP8). HP6 and HP8 are each composed of an amino-terminal clip domain and a carboxyl-terminal proteinase domain. HP6 is an apparent ortholog of Drosophila Persephone, whereas HP8 is most similar to Drosophila and Tenebrio spätzle-activating enzymes, all of which activate the Toll pathway. proHP6 and proHP8 are expressed constitutively in fat body and hemocytes and secreted into plasma, where they are activated by proteolytic cleavage in response to infection. To investigate activation and biological activity of HP6 and HP8, we purified recombinant proHP8, proHP6, and mutants of proHP6 in which the catalytic serine was replaced with alanine, and/or the activation site was changed to permit activation by bovine factor Xa. HP6 was found to activate proPO-activating proteinase (proPAP1) in vitro and induce proPO activation in plasma. HP6 was also determined to activate proHP8. Active HP6 or HP8 injected into larvae induced expression of antimicrobial peptides and proteins, including attacin, cecropin, gloverin, moricin, and lysozyme. Our results suggest that proHP6 becomes activated in response to microbial infection and participates in two immune pathways; activation of PAP1, which leads to proPO activation and melanin synthesis, and activation of HP8, which stimulates a Toll-like pathway.Innate immune systems of mammals and arthropods include extracellular serine proteinase cascade pathways, which rapidly amplify responses to infection and stimulate killing of pathogens. These proteinase-driven processes include the complement system of vertebrates (1, 2) and pathways in arthropods involving proteinases containing amino-terminal clip domains (3). Clip domain proteinases function in blood coagulation (4, 5), activation of prophenoloxidase (proPO) that leads to melanin synthesis (6 -9), and stimulation of the Toll pathway to promote synthesis of antimicrobial peptides/proteins (AMPs) 2 secreted into the hemolymph (10, 11).The serine proteinase systems best characterized in arthropods are the horseshoe crab hemolymph coagulation pathway and the cascade leading to activation of the Toll pathway in dorsal-ventral development in Drosophila (12-14). Recent research also has led to better characterization of the proPO activation pathway in Manduca sexta (7,15,16) and the Tollsignaling pathway in the Drosophila immune response (17, 18) and to both the proPO and Toll pathways in the beetle Tenebrio molitor (11,19).In the proPO activation pathway, soluble pattern recognition proteins initially recognize pathogen-associated molecular patterns such as bacterial peptidoglycan or fungal ␤-1,3-glucan (...

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Section: Discussionmentioning

confidence: 99%

Functions of Manduca sexta Hemolymph Proteinases HP6 and HP8 in Two Innate Immune Pathways

Ishibashi

Ragan

et al. 2009

Journal of Biological Chemistry

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150

176

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“…Melanization requires the limited proteolysis of inactive PPO to active PO by PPAEs, trypsin-like clip domain serine proteases similar to the Drosophila Easter protease. Much is known on the biochemistry of PPO activation by CLIPs from in vitro reconstitution studies using proteins purified from the cuticle or plasma of several large insect species, including B. mori (28), M. sexta (27,29,30), H. diomphalia (5, 6), and Hyalophora cecropia (31). However, the involvement and specific contribution of PPAE-like CLIPs to immune responses to microbes and parasites has not been genetically investigated, primarily because of the absence of genetic tools for rapid analysis of gene function in the insect species that serve as models for biochemical studies.…”

Section: Discussionmentioning

confidence: 99%

The Roles of Two Clip Domain Serine Proteases in Innate Immune Responses of the Malaria Vector Anopheles gambiae

Volz¹,

Osta²,

Kafatos

et al. 2005

Journal of Biological Chemistry

113

109

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The malaria vector Anopheles gambiae is capable of multiple immune responses against Plasmodium ookinetes. Accumulating evidence in several insect species suggests the involvement of serine protease cascades in the initiation and coordination of immune responses. We report molecular and reverse genetic characterization of two mosquito clip domain serine proteases, CLIPB14 and CLIPB15, which share structural similarity to proteases involved in prophenoloxidase activation in other insects. Both CLIPs are expressed in mosquito hemocytes and are transcriptionally induced by bacterial and Plasmodium challenges. Functional studies applying RNA interference revealed that both CLIPs are involved in the killing of Plasmodium ookinetes in Anopheles. Studies on parasite melanization demonstrated an additional role for CLIPB14 in the prophenoloxidase cascade. We further report that both CLIPs participate in defense toward Gram-negative bacteria. Our findings strongly suggest that clip domain serine proteases serve multiple functions and play distinctive roles in several immune pathways of A. gambiae.

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“…3B), confirmed to be the light chain by trypsinolytic peptide mass fingerprint analysis (data not shown). The faint bands at the 30 and 31 kDa positions represent the heavy chain of HP21 -polyclonal antibodies against clip-domain serine proteinases reacted with the clip domains stronger than with the catalytic domains (Jiang et al, ,2003b. Under nonreducing condition, active HP21 migrated as a series of bands around 50 kDa (Fig.…”

Section: Cleavage Activation Of Prohp21 By Hp14mentioning

confidence: 94%

“…Immunoblot analyses were performed using 1:2,000 diluted serpin-4 or HP21 antiserum as the first antibody. To determine the cleavage site in serpin-4, purified proHP14 (80 ng, 4 μl), proHP21 (80 ng, 4 μl), curdlan (20 μg, 2 μl), βGRP2 (80 ng, 4 μl), serpin-4 (0.4 μg, 4 μl), and buffer C (30 μl) were incubated at 37°C for 1 h. In one negative control, serpin-4 was substituted with 4 μl of buffer C whereas, in the other control, serpin-4 was incubated with 44 μl buffer C. After centrifugation, supernatants of the samples were subjected to MALDI-TOF mass spectrometry (Jiang et al, 2003b). The molecular mass of a major peak absent in the control spectra was compared with calculated masses of carboxylterminal peptides of M. sexta serpin-4 to deduce the position of cleavage.…”

Section: Inhibitory Regulation Of Hp21 By M Sexta Serpin-4mentioning

confidence: 99%

Reconstitution of a branch of the Manduca sexta prophenoloxidase activation cascade in vitro: Snake-like hemolymph proteinase 21 (HP21) cleaved by HP14 activates prophenoloxidase-activating proteinase-2 precursor

Wang¹,

Jiang²

2007

Insect Biochemistry and Molecular Biology

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Upon wounding or infection, a serine proteinase cascade in insect hemolymph leads to prophenoloxidase (proPO) activation and melanization, a defense response against invading microbes. In the tobacco hornworm Manduca sexta, this response is initiated via hemolymph proteinase 14 (HP14), a mosaic protein that interacts with bacterial peptidoglycan or fungal β-1,3-glucan to autoactivate. In this paper, we report the expression, purification, and functional analysis of M. sexta HP21 precursor, an HP14 substrate similar to Drosophila Snake. The recombinant proHP21 is a 51.1 kDa glycoprotein with an amino-terminal clip domain, a linker region, and a carboxyl-terminal serine proteinase domain. HP14, generated by incubating proHP14 with β-1,3-glucan and β-1,3-glucan recognition protein-2, activated proHP21 by limited proteolysis between Leu 152 and Ile 153 . Active HP21 formed an SDS-stable complex with M. sexta serpin-4, a physiological regulator of the proPO activation system. We determined the P1 site of serpin-4 to be Arg 355 and, thus, confirmed our prediction that HP21 has trypsin-like specificity. After active HP21 was added to the plasma, there was a major increase in PO activity. HP21 cleaved proPO activating proteinase-2 precursor (proPAP-2) after Lys 153 and generated an amidase activity which activated proPO in the presence of serine proteinase homolog-1 and 2. In summary, we have discovered and reconstituted a branch of the proPO activation cascade in vitro: β-1,3-glucan recognition -proHP14 autoactivation -proHP21 cleavage -PAP-2 generation -proPO activation -melanin formation.

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Prophenoloxidase-activating proteinase-3 (PAP-3) from Manduca sexta hemolymph: a clip-domain serine proteinase regulated by serpin-1J and serine proteinase homologs

Cited by 202 publications

References 27 publications

Functions of Manduca sexta Hemolymph Proteinases HP6 and HP8 in Two Innate Immune Pathways

Functions of Manduca sexta Hemolymph Proteinases HP6 and HP8 in Two Innate Immune Pathways

The Roles of Two Clip Domain Serine Proteases in Innate Immune Responses of the Malaria Vector Anopheles gambiae

Reconstitution of a branch of the Manduca sexta prophenoloxidase activation cascade in vitro: Snake-like hemolymph proteinase 21 (HP21) cleaved by HP14 activates prophenoloxidase-activating proteinase-2 precursor

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