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

Volz, Jennifer; Osta, Mike A.; Kafatos, Fotis C.; Müller, Hans‐Michael

doi:10.1074/jbc.m506191200

Cited by 115 publications

(118 citation statements)

References 37 publications

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“…While biochemical functions are unknown for most of these clip-domain serine proteinases, Northern blot and RT-PCR analyses revealed that the mRNA levels of HP2, HP13, HP18, HP21, and HP22 increased in the larval fat body and/or hemocytes after a bacterial injection, suggesting that these enzymes all play certain roles in defense responses . Due to higher sequence similarities with PAPs, current research on melanization in A. gambiae is focused on members of the B group (Volz et al, 2005;Paskewitz et al, 2006). RNA interference tests showed close associations of ClipB4, ClipB8, ClipB14, and ClipB15 with melanization of Sephadex beads or Plasmodium ookinetes.…”

Section: Discussionmentioning

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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Section: Discussionmentioning

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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“…Likewise, RNA interference studies indicate that more than one melanization pathway is induced in response to infection in A. gambiae (11,14).…”

Section: Discussionmentioning

confidence: 99%

Manduca sexta Hemolymph Proteinase 21 Activates Prophenoloxidase-activating Proteinase 3 in an Insect Innate Immune Response Proteinase Cascade

Gorman

Wang

Jiang

et al. 2007

Journal of Biological Chemistry

107

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Melanization, an insect immune response, requires a set of hemolymph proteins including pathogen recognition proteins that initiate the response, a cascade of mostly unknown serine proteinases, and phenoloxidase. Until now, only initial and final proteinases in the pathways have been conclusively identified. Four such proteinases have been purified from the larval hemolymph of Manduca sexta: hemolymph proteinase 14 (HP14), which autoactivates in the presence of microbial surface components, and three prophenoloxidase-activating proteinases (PAP1-3). In this study, we have used two complementary approaches to identify a serine proteinase that activates pro-PAP3. Partial purification from hemolymph of an activator of proPAP3 resulted in an active fraction with two abundant polypeptides of ϳ32 and ϳ37 kDa. Labeling of these polypeptides with a serine proteinase inhibitor, diisopropyl fluorophosphate, indicated that they were active serine proteinases. N-terminal sequencing revealed that both were cleaved forms of the previously identified hemolymph serine proteinase, HP21. Surprisingly, cleavage of proHP21 had occurred not at the predicted activation site but more N-terminal to it. In vitro reactions carried out with purified HP14 (which activates proHP21), proHP21, proPAP3, and site-directed mutant forms of the latter two proteinases confirmed that HP21 activates proPAP3 by limited proteolysis. Like the HP21 products purified from hemolymph, HP21 that was activated by HP14 in the in vitro reactions was not cleaved at its predicted activation site.

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“…hemocytes are recruited to the midgut and dorsal vessel during Plasmodium sp. infection (Volz et al, 2005;Rodrigues et al, 2010;King and Hillyer, 2012), and may mediate innate 'immune memory' (Rodrigues et al, 2010). However, despite the multi-faceted contribution of mosquito hemocytes to immunity against malaria parasites, little is known about their basic biology.…”

Section: Research Articlementioning

confidence: 99%

Blood feeding induces hemocyte proliferation and activation in the African malaria mosquito, Anopheles gambiae Giles

Bryant

Michel

2013

Journal of Experimental Biology

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Malaria is a global public health problem, especially in sub-Saharan Africa, where the mosquito Anopheles gambiae Giles serves as the major vector for the protozoan Plasmodium falciparum Welch. One determinant of malaria vector competence is the mosquito's immune system. Hemocytes are a critical component as they produce soluble immune factors that either support or prevent malaria parasite development. However, despite their importance in vector competence, understanding of their basic biology is just developing. Applying novel technologies to the study of mosquito hemocytes, we investigated the effect of blood meal on hemocyte population dynamics, DNA replication and cell cycle progression. In contrast to prevailing published work, the data presented here demonstrate that hemocytes in adult mosquitoes continue to undergo low basal levels of replication. In addition, blood ingestion caused significant changes in hemocytes within 24 h. Hemocytes displayed an increase in cell number, size, granularity and Ras-MAPK signaling as well as altered cell surface moieties. As these changes are well-known markers of immune cell activation in mammals and Drosophila melanogaster Meigen, we further investigated whether a blood meal changes the expression of hemocyte-derived immune factors. Indeed, hemocytes 24 h post-blood meal displayed higher levels of critical components of the complement and melanization immune reactions in mosquitoes. Taken together, this study demonstrates that the normal physiological process of a blood meal activates the innate immune response in mosquitoes. This process is likely in part regulated by Ras-MAPK signaling, highlighting a novel mechanistic link between blood feeding and immunity.

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

Cited by 115 publications

References 37 publications

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

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

Manduca sexta Hemolymph Proteinase 21 Activates Prophenoloxidase-activating Proteinase 3 in an Insect Innate Immune Response Proteinase Cascade

Blood feeding induces hemocyte proliferation and activation in the African malaria mosquito, Anopheles gambiae Giles

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