Organization and expression of the hemolin gene, a member of the immunoglobulin superfamily in an insect, Manduca sexta

Journal of Biological Chemistry

Gorman

et al. 2003

172

171

Many serine proteinase inhibitors of the serpin superfamily have evolved in vertebrates and invertebrates to regulate serine proteinase cascades that mediate the host defense responses. We have isolated an immuneresponsive serpin from the tobacco hornworm, Manduca sexta. This inhibitor, M. sexta serpin-3, contains a reactive site loop strikingly similar to the proteolytic activation site in prophenoloxidase (pro-PO). Molecular cloning and sequence comparison indicate that serpin-3 is orthologous to Drosophila melanogaster serpin 27A, a regulator of melanization. M. sexta serpin-3 is constitutively present in hemolymph at a low concentration of 5-12 g/ml and increases to 30 -75 g/ml after a microbial challenge. Recombinant serpin-3 efficiently blocks pro-PO activation in the hemolymph, and it forms SDS-stable acyl-enzyme complexes with purified pro-PO-activating proteinases (PAPs) from M. sexta. PAP-serpin-3 complexes were isolated by immunoaffinity chromatography from hemolymph activated by treatment with Micrococcus luteus. Kinetic analysis of PAP-serpin-3 association strongly suggests that serpin-3 is a physiological regulator of the pro-PO activation reaction.

Section: Methodsmentioning

confidence: 99%

Manduca sexta Serpin-3 Regulates Prophenoloxidase Activation in Response to Infection by Inhibiting Prophenoloxidase-activating Proteinases

Zhu

Journal of Biological Chemistry

Gorman

et al. 2003

172

171

“…To detect the amidase activity of PAP in these fractions, we used as a substrate acetyl-Ile-Glu-Ala-Arg (IEAR)-p-nitroanilide, which is similar to the amino-terminal side of the putative activation site in M. sexta proPO-p1 and proPO-p2 (Leu-AsnAsn-Arg) (19). Very low IEARase activity was present in the flow-through fractions (4-10), which contained most of the protein, whereas a large peak of IEARase activity was detected in fractions [25][26][27][28] (Fig. 1A).…”

Section: Methodsmentioning

confidence: 99%

Pro-phenol oxidase activating proteinase from an insect, Manduca sexta: A bacteria-inducible protein similar to Drosophila easter

Jiang

Proc. Natl. Acad. Sci. U.S.A.

Kanost

1998

246

206

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 aminoterminal amino acid sequence of the 31-kDa catalytic domain. PAP belongs to a family of arthropod serine proteinases containing a carboxyl-terminal proteinase domain and an amino-terminal ''clip'' domain. The member of this family most similar in sequence to PAP is the product of the easter gene from Drosophila melanogaster. PAP mRNA was present at a low level in larval hemocytes and fat body, but became much more abundant in fat body after insects were injected with Escherichia coli. Sequence data and 3 H-diisopropyl f luorphosphate labeling results suggest that the same PAP exists in hemolymph and cuticle.

“…Expression Analysis by Reverse Transcriptase-PCR-Fat body and hemocyte total RNA samples of both naive and bacteria-induced M. sexta larvae were prepared (23). First-strand cDNA synthesis was performed using 2-4 g of total RNA, 10 pmol of oligo(dT) 17 , and 200 units of Moloney murine leukemia virus reverse transcriptase (Invitrogen) at 37°C for 1 h. The M. sexta ribosomal protein S3 cDNA was used as an internal standard to adjust the template amounts in a preliminary PCR experiment.…”

Section: Methodsmentioning

confidence: 99%

A Pattern Recognition Serine Proteinase Triggers the Prophenoloxidase Activation Cascade in the Tobacco Hornworm, Manduca sexta

Journal of Biological Chemistry

Guo

et al. 2004

A serine proteinase cascade in insect hemolymph mediates prophenoloxidase activation, a defense mechanism against pathogen or parasite infection. Little is known regarding its initiating proteinase or how this enzyme is activated in response to invading microorganisms. We have isolated from the tobacco hornworm, Manduca sexta, a cDNA encoding a modular protein designated hemolymph proteinase 14 (HP14). It contains five low density lipoprotein receptor class A repeats, a Sushi domain, a unique Cys-rich region, and a proteinase-catalytic domain. The HP14 mRNA exists in fat body and hemocytes of the naive larvae, and its level increases significantly at 24 h after a bacterial challenge. We expressed proHP14 with a carboxyl-terminal hexahistidine tag in a baculovirus/insect cell system and detected the recombinant protein in two forms. The 87-kDa protein was primarily intracellular, whereas the 75-kDa form was present in the medium. Interaction with peptidoglycan resulted in proteolytic processing of the purified zymogen and generation of an amidase activity. Supplementation of hemolymph with proHP14 greatly enhanced prophenoloxidase activation in response to Micrococcus luteus. These data suggest that proHP14 is a pattern recognition protein that binds to bacteria and autoactivates and triggers the prophenoloxidase activation system in the hemolymph of M. sexta.Similar to other invertebrates, insects lack an adaptive immune system and rely solely on their innate immune mechanisms to fight against invading microorganisms (1-3). These defense mechanisms are mediated by hemocytes (e.g. phagocytosis and encapsulation), fat body (e.g. induced synthesis of antimicrobial peptides), and plasma factors (e.g. hemolymph coagulation and melanization). Accumulating evidence indicates that a complex serine proteinase network in insect hemolymph coordinates some of these responses (4). The prophenoloxidase (proPO) 1 activation cascade is probably composed of several serine proteinases that are sequentially activated in response to microbial infection and lead to the proteolytic activation of proPO to phenoloxidase (PO). PO catalyzes the formation of quinones that are reactive intermediates for melanin synthesis (5, 6). Quinones are also involved in cuticle sclerotization, wound healing, and sequestration of parasites or pathogens.Although the physiological importance of the proPO activation in insect immune system has been appreciated for many years, the molecular characterization of the cascade components was reported only recently. We isolated three proPOactivating proteinases from cuticular extract or hemolymph of M. sexta prepupae, and they all required a protein cofactor for proPO activation (7-10). Similar results were obtained from the beetles Holotrichia diomphalia and Tenebrio molitor (11-13). In contrast, the silkworm proPO-activating enzyme does not appear to need any auxiliary factor for proPO activation (14). Limited by substrate availability, current biochemical research is focused on the components at the end of th...