Drosophila Serpin-28D regulates hemolymph phenoloxidase activity and adult pigmentation

Scherfer, Christoph; Tang, Huaping; Kambris, Zakaria; Lhocine, Nouara; Hashimoto, Carl; Lemaître, Bruno

doi:10.1016/j.ydbio.2008.08.030

Cited by 101 publications

(69 citation statements)

References 40 publications

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“…A search of the raw MALDI-TOF peaks from trypsin-treated samples for predicted peptides from Lys 336 to the P1 residue for the remaining isoforms in putative complex bands (spots 16 -25) failed to reveal any additional isoform-specific information (data not shown). Of the four spots shown to contain HP8 by MS (spots 19,20,22,23), spot 19 contains serpin-1 isoform J based on PMF and MS/MS results. PMF results suggest that spots 19 and 20 contain serpin-1A, whereas spots 22 and 23 contain serpin-1E, but these identifications are more tentative because of the lack of MS/MS corroboration.…”

Section: Differential Expression Of Serpin-1 Isoform Mrnas-previ-mentioning

confidence: 99%

Analysis of Mutually Exclusive Alternatively Spliced Serpin-1 Isoforms and Identification of Serpin-1 Proteinase Complexes in Manduca sexta Hemolymph

Ragan

Yang

et al. 2010

Journal of Biological Chemistry

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Mutually exclusive alternative splicing produces transcripts for 12 serpin-1 isoforms in Manduca sexta that differ only in the region encoding the carboxyl-terminal 36 -40-amino acid residues. This variable region includes the reactive center loop, which determines the inhibitory selectivity of the serpin. We investigated mRNA levels of individual serpin-1 isoforms by quantitative PCR. The 12 isoforms were expressed at similar levels in hemocytes, but in fat body isoform B mRNA was present at significantly higher levels than isoforms C, D, E, F, G, J, K, and Z. To investigate the presence of individual serpin-1 isoforms in plasma we used immunoaffinity purification of serpin-1 isoforms from M. sexta plasma, followed by two-dimensional PAGE and identification of protein spots by digestion with a series of proteinases and analysis of the resulting peptides by MALDI-TOF/TOF. We identified nine of the 12 serpin-1 isoforms and, through analysis of putative serpin-1-proteinase complexes, identified three endogenous M. sexta proteinase targets of serpin-1. Our results suggest that M. sexta serpin-1 isoforms A, E, and J can inhibit hemolymph proteinase 8, which activates the cytokine spätzle. At least one isoform of serpin-1 can inhibit hemocyte proteinase 1, another M. sexta blood proteinase. In addition, a complex of serpin-1K in a complex with M. sexta midgut chymotrypsin was identified, suggesting serpin-1 isoforms may also function to protect insect tissues from digestive proteinases that may leak into the hemocoel.Serpins are a superfamily of proteins that are named for being serine proteinase inhibitors, a function that many serpins perform. Serpins contain an exposed reactive center loop, formed from a region near the carboxyl-terminal end of the protein. A proteinase that begins to cleave a serpin in the reactive center loop typically becomes covalently bound to the serpin and inactivated. The proteinase active site serine forms an ester bond with the acyl group of the serpin P1 residue at the amino-terminal side of the scissile bond, but cannot complete the hydrolysis because the serpin reactive center loop rapidly inserts into the A ␤-sheet, moving the proteinase ϳ70 Å and deforming the proteinase catalytic site (1, 2). Extracelluar serpins exert control over serine proteinase cascades in vertebrate blood, including complement activation, blood clotting, and fibrinolysis (3, 4). In insects, serine proteinase cascades initiated by microbial infection elicit antimicrobial peptide production and lead to activation of prophenoloxidase, which catalyzes the melanization response (5-13 In insects, serpin genes have evolved alternative exon splicing, which produces variation in the sequence of much of the reactive center loop, producing multiple functional serpins from a single gene. This was first described in M. sexta serpin-1, which has 12 different copies of exon 9 that undergo mutually exclusive alternative splicing to produce 12 putative protein isoforms. These isoforms differ in their carboxyl-terminal 39 -46 ...

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Section: Differential Expression Of Serpin-1 Isoform Mrnas-previ-mentioning

confidence: 99%

Analysis of Mutually Exclusive Alternatively Spliced Serpin-1 Isoforms and Identification of Serpin-1 Proteinase Complexes in Manduca sexta Hemolymph

Ragan

Yang

et al. 2010

Journal of Biological Chemistry

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“…Uncontrolled melanization generates excessive toxic intermediates that could kill the host, as demonstrated by the lethality resulting from mutations of serpins that inhibit melanization. [6][7][8][9] Indeed, phenol oxidase (PO), a key enzyme in melanin biosynthesis during melanization, is usually synthesized as an inactive zymogen called pro-phenoloxidase (PPO). Recognition of pathogens and/or injury leads to the activation of a serine protease cascade that culminates in proteolytic cleavage of inactive PPO to active PO.…”

Section: Introductionmentioning

confidence: 99%

Regulation and function of the melanization reaction in Drosophila

Tang

2009

Fly

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188

147

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The melanization reaction, involving the synthesis of melanin to encapsulate pathogens, is a prominent immune response in Drosophila, the mosquito and other insects and arthropods. Biochemical studies with large insects have defined a basic model for how melanization is activated and regulated upon microbial infection. In this model, recognition of a microorganism triggers a serine protease cascade that activates phenol oxidase (PO), a key enzyme in the melanin biosynthetic pathway, and serpin-type protease inhibitors are involved in inhibiting the cascade. In the past few years, genetic studies in Drosophila have identified serine proteases and serpins that regulate activation of PO and melanization in vivo. These studies, along with molecular genetic analysis of melanization in the mosquito, have provided new insight into the role that melanization plays in fighting microbial infection.

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“…SPN43Ac mutant flies accumulated cleaved Spätzle, resulting in constitutive activation of the Toll pathway and expression of AMPs (14). SPN27A and SPN28D are known to regulate the Toll pathway during early development (15)(16)(17) and are also involved in the melanin biosynthesis reaction (18,19). Another serpin, SPN77Ba, was identified as a negative regulator of melanization in the respiratory system (the trachea) of Drosophila (20).…”

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confidence: 99%

Three Pairs of Protease-Serpin Complexes Cooperatively Regulate the Insect Innate Immune Responses

Jiang

Kim

Gong

et al. 2009

Journal of Biological Chemistry

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Serpins are known to be necessary for the regulation of several serine protease cascades. However, the mechanisms of how serpins regulate the innate immune responses of invertebrates are not well understood due to the uncertainty of the identity of the serine proteases targeted by the serpins. We recently reported the molecular activation mechanisms of three serine protease-mediated Toll and melanin synthesis cascades in a large beetle, Tenebrio molitor. Here, we purified three novel serpins (SPN40, SPN55, and SPN48) from the hemolymph of T. molitor. These serpins made specific serpin-serine protease pairs with three Toll cascade-activating serine proteases, such as modular serine protease, Spätzle-processing enzyme-activating enzyme, and Spätzle-processing enzyme and cooperatively blocked the Toll signaling cascade and ␤-1,3-glucanmediated melanin biosynthesis. Also, the levels of SPN40 and SPN55 were dramatically increased in vivo by the injection of a Toll ligand, processed Spätzle, into Tenebrio larvae. This increase in SPN40 and SPN55 levels indicates that these serpins function as inducible negative feedback inhibitors. Unexpectedly, SPN55 and SPN48 were cleaved at Tyr and Glu residues in reactive center loops, respectively, despite being targeted by trypsin-like Spätzle-processing enzyme-activating enzyme and Spätzle-processing enzyme. These cleavage patterns are also highly similar to those of unusual mammalian serpins involved in blood coagulation and blood pressure regulation, and they may contribute to highly specific and timely inactivation of detrimental serine proteases during innate immune responses. Taken together, these results demonstrate the specific regulatory evidences of innate immune responses by three novel serpins.

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Drosophila Serpin-28D regulates hemolymph phenoloxidase activity and adult pigmentation

Cited by 101 publications

References 40 publications

Analysis of Mutually Exclusive Alternatively Spliced Serpin-1 Isoforms and Identification of Serpin-1 Proteinase Complexes in Manduca sexta Hemolymph

Analysis of Mutually Exclusive Alternatively Spliced Serpin-1 Isoforms and Identification of Serpin-1 Proteinase Complexes in Manduca sexta Hemolymph

Regulation and function of the melanization reaction in Drosophila

Three Pairs of Protease-Serpin Complexes Cooperatively Regulate the Insect Innate Immune Responses

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