A novel <i>Drosophila</i> serpin that inhibits serine proteases

Han, Jin-Hua; Zhang, Hongyan; Min, Gi–Sik; Kemler, Doris; Hashimoto, Carl

doi:10.1016/s0014-5793(00)01224-2

Cited by 48 publications

(42 citation statements)

References 24 publications

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“…Also, SPN55 and SPN48 only formed complexes with the active forms of SAE and SPE, respectively (lanes 7 and 12). Under the same conditions, recombinant Tenebrio SPN1, which shows high sequence identity (37%) with that of Drosophila SPN1 (25) and which was purified as a control serpin, did not form any complexes with the three Toll cascade-activating SPs (lanes 14 -16). These results suggest that the three purified serpins form highly specific serpin⅐protease complexes with the three Toll cascade-activating SPs.…”

Section: Three Purified Serpins Formed Specific Complexes With Threementioning

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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Section: Three Purified Serpins Formed Specific Complexes With Threementioning

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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“…The number of functional serpin proteins in Drosophila is likely Ͼ19, because invertebrate serpin genes often have multiple exons that code for reactive centers, allowing for mutually exclusive splicing (Jiang et al, 1994;Kruger et al, 2002). Of particular interest is Spn4, which was reported as one of six serpin-like genes expressed in Drosophila oocytes (Han et al, 2000). Spn4 protein shows significant similarity (30% identity on an amino acid level) to the vertebrate neuronal serpin neuroserpin (Osterwalder et al, 1996;Krueger et al, 1997;Schrimpf et al, 1997).…”

Section: Molecular Analysis Of the Spn4 Genementioning

confidence: 99%

“…Spn4 was initially identified as one of six serpins expressed in Drosophila oocytes (Han et al, 2000) and is the closest Drosophila homolog to neuroserpin, a vertebrate neuronal serpin (Osterwalder et al, 1996;Schrimpf et al, 1997). We analyzed the Spn4 gene and describe two of its alternative splice isoforms, referred to as Spn4.1 and Spn4.2.…”

Section: Introductionmentioning

confidence: 99%

DrosophilaSerpin 4 Functions as a Neuroserpin-Like Inhibitor of Subtilisin-Like Proprotein Convertases

Osterwalder

Kuhnen²,

Leiserson³

et al. 2004

J. Neurosci.

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The proteolytic processing of neuropeptide precursors is believed to be regulated by serine proteinase inhibitors, or serpins. Here we describe the molecular cloning and functional expression of a novel member of the serpin family, Serine protease inhibitor 4 (Spn4), that we propose is involved in the regulation of peptide maturation in Drosophila. The Spn4 gene encodes at least two different serpin proteins, generated by alternate splicing of the last coding exon. The closest vertebrate homolog to Spn4 is neuroserpin. Like neuroserpin, one of the Spn4 proteins (Spn4.1) features a unique C-terminal extension, reminiscent of an endoplasmic reticulum (ER) retention signal; however, Spn4.1 and neuroserpin have divergent reactive site loops, with Spn4.1 showing a generic recognition site for furin/SPC1, the founding member of the intracellularly active family of subtilisin-like proprotein convertases (SPCs). In vitro, Spn4.1 forms SDS-stable complexes with the SPC furin and directly inhibits it. When Spn4.1 is overexpressed in specific peptidergic cells of Drosophila larvae, the animals exhibit a phenotype consistent with disrupted neuropeptide processing. This observation, together with the unique combination of an ER-retention signal, a target sequence for SPCs in the reactive site loop, and the in vitro inhibitory activity against furin, strongly suggests that Spn4.1 is an intracellular regulator of SPCs.

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“…Preliminary evidence showed that this trypsin-binding protein was a member of the serpin family of antiproteases; purification and sequencing allowed us to identify it as Spn5, a Drosophila serpin first described by Han et al (Han et al, 2000). Spn5 is known to be expressed in the ovary and in embryos and to encode a protein of 427 amino acids, with a predicted signal Northern blot analysis of total RNA isolated from control UAS-Spn5-IR R larvae (oddnumbered lanes), and from Spn5 RNAi expressing larvae carrying the daGal4 driver and the UAS-Spn5-IR R construct (even-numbered lanes).…”

Section: Discussionmentioning

confidence: 99%

“…This band was electroblotted onto a polyvinylidene fluoride (PVDF) membrane and subjected to amino-terminal sequence analysis. The Nterminal sequence of the purified ligand was determined and sequence comparison analysis in the Swiss-Prot data bank revealed that the peptide was identical to a portion of the Drosophila serpin, Spn5, previously identified as a serpin gene with maternal and zygotic expression (Han et al, 2000). The Cterminal region of Spn5 contains highly conserved residues forming the reactive center loop (RCL) with a putative cleavage site for tryptic enzymes as its target proteases.…”

Section: Purification Of the Ligand And Identification As Spn5mentioning

confidence: 98%

The serpin Spn5 is essential for wing expansion in Drosophila melanogaster

Charron¹,

Madani²,

Combepine³

et al. 2008

Int. J. Dev. Biol.

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Serpins, a superfamily of protease inhibitors, control proteolytic cascades in many physiological processes. Genomic studies have revealed the presence of a high number of serpinencoding genes in Drosophila melanogaster, but their functions remain largely unknown. In a biochemical screen designed to detect protease inhibitors that may be implicated in early Drosophila development, we identified in embryos a ligand that forms a 67 kDa SDS-stable complex with the broad spectrum protease trypsin. Characterization of this ligand revealed it to be the recently described serpin, Spn5. Expression analysis by in situ and Northern blot hybridization indicated maternal transmission of the transcript as well as zygotic expression in many larval, pupal and adult tissues. Targeted repression by RNA interference did not alter early embryogenesis but resulted in a complete defect in the unfolding and expansion of the wings of freshly eclosed mutant flies, without other detectable effects on development.

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A novel Drosophila serpin that inhibits serine proteases

Cited by 48 publications

References 24 publications

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

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

DrosophilaSerpin 4 Functions as a Neuroserpin-Like Inhibitor of Subtilisin-Like Proprotein Convertases

The serpin Spn5 is essential for wing expansion in Drosophila melanogaster

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