Insect immunity: Two proteinase inhibitors from hemolymph of Locusta migratoria

Ra, Boigegrain; Mattras, Hélène; Brehélin, Michel; Paroutaud, Pierre; Ma, Coletti-Previero

doi:10.1016/0006-291x(92)92271-x

Cited by 77 publications

(65 citation statements)

References 12 publications

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“…3B). The PLDs share significant sequence similarities with the low molecular weight proteinase inhibitors LICM I, LICM II (11,12), and HI (14) isolated from the grasshopper L. migratoria (Fig. 3C).…”

Section: Methodsmentioning

confidence: 99%

“…The heavy chain has sequence similarity to transferrins, but in contrast to other transferrins, which have two lobes (8 -10), the pacifastin heavy chain has a three-lobe structure. The light chain contains nine homologous cysteine-rich domains with significant similarities to three proteinase inhibitors from L. migratoria (11)(12)(13)(14). This is the first known protein that has been found to contain both a transferrin chain and a proteinase inhibitor chain and we have named it pacifastin.…”

mentioning

confidence: 99%

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Pacifastin, a novel 155-kDa heterodimeric proteinase inhibitor containing a unique transferrin chain

Liang

Sottrup‐Jensen

Aspán

et al. 1997

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

128

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A 155-kDa proteinase inhibitor, pacifastin, from plasma of the freshwater crayfish, Pacifastacus leniusculus, was found to be composed of two covalently linked subunits. The two subunits are encoded by two different mRNAs, which were cloned and sequenced. The heavy chain of pacifastin (105 kDa) is related to transferrins, containing three transferrin lobes, two of which seem to be active for iron binding. The light chain of pacifastin (44 kDa) is the inhibitory subunit, and has nine cysteine-rich inhibitory domains that are homologous to each other and to low molecular weight proteinase inhibitors isolated from the grasshopper, Locusta migratoria. The nine light chain domains and the Locusta inhibitors share a characteristic cysteine array (Cys-Xaa 9 -12 -Cys-Xaa 2 -Cys-Xaa-Cys-Xaa 6 -8 -Cys-Xaa 4 -Cys) distinct from any described proteinase inhibitor family, suggesting that they constitute a new family of proteinase inhibitors. Pacifastin is the first known protein that has combined properties of a transferrin-like molecule and a proteinase inhibitor.

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Section: Methodsmentioning

confidence: 99%

mentioning

confidence: 99%

Pacifastin, a novel 155-kDa heterodimeric proteinase inhibitor containing a unique transferrin chain

Liang

Sottrup‐Jensen

Aspán

et al. 1997

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

128

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“…While this work was in progress, Boigegrain et al (22) have isolated from the hemolymph of L. migratoria two peptides with sequences identical with PMP-C and PMP-D2. They have shown that both PMP-C and PMP-D2 are powerful inhibitors of ␣-chymotrypsin (K i of 0.25 nM and 0.12 nM, respectively) and weak to medium inhibitors of HLE (K i Ͼ 0.1 M and of 18 nM, respectively).…”

Section: Discussionmentioning

confidence: 99%

Serine Protease Inhibition by Insect Peptides Containing a Cysteine Knot and a Triple-stranded β-Sheet

Kellenberger

Boudier

Bermúdez

et al. 1995

Journal of Biological Chemistry

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Three insect peptides showing high sequence similarity and belonging to the same structural family incorporating a cysteine knot and a short three-stranded antiparallel ␤-sheet were studied. Their inhibitory effect on two serine proteases (bovine ␣-chymotrypsin and human leukocyte elastase) is reported. One of them, PMP-C, is a strong ␣-chymotrypsin inhibitor (K i ‫؍‬ 0.2 nM) and interacts with leukocyte elastase with a K i of 0.12 M. The other two peptides, PMP-D2 and HI, interact only weakly with ␣-chymotrypsin and do not inhibit leukocyte elastase. Synthetic variants of these peptides were prepared by solid-phase synthesis, and their action toward serine proteases was evaluated. This enabled us to locate the P1 residues within the reactive sites (Leu-30 for PMP-C and Arg-29 for PMP-D2 and HI), and, interestingly, variants of PMP-D2 and HI were converted into powerful inhibitors of both ␣-chymotrypsin and leukocyte elastase, the most potent elastase inhibitor obtained in this study having a K i of 3 nM.In the last decade, naturally occurring serine protease inhibitors (1) have been the focus of many studies, mainly for two reasons: first, the target proteins control functions in a variety of fundamental proteolytic processes in humans and mammals (blood clotting, digestion, inflammation, fibrinolysis), in invertebrates such as insects (immune system, digestion, protection against their predators) or worms (protection against their host), and plants (protection against insect attack); second, low molecular weight inhibitors of serine proteases have been attractive tools for studying the general aspects of protein conformation and protein-protein interactions (2). In the present study, we report the inhibitory properties of three homologous peptides (primary and tertiary structures) toward ␣-chymotrypsin, trypsin, and human leukocyte elastase.We have previously isolated two peptides, PMP-C and PMP-D2, from the brain and the fat body of the insect Locusta migratoria (3). These peptides are composed of 36 and 35 residues, respectively, and are cross-linked by three disulfide bonds. The Thr-9 of PMP-C has an uncommon O-glycosidic linkage to a single fucose moiety. There is 40% strict identity between PMP-C and PMP-D2 with conservation of the Cys positions (3). Moreover, they are located on the same peptidic precursor, and, by Northern blot analysis, it has been shown that the gene encoding this precursor is mainly transcribed in the fat body (4). In this paper, we describe the isolation and characterization of HI, a novel locust peptide.Because the isolation from insect extracts is time-consuming and yields only small amounts of peptides, we have prepared, at a reasonable scale (5-10 mg), by solid-phase synthesis, PMP-D2 (5) and PMP-C with and without the fucose moiety. 1Although they are small peptides with a high disulfide content, no sequence similarities could be found when comparing them with small toxins or small protease inhibitors. However, the milligram quantities of PMP-D2 obtained by solid-phase synthesi...

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“…One of the novel families that has emerged since then is the grasshopper family (2). The first members were characterized from the brain (pars intercerebralis) and the hemolymph of the insect Locusta migratoria (3)(4)(5). Furthermore, similar peptides (named SGPI) were isolated from Schistocerca gregaria (6); they share 40 -80% homology (including the six conserved cysteines) with the Locusta peptides.…”

mentioning

confidence: 99%

Complexation of Two Proteic Insect Inhibitors to the Active Site of Chymotrypsin Suggests Decoupled Roles for Binding and Selectivity

Roussel¹,

Mathieu²,

Dobbs³

et al. 2001

Journal of Biological Chemistry

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The crystal structures of two homologous inhibitors (PMP-C and PMP-D2v) from the insect Locusta migratoria have been determined in complex with bovine ␣-chymotrypsin at 2.1-and 3.0-Å resolution, respectively. PMP-C is a potent bovine ␣-chymotrypsin inhibitor whereas native PMP-D2 is a weak inhibitor of bovine trypsin. One unique mutation at the P1 position converts PMP-D2 into a potent bovine ␣-chymotrypsin inhibitor. The two peptides have a similar overall conformation, which consists of a triple-stranded antiparallel ␤-sheet connected by three disulfide bridges, thus defining a novel family of serine protease inhibitors. They have in common the protease interaction site, which is composed of the classical protease binding loop (position P5 to P4, corresponding to residues 26 -34) and of an internal segment (residues 15-18), held together by two disulfide bridges. Structural divergences between the two inhibitors result in an additional interaction site between PMP-D2v (position P10 to P6, residues 21-25) and the residues 172-175 of ␣-chymotrypsin. This unusual interaction may be responsible for species selectivity. A careful comparison of data on bound and free inhibitors (from this study and previous NMR studies, respectively) suggests that complexation to the protease stabilizes the flexible binding loop (from P5 to P4).Small canonical serine protease inhibitors are widely distributed among living organisms. They have been classified by Bode and Huber (1) into 16 structural families. One of the novel families that has emerged since then is the grasshopper family (2). The first members were characterized from the brain (pars intercerebralis) and the hemolymph of the insect Locusta migratoria (3-5). Furthermore, similar peptides (named SGPI) were isolated from Schistocerca gregaria (6); they share 40 -80% homology (including the six conserved cysteines) with the Locusta peptides. More recently, the same sequence motif was also identified in Pacifastin, a 155-kDa protein from the crayfish Pacifastacus leniusculus and composed of two domains with different activities. One of these domains contains nine repeats similar to the locust peptides and has a protease inhibitory activity (7).We have carried out extensive investigations on two locust peptides, PMP-C and PMP-D2 (pars intercerebralis major peptide). They consist of 36 and 35 residues, respectively, have three disulfide bridges, and are 40% identical in sequence. The three-dimensional structures of PMP-D2 and PMP-C were determined by 1 H NMR (8, 9). These peptides display a new fold with an unusual disulfide bond pattern. PMP-C is a very potent bovine ␣-chymotrypsin inhibitor (K i 0.13 nM) and a weak human leukocyte elastase inhibitor (K i 180 nM) and is devoid of activity toward porcine trypsin. The nature of its P1 residue (nomenclature according to Schechter and Berger (10)), Leu 30 , is in accordance with the literature and its inhibitory properties. Indeed, chymotrypsin inhibitors have bulky and aromatic residues such as Phe, Leu, or Met as their P1 res...

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Insect immunity: Two proteinase inhibitors from hemolymph of Locusta migratoria

Cited by 77 publications

References 12 publications

Pacifastin, a novel 155-kDa heterodimeric proteinase inhibitor containing a unique transferrin chain

Pacifastin, a novel 155-kDa heterodimeric proteinase inhibitor containing a unique transferrin chain

Serine Protease Inhibition by Insect Peptides Containing a Cysteine Knot and a Triple-stranded β-Sheet

Complexation of Two Proteic Insect Inhibitors to the Active Site of Chymotrypsin Suggests Decoupled Roles for Binding and Selectivity

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