Proteinase inhibitors from desert locust, Schistocerca gregaria: engineering of both P1 and P1′ residues converts a potent chymotrypsin inhibitor to a potent trypsin inhibitor

Malik, Zulfiquar; Amir, Sumaira; Pál, Gábor; Buzás, Zsuzsa; Várallyay, Éva; Antal, József; Szilágyi, Zoltán; Vékey, Károly; Asbóth, Bence; Patthy, András; Gráf, László

doi:10.1016/s0167-4838(99)00167-3

Cited by 46 publications

(78 citation statements)

References 21 publications

Supporting

Mentioning

Contrasting

Unclassified

Order By: Relevance

“…After induction by IPTG, these produced the protein in sufficient quantities in a soluble form. After isolation and purification (Szenthe et al 2004), the double-headed inhibitor displayed K i values both versus bovine chymotrypsin and trypsin identical to those obtained earlier for the individual peptides (Malik et al 1999). This result indicated that the peptide was correctly folded in E. coli.…”

Section: Cloning Of the Lipsupporting

confidence: 78%

“…We have isolated and characterised two small serine protease inhibitor peptides (Schistocerca gregaria chymotrypsin inhibitor, SGCI, and S. gregaria trypsin inhibitor, SGTI) from the haemolymph of locust S. gregaria (Malik et al 1999). SGCI proved to be a very good inhibitor of bovine chymotrypsin whereas SGTI was a reasonable inhibitor of bovine trypsin.…”

Section: Introductionmentioning

confidence: 99%

See 1 more Smart Citation

Inhibition of Colorado potato beetle larvae by a locust proteinase inhibitor peptide expressed in potato

et al. 2005

Self Cite

View full text Add to dashboard Cite

The cDNA for a 73-mer peptide containing two locust serine proteinase inhibitors was cloned, fused to the constitutive CaMV35S promoter and introduced into potato by Agrobacterium-mediated transformation. From 23 independent transgenic lines, three with high mRNA level and proteinase inhibitory activity were propagated in vitro and transferred to pots. The peptide from the leaves was identified by its N-terminal sequence and by K(i) values against chymotrypsin and trypsin. Colorado potato beetle larvae reared on transgenic plants grew slightly but significantly more slowly than those on control plants. This supports the notion that expression of multifunctional proteinase inhibitors of insect origin might be a good strategy to improve insect resistance in plants.

show abstract

Section: Cloning Of the Lipsupporting

confidence: 78%

Section: Introductionmentioning

confidence: 99%

Inhibition of Colorado potato beetle larvae by a locust proteinase inhibitor peptide expressed in potato

et al. 2005

Self Cite

View full text Add to dashboard Cite

show abstract

“…As a result, subdomain II of PMP-D2v is shifted closer to chymotrypsin and may be responsible of additional contacts (either favorable interaction or steric hindrance) and could therefore be considered as an element for discriminating between protease targets. PMP-C may be regarded as more permissive toward various enzymes, as also observed with S. gregaria inhibitors (20).…”

Section: Resultsmentioning

confidence: 60%

“…1C). Although unrelated to grasshopper inhibitors, Ecotin (18, 19) displays a segment (residues 47-56) similar to that of PMP-C (residues [11][12][13][14][15][16][17][18][19][20], which is also connected to the binding site by a disulfide bridge (Cys 50 -Cys 87 ). However, the second disulfide bridge present in PMP-C (Cys 17 -Cys 28 ) is missing, and the cysteines are replaced by His 53 and Ser 82 , respectively.…”

Section: Resultsmentioning

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

View full text Add to dashboard Cite

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...

show abstract

“…The P1Ј preference of human CTRC resembles that of trypsin rather than chymotrypsin. Thus, bovine cationic and rat anionic trypsin prefer Met side chains at P1Ј, whereas bovine and rat chymotrypsin prefer positively charged Arg or Lys side chains at this position (30,34,(51)(52)(53)(54). The characteristic Arg/Lys P1Ј preference of chymotrypsin is attributed to electrostatic interactions with two negatively charged residues, Asp 35 and Asp 64 (chymotrypsin numbering), which are not conserved in human CTRC (34).…”

Section: Discussionmentioning

confidence: 95%

High Affinity Small Protein Inhibitors of Human Chymotrypsin C (CTRC) Selected by Phage Display Reveal Unusual Preference for P4′ Acidic Residues

Szabó

Héja

Szakács

et al. 2011

Journal of Biological Chemistry

Self Cite

View full text Add to dashboard Cite

Human chymotrypsin C (CTRC) is a pancreatic protease that participates in the regulation of intestinal digestive enzyme activity. Other chymotrypsins and elastases are inactive on the regulatory sites cleaved by CTRC, suggesting that CTRC recognizes unique sequence patterns. To characterize the molecular determinants underlying CTRC specificity, we selected high affinity substrate-like small protein inhibitors against CTRC from a phage library displaying variants of SGPI-2, a natural chymotrypsin inhibitor from Schistocerca gregaria. On the basis of the sequence pattern selected, we designed eight inhibitor variants in which amino acid residues in the reactive loop at P1 (Met or Leu), P2 (Leu or Asp), and P4 (Glu, Asp, or Ala) were varied. Binding experiments with CTRC revealed that (i) inhibitors with Leu at P1 bind 10-fold stronger than those with P1 Met; (ii) Asp at P2 (versus Leu) decreases affinity but increases selectivity, and (iii) Glu or Asp at P4 (versus Ala) increase affinity 10-fold. The highest affinity SGPI-2 variant (K D 20 pM) bound to CTRC 575-fold tighter than the parent molecule. The most selective inhibitor variant exhibited a K D of 110 pM and a selectivity ranging from 225-to 112,664-fold against other human chymotrypsins and elastases. Homology modeling and mutagenesis identified a cluster of basic amino acid residues (Lys 51 , Arg 56 , and Arg 80 ) on the surface of human CTRC that interact with the P4 acidic residue of the inhibitor. The acidic preference of CTRC at P4 is unique among pancreatic proteases and might contribute to the high specificity of CTRC-mediated digestive enzyme regulation.Digestion of dietary proteins in the small intestine is catalyzed by proteases secreted from the pancreas. These proteases are produced as inactive proenzymes (zymogens) and their activation is spatially restricted to the duodenum and proceeds in a cascade-like manner. The membrane-localized serine protease enteropeptidase (enterokinase) activates trypsinogens to active trypsins, which, in turn, activate all other proteolytic proenzymes; in the human these include chymotrypsinogens B1, B2, C, and L1, proelastases 2A, 3A, and 3B, and procarboxypeptidases A1, A2, and B1. This classic textbook paradigm of digestive enzyme activation has been recently revised by the discovery of interactions between digestive proteases that can modulate the ultimate levels of active enzymes. In this regard, we demonstrated that human chymotrypsin C (CTRC) 4 regulates activation and degradation of human cationic trypsinogen and trypsin (1, 2). CTRC facilitates trypsin-mediated activation (autoactivation) of cationic trypsinogen by processing the trypsinogen activation peptide, at the Phe 18 -Asp 19 peptide bond, to a shorter form that is more readily cleaved by trypsin (1). Mutation A16V in the trypsinogen activation peptide stimulates CTRC-mediated processing and subjects with this mutation are at increased risk of developing chronic pancreatitis (1, 3). CTRC can also promote degradation of human cationic trypsinogen a...

show abstract

Proteinase inhibitors from desert locust, Schistocerca gregaria: engineering of both P1 and P1′ residues converts a potent chymotrypsin inhibitor to a potent trypsin inhibitor

Cited by 46 publications

References 21 publications

Inhibition of Colorado potato beetle larvae by a locust proteinase inhibitor peptide expressed in potato

Inhibition of Colorado potato beetle larvae by a locust proteinase inhibitor peptide expressed in potato

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

High Affinity Small Protein Inhibitors of Human Chymotrypsin C (CTRC) Selected by Phage Display Reveal Unusual Preference for P4′ Acidic Residues

Contact Info

Product

Resources

About