When the Surface Tells What Lies Beneath: Combinatorial Phage-display Mutagenesis Reveals Complex Networks of Surface–Core Interactions in the Pacifastin Protease Inhibitor Family

Szenthe, Borbála; Patthy, András; Gáspári, Zoltán; Kékesi, Katalin A.; Gráf, László; Pál, Gábor

doi:10.1016/j.jmb.2007.04.029

Cited by 21 publications

(18 citation statements)

References 48 publications

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“…SGPI Library Construction-Library construction was based on the Tag-wtSGPI-2-pGP8 phagemid vector (14), which monovalently displays SGPI-2 on the p8 coat protein of the M13 phage. The library was produced in two successive Kunkel mutagenesis (15) steps to avoid wild-type SGPI-2 contamination.…”

Section: Methodsmentioning

confidence: 99%

“…1A) (29 -32). The 35-amino acid SGPI-2, which belongs to the pacifastin inhibitor family, had already been efficiently displayed on the surface of M13 phage (14). Wild-type SGPI-2 inhibited human chymotrypsins and elastases with K D values ranging from 0.2 nM to 2 M, and generally stronger inhibition was apparent for chymotrypsins (Table 2).…”

Section: Analysis Of Ctrc Specificity Through Phage Display Selectionmentioning

confidence: 99%

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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

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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...

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Section: Methodsmentioning

confidence: 99%

Section: Analysis Of Ctrc Specificity Through Phage Display Selectionmentioning

confidence: 99%

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

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“…In addition to pIII signal peptide and pVIII signal peptide, the majority of the commonly used signal peptides are ones from outer membrane proteins, such as pelB leader, 34,35,37 stII signal sequence, 36 CAT leader, 39 Omp A signal peptide, 42 lipoprotein signal sequence, 45,47 signal peptide of Omp T, 54 bacterial leader sequence, 64 signal peptide of maltose binding protein, 65,81 leader sequence of TorA 82 and signal peptide of PhoA. 83 Since different secretion systems in host cells can direct processing of proteins or peptides, the signal peptides are very crucial elements for enhancing the level of display. As illustrated in Fig.…”

Section: Signal Peptidesmentioning

confidence: 99%

Phagemid Vectors for Phage Display: Properties, Characteristics and Construction

Qiu

et al. 2012

Journal of Molecular Biology

132

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“…In a previous study, we demonstrated that the display of a chymotrypsin inhibitor peptide, SGCI, on the p8 coat protein is monovalent (31). Therefore, we fused the SFTI library N-terminal to the SGCI-p8 construct, in which-in terms of MASP inhibition-SGCI is an indifferent module, ensuring monovalency (Fig.…”

Section: Design and Construction Of The Inhibitory Peptide-phage Librarymentioning

confidence: 99%

Selective Inhibition of the Lectin Pathway of Complement with Phage Display Selected Peptides against Mannose-Binding Lectin-Associated Serine Protease (MASP)-1 and -2: Significant Contribution of MASP-1 to Lectin Pathway Activation

Kocsis

Kékesi

Szász

et al. 2010

The Journal of Immunology

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The complement system, an essential part of the innate immune system, can be activated through three distinct routes: the classical, the alternative, and the lectin pathways. The contribution of individual activation pathways to different biological processes can be assessed by using pathway-selective inhibitors. In this paper, we report lectin pathway-specific short peptide inhibitors developed by phage display against mannose-binding lectin-associated serine proteases (MASPs), MASP-1 and MASP-2. On the basis of the selected peptide sequences, two 14-mer peptides, designated as sunflower MASP inhibitor (SFMI)-1 and SFMI-2, were produced and characterized. SFMI-1 inhibits both MASP-1 and MASP-2 with a KI of 65 and 1030 nM, respectively, whereas SFMI-2 inhibits only MASP-2 with a KI of 180 nM. Both peptides block the lectin pathway activation completely while leaving the classical and the alternative routes intact and fully functional, demonstrating that of all complement proteases only MASP-1 and/or MASP-2 are inhibited by these peptides. In a C4 deposition inhibitor assay using preactivated MASP-2, SFMI-2 is 10-fold more effective than SFMI-1 in accordance with the fact that SFMI-2 is a more potent inhibitor of MASP-2. Surprisingly, however, out of the two peptides, SFMI-1 is much more effective in preventing C3 and C4 deposition when normal human serum containing zymogen MASPs is used. This suggests that MASP-1 has a crucial role in the initiation steps of lectin pathway activation most probably by activating MASP-2. Because the lectin pathway has been implicated in several life-threatening pathological states, these inhibitors should be considered as lead compounds toward developing lectin pathway blocking therapeutics.

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When the Surface Tells What Lies Beneath: Combinatorial Phage-display Mutagenesis Reveals Complex Networks of Surface–Core Interactions in the Pacifastin Protease Inhibitor Family

Cited by 21 publications

References 48 publications

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

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

Phagemid Vectors for Phage Display: Properties, Characteristics and Construction

Selective Inhibition of the Lectin Pathway of Complement with Phage Display Selected Peptides against Mannose-Binding Lectin-Associated Serine Protease (MASP)-1 and -2: Significant Contribution of MASP-1 to Lectin Pathway Activation

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