Amino acids critical for binding of autoantibody to an immunodominant conformational epitope of the pyruvate dehydrogenase complex subunit E2: Identification by phage display and site-directed mutagenesis

Abstract The genus Listeria comprises ubiquitous bacteria, commonly present in foods and food production facilities. In this study, three different phage display technologies were employed to discover targets, and to generate and characterize novel antibodies against Listeria: antibody display for biomarker discovery and antibody generation; ORFeome display for target identification; and single-gene display for epitope characterization. With this approach, pyruvate dehydrogenase complex—enzyme 2 (PDC-E2) was defined as a new detection target for Listeria, as confirmed by immunomagnetic separation-mass spectrometry (IMS-MS). Immunoblot and fluorescence microscopy showed that this protein is accessible on the bacterial cell surface of living cells. Recombinant PDC-E2 was produced in E. coli and used to generate 16 additional antibodies. The resulting set of 20 monoclonal scFv-Fc was tested in indirect ELISA against 17 Listeria and 16 non-Listeria species. Two of them provided 100% sensitivity (CI 82.35–100.0%) and specificity (CI 78.20–100.0%), confirming PDC-E2 as a suitable target for the detection of Listeria. The binding region of 18 of these antibodies was analyzed, revealing that ≈ 90% (16/18) bind to the lipoyl domains (LD) of the target. The novel target PDC-E2 and highly specific antibodies against it offer new opportunities to improve the detection of Listeria.

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Pyruvate dehydrogenase kinase from Arabidopsis thaliana: a protein histidine kinase that phosphorylates serine residues

Biochemical Journal ◽

10.1042/bj3490195 ◽

2000 ◽

Vol 349 (1) ◽

pp. 195-201 ◽

Cited By ~ 21

Author(s):

Jay J. THELEN ◽

Jan A. MIERNYK ◽

Douglas D. RANDALL

Keyword(s):

Arabidopsis Thaliana ◽

Pyruvate Dehydrogenase ◽

Histidine Kinase ◽

Pyruvate Dehydrogenase Complex ◽

Pyruvate Dehydrogenase Kinase ◽

Site Directed Mutagenesis ◽

Directed Mutagenesis ◽

Primary Amino Acid Sequence ◽

N Terminus ◽

Primary Amino

Pyruvate dehydrogenase kinase (PDK) is the primary regulator of flux through the mitochondrial pyruvate dehydrogenase complex (PDC). Although PDKs inactivate mitochondrial PDC by phosphorylating specific Ser residues, the primary amino acid sequence indicates that they are more closely related to prokaryotic His kinases than to eukaryotic Ser/Thr kinases. Unlike Ser/Thr kinases, His kinases use a conserved His residue for phosphotransfer to Asp residues. To understand these unique kinases better, a presumptive PDK from Arabidopsis thaliana was heterologously expressed and purified for this investigation. Purified, recombinant A. thaliana PDK could inactivate kinase-depleted maize mitochondrial PDC by phosphorylating Ser residues. Additionally, A. thaliana PDK was capable of autophosphorylating Ser residues near its N-terminus, although this reaction is not part of the phosphotransfer pathway. To elucidate the mechanism involved, we performed site-directed mutagenesis of the canonical His residue likely to be involved in phosphotransfer. When His-121 was mutated to Ala or Gln, Ser-autophosphorylation was decreased by 50% and transphosphorylation of PDC was decreased concomitantly. We postulate that either (1) His-121 is not the sole phosphotransfer His residue or (2) mutagenesis of His-121 exposes an additional otherwise cryptic phosphotransfer His residue. Thus His-121 is one residue involved in kinase function.

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Harnessing the functional diversity of plant cystatins to design inhibitor variants highly active against herbivorous arthropod digestive proteases

10.1101/2021.07.22.453419 ◽

2021 ◽

Author(s):

Jonathan Tremblay ◽

Marie-Claire Goulet ◽

Juan Vorster ◽

Charles Goulet ◽

Dominique Michaud

Keyword(s):

Amino Acids ◽

Phage Display ◽

Site Directed Mutagenesis ◽

Directed Mutagenesis ◽

Structural Elements ◽

New Approach ◽

Digestive Proteases ◽

Highly Active ◽

Plant Cystatin ◽

Selection Of

Protein engineering approaches have been proposed to improve the inhibitory properties of plant cystatins against herbivorous arthropod digestive proteases. These approaches typically involve the site-directed mutagenesis of functionally relevant amino acids, the production and selection of improved inhibitory variants by molecular phage display procedures, or the design of bi/multifunctional translational fusions integrating one or several cystatin inhibitory domains. Here, we propose a new approach where the function-related structural elements of a cystatin are substituted by the corresponding elements of an alternative cystatin. Cys protease inhibitory assays were first performed with 20 representative plant cystatins and model Cys proteases, including herbivorous arthropod digestive proteases, to appreciate the extent of functional variability among plant cystatin protein family members. The most, and less, potent of these cystatins were then used as donors of structural elements to create hybrids of tomato cystatin SlCYS8 used as a model recipient inhibitor. Our data confirm the wide variety of cystatin protease inhibitory profiles among plant taxa. They also demonstrate the usefulness of these proteins as a pool of discrete structural elements for the design of cystatin variants with improved potency against herbivorous pest digestive Cys proteases.

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