Correction: Phage Display of the Serpin Alpha-1 Proteinase Inhibitor Randomized at Consecutive Residues in the Reactive Centre Loop and Biopanned with or without Thrombin.
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ABSTRACT:
SUBMITTER: Scott BM
PROVIDER: S-EPMC7482966 | biostudies-literature | 2020
REPOSITORIES: biostudies-literature
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Correction: Phage Display of the Serpin Alpha-1 Proteinase Inhibitor Randomized at Consecutive Residues in the Reactive Centre Loop and Biopanned with or without Thrombin.
Scott Benjamin M BM Matochko Wadim L WL Gierczak Richard F RF Bhakta Varsha V Derda Ratmir R Sheffield William P WP
PloS one 20200910 9
[This corrects the article DOI: 10.1371/journal.pone.0084491.]. ...[more]
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Project description:In spite of the power of phage display technology to identify variant proteins with novel properties in large libraries, it has only been previously applied to one member of the serpin superfamily. Here we describe phage display of human alpha-1 proteinase inhibitor (API) in a T7 bacteriophage system. API M358R fused to the C-terminus of T7 capsid protein 10B was directly shown to form denaturation-resistant complexes with thrombin by electrophoresis and immunoblotting following exposure of intact phages to thrombin. We therefore developed a biopanning protocol in which thrombin-reactive phages were selected using biotinylated anti-thrombin antibodies and streptavidin-coated magnetic beads. A library consisting of displayed API randomized at residues 357 and 358 (P2-P1) yielded predominantly Pro-Arg at these positions after five rounds of thrombin selection; in contrast the same degree of mock selection yielded only non-functional variants. A more diverse library of API M358R randomized at residues 352-356 (P7-P3) was also probed, yielding numerous variants fitting a loose consensus of DLTVS as judged by sequencing of the inserts of plaque-purified phages. The thrombin-selected sequences were transferred en masse into bacterial expression plasmids, and lysates from individual colonies were screening for API-thrombin complexing. The most active candidates from this sixth round of screening contained DITMA and AAFVS at P7-P3 and inhibited thrombin 2.1-fold more rapidly than API M358R with no change in reaction stoichiometry. Deep sequencing using the Ion Torrent platform confirmed that over 800 sequences were significantly enriched in the thrombin-panned versus naïve phage display library, including some detected using the combined phage display/bacterial lysate screening approach. Our results show that API joins Plasminogen Activator Inhibitor-1 (PAI-1) as a serpin amenable to phage display and suggest the utility of this approach for the selection of "designer serpins" with novel reactivity and/or specificity.
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Project description:Serine proteinase inhibitors (serpins), typically fold to a metastable native state and undergo a major conformational change in order to inhibit target proteases. However, conformational lability of the native serpin fold renders them susceptible to misfolding and aggregation, and underlies misfolding diseases such as ?1-antitrypsin deficiency. Serpin specificity towards its protease target is dictated by its flexible and solvent exposed reactive centre loop (RCL), which forms the initial interaction with the target protease during inhibition. Previous studies have attempted to alter the specificity by mutating the RCL to that of a target serpin, but the rules governing specificity are not understood well enough yet to enable specificity to be engineered at will. In this paper, we use conserpin, a synthetic, thermostable serpin, as a model protein with which to investigate the determinants of serpin specificity by engineering its RCL. Replacing the RCL sequence with that from ?1-antitrypsin fails to restore specificity against trypsin or human neutrophil elastase. Structural determination of the RCL-engineered conserpin and molecular dynamics simulations indicate that, although the RCL sequence may partially dictate specificity, local electrostatics and RCL dynamics may dictate the rate of insertion during protease inhibition, and thus whether it behaves as an inhibitor or a substrate. Engineering serpin specificity is therefore substantially more complex than solely manipulating the RCL sequence, and will require a more thorough understanding of how conformational dynamics achieves the delicate balance between stability, folding and function required by the exquisite serpin mechanism of action.
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Project description:Extracellular serine proteinase cascades stimulate prophenoloxidase (proPO) activation and antimicrobial peptide production in insect innate immune responses. Serpins in plasma regulate such cascades by selective inhibition of proteinases, in reactions which result in the formation of covalent serpin-proteinase complexes. We carried out experiments to identify plasma proteinases that are inhibited by Manduca sexta serpin-3, an immune-inducible serpin known to regulate proPO activation. Immunoaffinity chromatography, using antiserum to serpin-3, yielded serpin-3 complexes with proteinases identified by immunoblot analysis as prophenoloxidase-activating proteinase (PAP)-1, PAP-2, PAP-3, and hemolymph proteinase 8 (HP8). HP8 can cleave and activate the Toll ligand, Spätzle, leading to synthesis of antimicrobial peptides. Analysis by mass spectrometry of tryptic peptides derived from the serpin-3 complexes confirmed the presence of PAP-1, PAP-3, and HP8. Purified recombinant serpin-3 and active HP8 formed an SDS-stable complex in vitro. Identification of serpin-3-proteinase complexes in plasma provides insight into proteinase targets of serpin-3 and extends the understanding of serpin/proteinase function in the immune response of M. sexta.
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Project description:BackgroundA33 is a type II integral membrane protein expressed on the extracellular enveloped form of vaccinia virus (VACV). Passive transfer of A33-directed monoclonal antibodies or vaccination with an A33 subunit vaccine confers protection against lethal poxvirus challenge in animal models. Homologs of A33 are highly conserved among members of the Orthopoxvirus genus and are potential candidates for inclusion in vaccines or assays targeting extracellular enveloped virus activity. One monoclonal antibody directed against VACV A33, MAb-1G10, has been shown to target a conformation-dependent epitope. Interestingly, while it recognizes VACV A33 as well as the corresponding variola homolog, it does not bind to the monkeypox homolog. In this study, we utilized a random phage display library to investigate the epitope recognized by MAb-1G10 that is critical for facilitating cell-to-cell spread of the vaccinia virus.ResultsBy screening with linear or conformational random phage libraries, we found that phages binding to MAb-1G10 display the consensus motif CEPLC, with a disulfide bond formed between two cysteine residues required for MAb-1G10 binding. Although the phage motif contained no linear sequences homologous to VACV A33, structure modeling and analysis suggested that residue D115 is important to form the minimal epitope core. A panel of point mutants expressing the ectodomain of A33 protein was generated and analyzed by either binding assays such as ELISA and immunoprecipitation or a functional assessment by blocking MAb-1G10 mediated comet inhibition in cell culture.ConclusionsThese results confirm L118 as a component of the MAb-1G10 binding epitope, and further identify D115 as an essential residue. By defining the minimum conformational structure, as well as the conformational arrangement of a short peptide sequence recognized by MAb-1G10, these results introduce the possibility of designing small molecule mimetics that may interfere with the function of A33 in vivo. This information will also be useful for designing improved assays to evaluate the potency of monoclonal and polyclonal products that target A33 or A33-modulated EV dissemination.
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Project description:Mutually exclusive alternative splicing produces transcripts for 12 serpin-1 isoforms in Manduca sexta that differ only in the region encoding the carboxyl-terminal 36-40-amino acid residues. This variable region includes the reactive center loop, which determines the inhibitory selectivity of the serpin. We investigated mRNA levels of individual serpin-1 isoforms by quantitative PCR. The 12 isoforms were expressed at similar levels in hemocytes, but in fat body isoform B mRNA was present at significantly higher levels than isoforms C, D, E, F, G, J, K, and Z. To investigate the presence of individual serpin-1 isoforms in plasma we used immunoaffinity purification of serpin-1 isoforms from M. sexta plasma, followed by two-dimensional PAGE and identification of protein spots by digestion with a series of proteinases and analysis of the resulting peptides by MALDI-TOF/TOF. We identified nine of the 12 serpin-1 isoforms and, through analysis of putative serpin-1-proteinase complexes, identified three endogenous M. sexta proteinase targets of serpin-1. Our results suggest that M. sexta serpin-1 isoforms A, E, and J can inhibit hemolymph proteinase 8, which activates the cytokine spätzle. At least one isoform of serpin-1 can inhibit hemocyte proteinase 1, another M. sexta blood proteinase. In addition, a complex of serpin-1K in a complex with M. sexta midgut chymotrypsin was identified, suggesting serpin-1 isoforms may also function to protect insect tissues from digestive proteinases that may leak into the hemocoel.
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Project description:A mutant ovalbumin (R339T), but not the wild-type protein, is transformed into the canonical loop-inserted, thermostabilized form after the P1-P1' cleavage [Yamasaki, Arii, Mikami and Hirose (2002) J. Mol. Biol. 315, 113-120]. The loop-insertion mechanism in the ovalbumin mutant was investigated by proteolytic-cleavage kinetics. The nature of the inserted loop prevented further cleavage of the P1-P1' pre-cleaved R339T mutant by subtilisin, which cleaved the second P8-P7 loop site in the P1-P1' pre-cleaved wild-type protein. After subtilisin proteolysis of the intact R339T, however, two final products that corresponded to the single P1-P1' and double P1-P1'/P8-P7 cleavages were generated with variable ratios depending on the proteolysis conditions. This was accounted for by the occurrence of two mutually competitive reactions: the loop-insertion reaction and the proteolytic cleavage of the second P8-P7 site in the immediate intermediate after the P1-P1' cleavage. The competitive nature of the two reactions enabled us to establish a kinetic method to determine the rate constants of the reactions. The first-order rate constant for the loop insertion was determined to be 4.0 x 10(-3)/s in the R339T mutant. The second-order rate constant for the P8-P7 cleavage in the immediate P1-P1' cleavage product for the R339T mutant was >10 times compared with that for its wild-type counterpart. This highly accessible loop nature may play a crucial role in the loop-insertion mechanism for R339T mutant ovalbumin.
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Project description:The serpin mechanism of protease inhibition involves the rapid and stable incorporation of the reactive center loop (RCL) into central ?-sheet A. Serpins therefore require a folding mechanism that bypasses the most stable "loop-inserted" conformation to trap the RCL in an exposed and metastable state. This unusual feature of serpins renders them highly susceptible to point mutations that lead to the accumulation of hyperstable misfolded polymers in the endoplasmic reticulum of secretory cells. The ordered and stable protomer-protomer association in serpin polymers has led to the acceptance of the "loop-sheet" hypothesis of polymerization, where a portion of the RCL of one protomer incorporates in register into sheet A of another. Although this mechanism was proposed 20 years ago, no study has ever been conducted to test its validity. Here, we describe the properties of a variant of ?(1)-antitrypsin with a critical hydrophobic section of the RCL substituted with aspartic acid (P8-P6). In contrast to the control, the variant was unable to polymerize when incubated with small peptides or when cleaved in the middle of the RCL (accepted models of loop-sheet polymerization). However, when induced by guanidine HCl or heat, the variant polymerized in a manner indistinguishable from the control. Importantly, the Asp mutations did not affect the ability of the Z or Siiyama ?(1)-antitrypsin variants to polymerize in COS-7 cells. These results argue strongly against the loop-sheet hypothesis and suggest that, in serpin polymers, the P8-P6 region is only a small part of an extensive domain swap.
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Project description:The thrombin generation (TG) test is useful for characterizing global hemostasis potential, but fluorescence substrate artifacts, such as thrombin-α2macroglobulin (T-α2MG) signal, inner filter effect (IFE), substrate consumption, and calibration algorithms have been suggested as sources of intra- and inter-laboratory variance, which may limit its clinical utility. Effects of internal vs. external normalization, IFE and T-α2MG on TG curves in normal plasma supplemented with coagulation factors, thrombomodulin, and tissue factor were studied using the Calibrated Automated Thrombinography (CAT; Diagnostica Stago, Parsippany, NJ, USA) and in-house software. The various calibration methods demonstrated no significant difference in producing TG curves, nor increased the robustness of the TG assay. Several TG parameters, including thrombin peak height (TPH), produced from internal linear calibration did not differ significantly from uncalibrated TG parameters. Further, TPH values from internal linear and nonlinear calibration with or without T-α2MG correction correlated well with TPH from external calibration. Higher coefficients of variation (CVs) for TPH values were observed in both platelet-free and platelet-rich plasma with added thrombomodulin. Our work suggests minimal differences between distinct computational approaches toward calibrating and correcting fluorescence signals into TG levels, with most samples returning similar or equivalent TPH results.
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