Cellulose-bound Peptide Research Articles

Immunoinformatics and Pepscan strategies on the path of a peptide-based serological diagnosis of COVID19

Several diagnostic tools have been developed for clinical and epidemiological assays. RT-PCR and antigen detection tests are more useful for diagnosis of acute disease, while antibody tests allow the estimation of exposure in the population. Currently, there is an urgent need for the development of diagnostic tests for COVID-19 that can be used for large-scale epidemiological sampling. Through a comprehensive strategy, potential 16 mer antigenic peptides suited for antibody-based SARS-CoV-2 diagnosis were identified. A systematic scan of the three structural proteins (S,N and M) and the non-structural proteins (ORFs) present in the SARS-CoV-2 virus was conducted through the combination of immunoinformatic methods, peptide SPOT synthesis and an immunoassay with cellulose-bound peptides (Pepscan). The Pepscan filter paper sheets with synthetic peptides were tested against pools of sera of COVID-19 patients. Antibody recognition showed a strong signal for peptides corresponding to the S, N and M proteins of SARS-CoV-2 virus, but not for the ORFs proteins. The peptides exhibiting higher signal intensity were found in the C-terminal region of the N protein. Several peptides of this region showed strong recognition with all three immunoglobulins in the pools of sera. The differential reactivity observed between the different immunoglobulin isotypes (IgA, IgM and IgG) within different regions of the S and N proteins, can be advantageous for ensuring accurate diagnosis of all infected patients, with different times of exposure to infection. Few peptides of the M protein showed antibody recognition and no recognition was observed for peptides of the ORFs proteins.

Peptide array on cellulose support--a screening tool to identify peptides with dipeptidyl-peptidase IV inhibitory activity within the sequence of α-lactalbumin.

The inhibition of the enzyme dipeptidyl-peptidase IV (DPP-IV) is an effective pharmacotherapeutic approach for the management of type 2 diabetes. Recent findings have suggested that dietary proteins, including bovine α-lactalbumin, could be precursors of peptides able to inhibit DPP-IV. However, information on the location of active peptide sequences within the proteins is far from being comprehensive. Moreover, the traditional approach to identify bioactive peptides from foods can be tedious and long. Therefore, the objective of this study was to use peptide arrays to screen α-lactalbumin-derived peptides for their interaction with DPP-IV. Deca-peptides spanning the entire α-lactalbumin sequence, with a frame shift of 1 amino acid between successive sequences, were synthesized on cellulose membranes using “SPOT” technology, and their binding to and inhibition of DPP-IV was studied. Among the 114 α-lactalbumin-derived decamers investigated, the peptides 60WCKDDQNPHS69 (αKi = 76 µM), 105LAHKALCSEK114 (Ki = 217 µM) and 110LCSEKLDQWL119 (Ki = 217 µM) were among the strongest DPP-IV inhibitors. While the SPOT- and traditionally-synthesized peptides showed consistent trends in DPP-IV inhibitory activity, the cellulose-bound peptides’ binding behavior was not correlated to their ability to inhibit the enzyme. This research showed, for the first time, that peptide arrays are useful screening tools to identify DPP-IV inhibitory peptides from dietary proteins.

Generation and molecular characterization of a monoclonal antibody reactive with conserved epitope in sphingomyelinases D from Loxosceles spider venoms

We report the production of a neutralizing monoclonal antibody able to recognize the venoms of three major medically important species of Loxosceles spiders in Brazil. The mAb was produced by immunization of mice with a toxic recombinant L. intermedia sphingomyelinase D {SMases D isoform (rLiD1)} [1] and screened by enzyme-linked immunosorbent assay (ELISA) using L. intermedia, L. laeta and L. gaucho venoms as antigens. One clone (LiD1mAb16) out of seventeen anti-rLiD1 hybridomas was cross-reactive with the three whole Loxosceles venoms. 2D Western blot analysis indicated that LiD1mAb16 was capable of interacting with 34 proteins of 29–36kDa in L. intermedia, 33 in L. gaucho and 27 in L. laeta venoms. The results of immunoassays with cellulose-bound peptides revealed that the LiD1mAb16 recognizes a highly conserved linear epitope localized in the catalytic region of SMases D toxins. The selected mAb displayed in vivo protective activity in rabbits after challenge with rLiD1. These results show the potential usefulness of monoclonal antibodies for future therapeutic approaches and also opens up the perspective of utilization of these antibodies for immunodiagnostic assays in loxoscelism.

Identification and Characterization of Mitochondrial Targeting Sequence of Human Apurinic/Apyrimidinic Endonuclease 1

Dually targeted mitochondrial proteins usually possess an unconventional mitochondrial targeting sequence (MTS), which makes them difficult to predict by current bioinformatics approaches. Human apurinic/apyrimidinic endonuclease (APE1) plays a central role in the cellular response to oxidative stress. It is a dually targeted protein preferentially residing in the nucleus with conditional distribution in the mitochondria. However, the mitochondrial translocation mechanism of APE1 is not well characterized because it harbors an unconventional MTS that is difficult to predict by bioinformatics analysis. Two experimental approaches were combined in this study to identify the MTS of APE1. First, the interactions between the peptides from APE1 and the three purified translocase receptors of the outer mitochondrial membrane (Tom) were evaluated using a peptide array screen. Consequently, the intracellular distribution of green fluorescent protein-tagged, truncated, or mutated APE1 proteins was traced by tag detection. The results demonstrated that the only MTS of APE1 is harbored within residues 289-318 in the C terminus, which is normally masked by the intact N-terminal structure. As a dually targeted mitochondrial protein, APE1 possesses a special distribution pattern of different subcellular targeting signals, the identification of which sheds light on future prediction of MTSs.

The Brichos domain of prosurfactant protein C can hold and fold a transmembrane segment

Prosurfactant protein C (proSP-C) is a 197-residue integral membrane protein, in which the C-terminal domain (CTC, positions 59-197) is localized in the endoplasmic reticulum (ER) lumen and contains a Brichos domain (positions 94-197). Mature SP-C corresponds largely to the transmembrane (TM) region of proSP-C. CTC binds to SP-C, provided that it is in nonhelical conformation, and can prevent formation of intracellular amyloid-like inclusions of proSP-C that harbor mutations linked to interstitial lung disease (ILD). Herein it is shown that expression of proSP-C (1-58), that is, the N-terminal propeptide and the TM region, in HEK293 cells results in virtually no detectable protein, while coexpression of CTC in trans yields SDS-soluble monomeric proSP-C (1-58). Recombinant human (rh) CTC binds to cellulose-bound peptides derived from the nonpolar TM region, but not the polar cytosolic part, of proSP-C, and requires >/=5-residues for maximal binding. Binding of rhCTC to a nonhelical peptide derived from SP-C results in alpha-helix formation provided that it contains a long TM segment. Finally, rhCTC and rhCTC Brichos domain shows very similar substrate specificities, but rhCTC(L188Q), a mutation linked to ILD is unable to bind all peptides analyzed. These data indicate that the Brichos domain of proSP-C is a chaperone that induces alpha-helix formation of an aggregation-prone TM region.

Secreted Cyclophilin A, a Peptidylprolyl cis-trans Isomerase, Mediates Matrix Assembly of Hensin, a Protein Implicated in Epithelial Differentiation

Hensin is a rabbit ortholog of DMBT1, a multifunctional, multidomain protein implicated in the regulation of epithelial differentiation, innate immunity, and tumorigenesis. Hensin in the extracellular matrix (ECM) induced morphological changes characteristic of terminal differentiation in a clonal cell line (clone C) of rabbit kidney intercalated cells. Although hensin is secreted in monomeric and various oligomeric forms, only the polymerized ECM form is able to induce these phenotypic changes. Here we report that hensin secretion and matrix assembly were inhibited by the peptidylprolyl cis-trans isomerase (PPIase) inhibitors cyclosporin A (CsA) and a derivative of cyclosporin A with modifications in the d-Ser side chain (Cs9) but not by the calcineurin pathway inhibitor FK506. PPIase inhibition led to failure of hensin polymerization in the medium and ECM, plus the loss of apical cytoskeleton, apical microvilli, and the columnar epithelial shape of clone C cells. Cyclophilin A was produced and secreted into the media to a much greater extent than cyclophilins B and C. Our results also identified the direct CsA-sensitive interaction of cyclophilin A with hensin, suggesting that cyclophilin A is the PPIase that mediates the polymerization and matrix assembly of hensin. These results are significant because this is the first time a direct role of peptidylprolyl cis-trans isomerase activity has been implicated in the process of epithelial differentiation.

SAP18 Promotes Krüppel-dependent Transcriptional Repression by Enhancer-specific Histone Deacetylation

Body pattern formation during early embryogenesis of Drosophila melanogaster relies on a zygotic cascade of spatially restricted transcription factor activities. The gap gene Krüppel ranks at the top level of this cascade. It encodes a C2H2 zinc finger protein that interacts directly with cis-acting stripe enhancer elements of pair rule genes, such as even skipped and hairy, at the next level of the gene hierarchy. Krüppel mediates their transcriptional repression by direct association with the corepressor Drosophila C terminus-binding protein (dCtBP). However, for some Krüppel target genes, deletion of the dCtBP-binding sites does not abolish repression, implying a dCtBP-independent mode of repression. We identified Krüppel-binding proteins by mass spectrometry and found that SAP18 can both associate with Krüppel and support Krüppel-dependent repression. Genetic interaction studies combined with pharmacological and biochemical approaches suggest a site-specific mechanism of Krüppel-dependent gene silencing. The results suggest that Krüppel tethers the SAP18 bound histone deacetylase complex 1 at distinct enhancer elements, which causes repression via histone H3 deacetylation.

Phage display screening for peptidic chitinase inhibitors

A phage display library with disulfide-cyclized peptides was screened for peptides binding to chitinases from Serratia marcescens. One of those peptides was found to efficiently inhibit chitinase A and two others were inhibitors of chitinase B. Complete substitutional analysis of all three peptides using cellulose-bound peptide spot synthesis revealed key interaction positions and allowed optimization of the chitinase B inhibitory peptides towards higher affinity, with inhibitory constants in the lower nanomolar range. Inhibition by all peptides proved to be competitive and highly specific for the chitinase used to select them, as shown with a series of chitinases from different organisms.

Cellulose-bound Peptide Arrays: Preparation and Applications

The way of the development of the cellulose-bound peptide arrays leads via the synthesis of oligonucleotides on cellulose pieces (Frank et al., 1983) and multiple peptide synthesis on cellulose paper discs in columns (Frank and Doring, 1988). Probably inspired by the addressability of the multipin method (Geysen et al., 1984), Ronald Frank and co-workers developed SPOT synthesis and first presented the method in 1990 at the 21st European Peptide Symposium (Frank et al., 1991). Today,

Binding Specificity of an α-Helical Protein Sequence to a Full-Length Hsp70 Chaperone and Its Minimal Substrate-Binding Domain

Hsp70 chaperones are involved in the prevention of misfolding, and possibly the folding, of newly synthesized proteins. The members of this chaperone family are capable of interacting with polypeptide chains both co- and posttranslationally, but it is currently not clear how different structural domains of the chaperone affect binding specificity. We explored the interactions between the bacterial Hsp70, DnaK, and the sequence of a model all-alpha-helical globin (apoMb) by cellulose-bound peptide scanning. The binding specificity of the full-length chaperone was compared with that of its minimal substrate-binding domain, DnaK-beta. Six specific chaperone binding sites evenly distributed along the apoMb sequence were identified. Binding site locations are identical for the full-length chaperone and its substrate-binding domain, but relative affinities differ. The binding specificity of DnaK-beta is only slightly decreased relative to that of full-length DnaK. DnaK's binding motif is known to comprise hydrophobic regions flanked by positively charged residues. We found that the simple fractional mean buried area correlates well with Hsp70's binding site locations along the apoMb sequence. In order to further characterize the properties of the minimal binding host, the stability of DnaK-beta upon chemical denaturation by urea and protons was investigated. Urea unfolding titrations yielded an apparent folding DeltaG degrees of 3.1 +/- 0.9 kcal mol-1 and an m value of 1.7 +/- 0.4 kcal mol-1 M-1.

Structural Determinants of HscA Peptide-Binding Specificity

The Hsp70-class molecular chaperone HscA interacts specifically with a conserved (99)LPPVK(103) motif of the iron-sulfur cluster scaffold protein IscU. We used a cellulose-bound peptide array to perform single-site saturation substitution of peptide residues corresponding to Glu(98)-Ile(104) of IscU to determine positional amino acid requirements for recognition by HscA. Two mutant chaperone forms, HscA(F426A) with a DnaK-like arch structure and HscA(M433V) with a DnaK-like substrate-binding pocket, were also studied. Wild-type HscA and HscA(F426A) exhibited a strict preference for proline in the central peptide position (ELPPVKI), whereas HscA(M433V) bound a peptide containing a Pro-->Leu substitution at this location (ELPLVKI). Contributions of Phe(426) and Met(433) to HscA peptide specificity were further tested in solution using a fluorescence-based peptide-binding assay. Bimane-labeled HscA and HscA(F426A) bound ELPPVKI peptides with higher affinity than leucine-substituted peptides, whereas HscA(M433V) favored binding of ELPLVKI peptides. Fluorescence-binding studies were also carried out with derivatives of the peptide NRLLLTG, a model substrate for DnaK. HscA and HscA(F426A) bound NRLLLTG peptides weakly, whereas HscA(M433V) bound NRLLLTG peptides with higher affinity than IscU-derived peptides ELPPVKI and ELPLVKI. These results suggest that the specificity of HscA for the LPPVK recognition sequence is determined in part by steric obstruction of the hydrophobic binding pocket by Met(433) and that substitution with the Val(433) sidechain imparts a broader, more DnaK-like, substrate recognition pattern.

Topography of the Surface of the Signal-transducing Protein EIIAGlc That Interacts with the MalK Subunits of the Maltose ATP-binding Cassette Transporter (MalFGK2) of Salmonella typhimurium

The signal-transducing protein EIIA(Glc), a component of the phosphoenolpyruvate-glucose phosphotransferase system, plays a key role in carbon regulation in enteric bacteria, such as Escherichia coli and Salmonella typhimurium. The phosphorylation state of EIIA(Glc) governs transport and metabolism of a number of carbohydrates. When glucose as preferred carbon source is transported, EIIA(Glc) becomes predominantly unphosphorylated and allosterically inhibits several permeases, including the maltose ATP-binding cassette transport system (MalFGK2) in a process termed "inducer exclusion." We have mapped the binding surface of EIIA(Glc) that interacts with the MalK subunits by using synthetic cellulose-bound peptide arrays like pep scan- and substitutional analyses. Three regions constituting two binding sites were identified encompassing residues 69-79 (I), 87-91 (II), and 118-127 (III). Region III is MalK-specific, whereas residues from regions I and II partly overlap but are not identical to the binding interfaces for interaction with glycerol kinase and lactose permease. These results were fully verified by studying the inhibitory effect of purified EIIA(Glc) variants carrying mutations at positions representative of each of the three regions on the ATPase activity of the purified maltose transport complex reconstituted into proteoliposomes. Moreover, a synthetic peptide encompassing residues 69-91 was demonstrated to partially inhibit ATPase activity. We also show for the first time that the N-terminal domain of EIIA(Glc) is essential for inducer exclusion.

Probing the Ligand‐Binding Specificity and Analyzing the Folding State of SPOT‐Synthesized FBP28 WW Domain Variants

The WW domains are known as the smallest naturally occurring, monomeric, triple-stranded, antiparallel beta-sheet domains. Hence, we chose the FBP28 WW domain as a model to investigate the stability of the beta-sheet structure at the amino acid level in the context of its function (ligand binding). The structure-function relationship was investigated through a complete substitution analysis of the FBP28 WW domain, with variants synthesized as a cellulose-bound peptide array. The functionality of the FBP28 WW domain variants was examined by probing the peptide array for ligand binding. In addition, selected FBP28 WW domain variants were investigated by CD measurements to determine the stability of the antiparallel beta-sheet structure. We discuss the correlation between structure stability and functionality for the FBP28 WW domain, as well as the effect of ligand-induced structure stabilization.

Complete Substitutional Analysis of a Sunflower Trypsin Inhibitor with Different Serine Proteases

Here we present a method to simultaneously characterize and/or optimize both the binding loop towards the protease and a cysteine-stabilized scaffold. The small peptidic sunflower trypsin inhibitor (SFTI-1) was chosen as a model system for these experiments. The inhibitor was investigated for positional specificity against trypsin, elastase and proteinase K using complete substitutional analyses based on cellulose-bound peptide spot synthesis. Inhibitor variants optimized for elastase or proteinase K inhibition by several rounds of substitutional analyses exhibit K(i) values in the micromolar range and high specificity for the corresponding protease. The results of this easy-to-perform assay can be used to design an improved peptide library using classical methods.

The Periplasmic Chaperone SurA Exploits Two Features Characteristic of Integral Outer Membrane Proteins for Selective Substrate Recognition

The Escherichia coli periplasmic chaperone and peptidyl-prolyl isomerase (PPIase) SurA facilitates the maturation of outer membrane porins. Although the PPIase activity exhibited by one of its two parvulin-like domains is dispensable for this function, the chaperone activity residing in the non-PPIase regions of SurA, a sizable N-terminal domain and a short C-terminal tail, is essential. Unlike most cytoplasmic chaperones SurA is selective for particular substrates and recognizes outer membrane porins synthesized in vitro much more efficiently than other proteins. Thus, SurA may be specialized for the maturation of outer membrane proteins. We have characterized the substrate specificity of SurA based on its natural, biologically relevant substrates by screening cellulose-bound peptide libraries representing outer membrane proteins. We show that two features are critical for peptide binding by SurA: specific patterns of aromatic residues and the orientation of their side chains, which are found more frequently in integral outer membrane proteins than in other proteins. For the first time this sufficiently explains the capability of SurA to discriminate between outer membrane protein and non-outer membrane protein folding intermediates. Furthermore, peptide binding by SurA requires neither an active PPIase domain nor the presence of proline, indicating that the observed substrate specificity relates to the chaperone function of SurA. Finally, we show that SurA is capable of associating with the outer membrane. Together, our data support a model in which SurA is specialized to interact with non-native periplasmic outer membrane protein folding intermediates and to assist in their maturation from early to late outer membrane-associated steps.

Detection of oligomerisation and substrate recognition sites of small heat shock proteins by peptide arrays

Small heat shock proteins (sHsps) form large oligomers that are characterised by their dynamic behaviour, e.g., complex disassembly/reassembly and extensive subunit exchange. These processes are interrelated with sHsp/substrate interaction. sHsps bind a broad spectrum of unrelated substrate proteins under denaturing conditions. Detailed knowledge about the binding process and regions critical for sHsp/substrate interaction is missing. In this study, we screened cellulose-bound peptide spot libraries derived from a bacterial sHsp and the model-substrate citrate synthase to detect oligomerisation and substrate interaction sites, respectively. In line with previous results, it was demonstrated that multiple contacts involving the N- and C-terminal extensions and the central α-crystallin domain are required for oligomerisation. Incubation of the citrate synthase membrane with sHsps revealed a putative substrate interaction site. A soluble peptide with the sequence RTKYWELIYEDCMDL (CS 191–205) corresponding to that site inhibited chaperone activity of sHsps, presumably by blocking their substrate-binding sites.

Profiling the immune responses of human patients treated with recombinant streptokinase after myocardial infarct.

The SPOT synthesis of peptide arrays on continuous cellulose membranes should be generally applicable in the analysis of sequential antibody binding sites using the enzyme-substrate or other standard detection protocols. The use of total serum is limited by the occurrence of high background levels. This may be overcome if affinity purified antibodies or sera with high antibody titers are used, which allows work at high dilutions and a consequent reduction of background level. Here we demonstrate the mapping of antigenic regions located on recombinant streptokinase SK-2 (Heberkinase) using cellulose-bound peptide scans and human total sera from patients treated with SK-2 (Heberkinase). Streptokinase (SK) is a 47 kDa protein produced by various strains of hemolytic streptococci and is a potent activator of the fibrinolytic enzyme system in humans. SK is in widespread clinical use to treat acute infarction because of its function as an activator of vascular fibrinolysis. Since streptococcal infections are common, normal individuals are immunized with SK and antibodies (Abs) to SK can be detected in most of them. This therapy generates significant T-cell responses to SK and the neutralizing capacity of the Abs rises significantly. Neutralizing Abs reduces the efficiency of thrombolytic therapy and may cause allergic reactions. The widespread use of SK in humans makes its antigenicity an important clinical problem. In this regard the study of the immunodominant regions of SK becomes an important aspect for the improvement of this thrombolytic agent.

Investigations of cyclophilin interactions with oligopeptides containing proline by affinity capillary electrophoresis.

Affinity capillary electrophoresis using mobility-shift analysis was utilized to characterize the binding of peptide ligands to cyclophilins, which are members of the enzyme family of peptidyl-prolyl cis/trans isomerases. Peptides derived from the human immunodeficiency virus capsid protein p24 exhibited different affinities to the isoenzymes cyclophilin18 and cyclophilin20. For the interaction of the peptide hormone bradykinin with cyclophilin18, a dissociation constant of 1.4 +/- 0.1 mM was determined. Finally, the affinity of cyclophilin20 to peptides from a cellulose-bound peptide library scanning the sequence of Drosophila melanogaster protein cappuccino was investigated. The affinities of selected peptides to cyclophilin20 and a green fluorescent fusion protein with cyclophilin20 were compared.

Specificity of elongation factor EF-TU for hydrophobic peptides

The elongation factor EF-Tu carries aminoacyl-tRNAs to the A-site of the ribosome during the elongation process of protein biosynthesis. We, and others, have recently reported that the Escherichia coli EF-Tu interacts with unfolded and denatured proteins and behaves like a chaperone in protein folding and protection against protein thermal denaturation. In this study, we have identified EF-Tu binding sites in protein substrates by screening cellulose-bound peptides scanning the sequences of several proteins. The binding motifs recognized by EF-Tu in protein substrates are also recognized by the chaperone DnaK and mainly consist of hydrophobic clusters. EF-Tu interacts as efficiently as DnaK with the membrane spanning sequence of the membrane protein phospholemman and with the signal sequence of alkaline phosphatase. It interacts less efficiently with several other hydrophobic clusters of lysozyme and alkaline phosphatase, which are also DnaK substrates and fails to bind to several DnaK binding sites. Our results suggest that EF-Tu, like DnaK, interacts albeit more weakly with the hydrophobic regions of substrate protein and are consistent with the hypothesis that it possesses chaperone properties.

Hsc66 Substrate Specificity Is Directed toward a Discrete Region of the Iron-Sulfur Cluster Template Protein IscU

Hsc66 and Hsc20 comprise a specialized chaperone system important for the assembly of iron-sulfur clusters in Escherchia coli. Only a single substrate, the Fe/S template protein IscU, has been identified for the Hsc66/Hsc20 system, but the mechanism by which Hsc66 selectively binds IscU is unknown. We have investigated Hsc66 substrate specificity using phage display and a peptide array of IscU. Screening of a heptameric peptide phage display library revealed that Hsc66 prefers peptides with a centrally located Pro-Pro motif. Using a cellulose-bound peptide array of IscU we determined that Hsc66 interacts specifically with a region (residues 99-103, LPPVK) that is invariant among all IscU family members. A synthetic peptide (ELPPVKIHC) corresponding to IscU residues 98-106 behaves in a similar manner to native IscU, stimulating the ATPase activity of Hsc66 with similar affinity as IscU, preventing Hsc66 suppression of bovine rhodanese aggregation, and interacting with the peptide-binding domain of Hsc66. Unlike native IscU, however, the synthetic peptide is not bound by Hsc20 and does not synergistically stimulate Hsc66 ATPase activity with Hsc20. Our results indicate that Hsc66 and Hsc20 recognize distinct regions of IscU and further suggest that Hsc66 will not bind LPPVK motifs with high affinity in vivo unless they are in the context of native IscU and can be directed to Hsc66 by Hsc20.