Epitope-resolved profiling of the SARS-CoV-2 antibody response identifies cross-reactivity with endemic human coronaviruses.

The SARS-CoV-2 proteome shares regions of conservation with endemic human coronaviruses (CoVs), but it remains unknown to what extent these may be cross-recognized by the antibody response. Here, we study cross-reactivity using a highly multiplexed peptide assay (PepSeq) to generate an epitope-resolved view of IgG reactivity across all human CoVs in both COVID-19 convalescent and negative donors. PepSeq resolves epitopes across the SARS-CoV-2 Spike and Nucleocapsid proteins that are commonly targeted in convalescent donors, including several sites also recognized in some uninfected controls. By comparing patterns of homologous reactivity between CoVs and using targeted antibody-depletion experiments, we demonstrate that SARS-CoV-2 elicits antibodies that cross-recognize pandemic and endemic CoV antigens at two Spike S2 subunit epitopes. We further show that these cross-reactive antibodies preferentially bind endemic homologs. Our findings highlight sites at which the SARS-CoV-2 response appears to be shaped by previous CoV exposures and which have the potential to raise broadly neutralizing responses.

Epitope-resolved profiling of the SARS-CoV-2 antibody response identifies cross-reactivity with an endemic human CoV.

A high-resolution understanding of the antibody response to SARS-CoV-2 is important for the design of effective diagnostics, vaccines and therapeutics. However, SARS-CoV-2 antibody epitopes remain largely uncharacterized, and it is unknown whether and how the response may cross-react with related viruses. Here, we use a multiplexed peptide assay ('PepSeq') to generate an epitope-resolved view of reactivity across all human coronaviruses. PepSeq accurately detects SARS-CoV-2 exposure and resolves epitopes across the Spike and Nucleocapsid proteins. Two of these represent recurrent reactivities to conserved, functionally-important sites in the Spike S2 subunit, regions that we show are also targeted for the endemic coronaviruses in pre-pandemic controls. At one of these sites, we demonstrate that the SARS-CoV-2 response strongly and recurrently cross-reacts with the endemic virus hCoV-OC43. Our analyses reveal new diagnostic and therapeutic targets, including a site at which SARS-CoV-2 may recruit common pre-existing antibodies and with the potential for broadly-neutralizing responses.

High-Throughput Multiplexed Peptide-Centric Profiling Illustrates Both Substrate Cleavage Redundancy and Specificity in the MMP Family.

Matrix metalloproteinases (MMPs) play incompletely understood roles in health and disease. Knowing the MMP cleavage preferences is essential for a better understanding of the MMP functions and design of selective inhibitors. To elucidate the cleavage preferences of MMPs, we employed a high-throughput multiplexed peptide-centric profiling technology involving the cleavage of 18,583 peptides by 18 proteinases from the main sub-groups of the MMP family. Our results enabled comparison of the MMP substrates on a global scale, leading to the most efficient and selective substrates. The data validated the accuracy of our cleavage prediction software. This software allows us and others to locate, with nearly 100% accuracy, the MMP cleavage sites in the peptide sequences. In addition to increasing our understanding of both the selectivity and the redundancy of the MMP family, our study generated a roadmap for the subsequent MMP structural-functional studies and efficient substrate and inhibitor design.

Structural and functional diversity of metalloproteinases encoded by the Bacteroides fragilis pathogenicity island.

Bacteroides fragilis causes the majority of anaerobic infections in humans. The presence of a pathogenicity island in the genome discriminates pathogenic and commensal B. fragilis strains. The island encodes metalloproteinase II (MPII), a potential virulence protein, and one of three homologous fragilysin isozymes (FRA; also termed B. fragilis toxin or BFT). Here, we report biochemical data on the structural-functional characteristics of the B. fragilis pathogenicity island proteases by reporting the crystal structure of MPII at 2.13 Å resolution, combined with detailed characterization of the cleavage preferences of MPII and FRA3 (as a representative of the FRA isoforms), identified using a high-throughput peptide cleavage assay with 18 583 substrate peptides. We suggest that the evolution of the MPII catalytic domain can be traced to human and archaebacterial proteinases, whereas the prodomain fold is a feature specific to MPII and FRA. We conclude that the catalytic domain of both MPII and FRA3 evolved differently relative to the prodomain, and that the prodomain evolved specifically to fit the B. fragilis pathogenicity. Overall, our data provide insights into the evolution of cleavage specificity and activation mechanisms in the virulent metalloproteinases.

Substrate cleavage profiling suggests a distinct function of Bacteroides fragilis metalloproteinases (fragilysin and metalloproteinase II) at the microbiome-inflammation-cancer interface.

Enterotoxigenic anaerobic Bacteroides fragilis is a significant source of inflammatory diarrheal disease and a risk factor for colorectal cancer. Two distinct metalloproteinase types (the homologous 1, 2, and 3 isoforms of fragilysin (FRA1, FRA2, and FRA3, respectively) and metalloproteinase II (MPII)) are encoded by the B. fragilis pathogenicity island. FRA was demonstrated to be important to pathogenesis, whereas MPII, also a potential virulence protein, remained completely uncharacterized. Here, we, for the first time, extensively characterized MPII in comparison with FRA3, a representative of the FRA isoforms. We employed a series of multiplexed peptide cleavage assays to determine substrate specificity and proteolytic characteristics of MPII and FRA. These results enabled implementation of an efficient assay of MPII activity using a fluorescence-quenched peptide and contributed to structural evidence for the distinct substrate cleavage preferences of MPII and FRA. Our data imply that MPII specificity mimics the dibasic Arg↓Arg cleavage motif of furin-like proprotein convertases, whereas the cleavage motif of FRA (Pro-X-X-Leu-(Arg/Ala/Leu)↓) resembles that of human matrix metalloproteinases. To the best of our knowledge, MPII is the first zinc metalloproteinase with the dibasic cleavage preferences, suggesting a high level of versatility of metalloproteinase proteolysis. Based on these data, we now suggest that the combined (rather than individual) activity of MPII and FRA is required for the overall B. fragilis virulence in vivo.

Enzymatic probing analysis of an engineered riboswitch reveals multiple off conformations.

We investigated the gene regulatory mechanism of a previously engineered riboswitch +thiMN(15)#19 that turns on gene expression in response to thiamine pyrophosphate (TPP). In vitro enzymatic probing was performed to identify the secondary structures of the OFF conformations predicted by Mfold. Interestingly, enzymatic probing data of the riboswitch and its variants indicated that the riboswitch in its OFF state adopts two distinct structures. Moreover, further in vivo experiments suggested that both OFF structures contribute to the riboswitch function. A deeper understanding of how riboswitches function at the molecular level should enhance our ability to design synthetic riboswitches with new or improved characteristics.

Biosynthesis of proteins containing modified lysines and fluorescent labels using non-natural amino acid mutagenesis.

The preparation of posttranslationally modified proteins is required to investigate the function and structure of modified proteins. However, homogeneously modified proteins are not easily isolated from natural sources or prepared using modification enzymes. Non-natural amino acid mutagenesis has enabled us to incorporate modified amino acids into specific positions of proteins in both cell-free and in-cell translation systems using tRNAs that are aminoacylated with modified amino acids. Here, we developed a method of double incorporation of modified amino acids and fluorescent non-natural amino acids in a quantitative, position-specific manner to obtain modified and fluorescently labeled proteins. To introduce methyllysine, dimethyllysine, trimethyllysine, and acetyllysine, frameshift and amber suppressor tRNAs aminoacylated with modified lysines were synthesized by chemical aminoacylation and supplied to an Escherichia coli cell-free translation system. The immunodetection of the translation products indicated that the modified lysines were incorporated into streptavidin and histone H3 in a quantitative, position-specific manner. Calmodulin derivatives containing a fluorescent non-natural amino acid at the N-terminal region and modified lysines at the Lys115 position were also synthesized, and their binding activity to a calmodulin-binding peptide was analyzed by fluorescence correlation spectroscopy. The results obtained here demonstrate that this method is useful in preparing and analyzing naturally occurring and non-natural modified proteins.

A synthetic riboswitch with chemical band-pass response.

We designed a synthetic riboswitch containing an OFF and an ON riboswitch units fused in tandem. The resulting complex riboswitch functions as a chemical band-pass filter circuit.

Mechanism-guided library design and dual genetic selection of synthetic OFF riboswitches.

After the recent discovery of bacterial riboswitches, synthetic riboswitches have been engineered by using natural and artificial RNA aptamers. In contrast to natural riboswitches, the majority of synthetic riboswitches in bacteria reported to date are ON switches that activate gene expression in response to the aptamer ligand. In this study, we adopted a mechanism-guided approach to design libraries predisposed to contain OFF riboswitches that respond to thiamine pyrophosphate (TPP). The first library design exploited a pseudo-Shine-Dalgarno (SD) sequence located near the 3'-end of the TPP aptamer, which would be less accessible to the ribosome when the aptamer is bound to TPP. In the second library, an SD sequence was strategically placed in the aptamer's P1 stem, which is stabilized upon ligand binding. OFF riboswitches were obtained by dual genetic selection of these libraries. The results underscore the importance of effective library design to achieve desired riboswitch functions.

An efficient platform for genetic selection and screening of gene switches in Escherichia coli.

Engineered gene switches and circuits that can sense various biochemical and physical signals, perform computation, and produce predictable outputs are expected to greatly advance our ability to program complex cellular behaviors. However, rational design of gene switches and circuits that function in living cells is challenging due to the complex intracellular milieu. Consequently, most successful designs of gene switches and circuits have relied, to some extent, on high-throughput screening and/or selection from combinatorial libraries of gene switch and circuit variants. In this study, we describe a generic and efficient platform for selection and screening of gene switches and circuits in Escherichia coli from large libraries. The single-gene dual selection marker tetA was translationally fused to green fluorescent protein (gfpuv) via a flexible peptide linker and used as a dual selection and screening marker for laboratory evolution of gene switches. Single-cycle (sequential positive and negative selections) enrichment efficiencies of >7000 were observed in mock selections of model libraries containing functional riboswitches in liquid culture. The technique was applied to optimize various parameters affecting the selection outcome, and to isolate novel thiamine pyrophosphate riboswitches from a complex library. Artificial riboswitches with excellent characteristics were isolated that exhibit up to 58-fold activation as measured by fluorescent reporter gene assay.

Four-base codon-mediated saturation mutagenesis in a cell-free translation system.

Saturation mutagenesis is a useful technique for the structural and functional analyses of proteins and for protein engineering. However, the extensive mutagenesis of genes and expression of mutated proteins are tedious and time-consuming. We have developed a simple and rapid method for the expression of mutated proteins with comprehensive single amino acid substitutions from single mutated genes having a four-base codon in a cell-free translation system. Twenty types of tRNA that were aminoacylated with one of the 20 proteinogenic amino acids and that contained a four-base anticodon were prepared by chemical aminoacylation. In the presence of one of the aminoacyl-tRNAs, a streptavidin mRNA with a four-base codon at the Tyr83 position was expressed in an Escherichia coli cell-free translation system. The N-terminus of the expressed proteins was fluorescently labeled using a fluorescent-labeled initiator Met-tRNA. Fluorescence imaging of an SDS-PAGE gel showed that all the amino acids are incorporated in response to the four-base codon; however, the incorporation efficiency was dependent on the structure of the side chains. Streptavidin mutants with comprehensive amino acid substitutions at the Tyr83, Arg84, and Tyr54 positions were used for analyzing their biotin-binding activity by dot blot analysis. These results demonstrate that this method is effective for the expression and analysis of mutated proteins with comprehensive amino acid substitutions at desired positions.

N-terminal specific fluorescence labeling of proteins through incorporation of fluorescent hydroxy acid and subsequent ester cleavage.

We have developed a novel method to attach a fluorescent label at the N terminus of proteins through a four-base codon-mediated incorporation of a fluorescent hydroxy acid and subsequent cleavage of the ester bond in a cell-free translation system. We found that a fluorescent-labeled p-amino-L-phenyllactic acid was successfully incorporated downstream of N-terminal tag peptides in response to a CGGG codon, and the tag peptides could be removed through ester cleavage to leave the fluorescent hydroxy acid at the N terminus of the proteins. Immunoprecipitation analysis revealed that ester cleavage occurred spontaneously during the translation reaction. The efficiency of the ester cleavage and the yield of the labeled proteins were dependent on the peptide tag sequence. We demonstrate that the insertion of an asparagine residue between the N-terminal T7 tag and the fluorescent hydroxy acid achieved both quantitative ester cleavage and efficient expression of the labeled proteins. The present method is a potential tool for N-terminal specific labeling of proteins with various compounds.

N-terminal specific fluorescence labeling of proteins through fourbase codon-mediated incorporation of fluorescent hydroxy acid.

Fluorescence labeling of proteins is a useful tool for protein structural and functional analysis. We developed here a novel method to attach a fluorescence labeling at the N terminus of proteins through the incorporation of a fluorescent hydroxy acid and subsequent hydrolysis of the ester bond in a cell-free translation system. We found that N-terminal tagged proteins containing p-(BODIPYFL-amino)-L-phenyllactic acid at the downstream of the tag peptides were efficiently synthesized and the resulting ester bonds were hydrolyzed during the translation reaction. These results indicate that the present method will become a useful tool for the N-terminal specific labeling of proteins.

Position-specific incorporation of biotinylated non-natural amino acids into a protein in a cell-free translation system.

Biotinylation is useful for the detection, purification and immobilization of proteins. It is performed by chemical modification, although position-specific and quantitative biotinylation is rarely achieved. We developed a position-specific biotinylation method using biotinylated non-natural amino acids. We showed that biotinylated p-aminophenylalanine derivatives were incorporated into a protein more efficiently than biotinylated lysine derivatives in a cell-free translation system. In addition, the biotinylated p-aminophenylalanines overcame the serious position-dependency observed for biotinylated lysines. The present method will be useful for detection and purification of proteins along with comprehensive exploration of surface-exposed residues and oriented immobilization of proteins.

Four-base codon mediated mRNA display to construct peptide libraries that contain multiple nonnatural amino acids.

In vitro selection and directed evolution of peptides from mRNA display are powerful strategies to find novel peptide ligands that bind to target biomolecules. In this study, we expanded the mRNA display method to include multiple nonnatural amino acids by introducing three different four-base codons at a randomly selected single position on the mRNA. Another nonnatural amino acid may be introduced by suppressing an amber codon that may appear from a (NNK)(n) nucleotide sequence on the mRNA. The mRNA display was expressed in an Escherichia coli in vitro translation system in the presence of three types of tRNAs carrying different four-base anticodons and a tRNA carrying an amber anticodon, the tRNAs being chemically aminoacylated with different nonnatural amino acids. The complexity of the starting mRNA-displayed peptide library was estimated to be 1.1 x 10(12) molecules. The effectiveness of the four-base codon mediated mRNA display method was demonstrated in the selection of biocytin-containing peptides on streptavidin-coated beads. Moreover, a novel streptavidin-binding nonnatural peptide containing benzoylphenylalanine was obtained from the nonnatural peptide library. The nonnatural peptide library from the four-base codon mediated mRNA display provides much wider functional and structural diversity than conventional peptide libraries that are constituted from 20 naturally occurring amino acids.

Position-specific incorporation of dansylated non-natural amino acids into streptavidin by using a four-base codon.

Novel non-natural amino acids carrying a dansyl fluorescent group were designed, synthesized, and incorporated into various positions of streptavidin by using a CGGG four-base codon in an Escherichia coli in vitro translation system. 2,6-Dansyl-aminophenylalanine (2,6-dnsAF) was found to be incorporated into the protein more efficiently than 1,5-dansyl-lysine, 2,6-dansyl-lysine, and 1,5-dansyl-aminophenylalanine. Fluorescence measurements indicate that the position-specific incorporation of the 2,6-dnsAF is a useful technique to probe protein structures. These results also indicate that well-designed non-natural amino acids carrying relatively large side chains can be accepted as substrates of the translation system.

Photoswitching of peroxidase activity by position-specific incorporation of a photoisomerizable non-natural amino acid into horseradish peroxidase.

Horseradish peroxidase mutants containing L-p-phenylazophenylalanine (azoAla) at various positions were synthesized by using an Escherichia coli in vitro translation system. Among the 15 mutants examined, four mutants containing a single azoAla unit at the 6th, 68th, 142nd, and 179th positions, respectively, retained the peroxidase activity. The activity of the Phe68azoAla mutant was higher when the azobenzene group was in the cis form than in the trans form. On the contrary, the activity of the Phe179azoAla mutant disappeared when the azobenzene group was photoisomerized to the cis form, but recovered in the trans form. In the latter mutant, therefore, an on/off photoswitching of the peroxidase activity was attained.