Single Amino Acid Difference Research Articles

Glycoprotein-Specific Polyclonal Antibodies Targeting Machupo Virus Protect Guinea Pigs against Lethal Infection.

Convalescent plasma has been shown to be effective at protecting humans against severe diseases caused by New World (NW) arenaviruses, including Junin virus (JUNV) and Machupo virus (MACV). This plasma contains antibodies against the full complement of structural proteins including the nucleocapsid and envelope glycoproteins (GPcs) consisting of GP1 and GP2. To gain insights into the protective and cross-protective properties of anti-GPc-specific polyclonal antibodies, we evaluated the ability of a DNA vaccine-produced anti-GPc rabbit antisera targeting MACV strain Carvallo to provide heterologous protection against another MACV strain termed Chicava in the Hartley guinea pig model. The neutralizing activity of the rabbit antisera against the heterologous MACV strains Chicava and Mallale was found to be 54-fold and 23-fold lower, respectively, compared to the titer against the homologous MACV strain Carvallo in the PRNT50 assay. Despite lower neutralizing activity against the strain Chicava, the rabbit antisera protected 100% of the guinea pigs from this strain when administered up to four days post-infection, whereas all the control animals succumbed to the disease. Using vesicular stomatitis virus (VSV) particles pseudotyped with MACV GPc, we identified a single amino acid difference at position 122 between the strains Chicava and Carvallo GPc that significantly influenced the neutralization activity of the rabbit antisera. These findings indicate that polyclonal antibodies targeting the MACV glycoproteins can protect against lethal infection in a post-challenge setting. These data will help guide future antibody-based therapeutics development against NW arenaviruses.

Peptidoglycan fragment release and NOD activation by commensal Neisseria species from humans and other animals.

Neisseria gonorrhoeae, a human restricted pathogen, releases inflammatory peptidoglycan (PG) fragments that contribute to the pathophysiology of pelvic inflammatory disease. The genus Neisseria is also home to multiple species of human- or animal-associated Neisseria that form part of the normal microbiota. Here we characterized PG release from the human-associated nonpathogenic species Neisseria lactamica and Neisseria mucosa and animal-associated Neisseria from macaques and wild mice. An N. mucosa strain and an N. lactamica strain were found to release limited amounts of the proinflammatory monomeric PG fragments. However, a single amino acid difference in the PG fragment permease AmpG resulted in increased PG fragment release in a second N. lactamica strain examined. Neisseria isolated from macaques also showed substantial release of PG monomers. The mouse colonizer Neisseria musculi exhibited PG fragment release similar to that seen in N. gonorrhoeae with PG monomers being the predominant fragments released. All the human-associated species were able to stimulate NOD1 and NOD2 responses. N. musculi was a poor inducer of mouse NOD1, but ldcA mutation increased this response. The ability to genetically manipulate N. musculi and examine effects of different PG fragments or differing amounts of PG fragments during mouse colonization will lead to a better understanding of the roles of PG in Neisseria infections. Overall, we found that only some nonpathogenic Neisseria have diminished release of proinflammatory PG fragments, and there are differences even within a species as to types and amounts of PG fragments released.

Sequence signatures within the genome of SARS-CoV-2 can be used to predict host source.

We conducted an in silico analysis to better understand the potential factors impacting host adaptation of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) in white-tailed deer, humans, and mink due to the strong evidence of sustained transmission within these hosts. Classification models trained on single nucleotide and amino acid differences between samples effectively identified white-tailed deer-, human-, and mink-derived SARS-CoV-2. For example, the balanced accuracy score of Extremely Randomized Trees classifiers was 0.984 ± 0.006. Eighty-eight commonly identified predictive mutations are found at sites under strong positive and negative selective pressure. A large fraction of sites under selection (86.9%) or identified by machine learning (87.1%) are found in genes other than the spike. Some locations encoded by these gene regions are predicted to be B- and T-cell epitopes or are implicated in modulating the immune response suggesting that host adaptation may involve the evasion of the host immune system, modulation of the class-I major-histocompatibility complex, and the diminished recognition of immune epitopes by CD8+ T cells. Our selection and machine learning analysis also identified that silent mutations, such as C7303T and C9430T, play an important role in discriminating deer-derived samples across multiple clades. Finally, our investigation into the origin of the B.1.641 lineage from white-tailed deer in Canada discovered an additional human sequence from Michigan related to the B.1.641 lineage sampled near the emergence of this lineage. These findings demonstrate that machine-learning approaches can be used in combination with evolutionary genomics to identify factors possibly involved in the cross-species transmission of viruses and the emergence of novel viral lineages.IMPORTANCESevere acute respiratory syndrome coronavirus 2 (SARS-CoV-2) is a highly transmissible virus capable of infecting and establishing itself in human and wildlife populations, such as white-tailed deer. This fact highlights the importance of developing novel ways to identify genetic factors that contribute to its spread and adaptation to new host species. This is especially important since these populations can serve as reservoirs that potentially facilitate the re-introduction of new variants into human populations. In this study, we apply machine learning and phylogenetic methods to uncover biomarkers of SARS-CoV-2 adaptation in mink and white-tailed deer. We find evidence demonstrating that both non-synonymous and silent mutations can be used to differentiate animal-derived sequences from human-derived ones and each other. This evidence also suggests that host adaptation involves the evasion of the immune system and the suppression of antigen presentation. Finally, the methods developed here are general and can be used to investigate host adaptation in viruses other than SARS-CoV-2.

Isolation of a feline-derived feline panleukopenia virus with an A300P substitution in the VP2 protein and confirmation of its pathogenicity in dogs

Feline panleukopenia virus (FPV) is a single-stranded DNA virus that can infect cats and cause feline panleukopenia, which is a highly contagious and fatal disease in felines. The sequence of FPV is highly variable, and mutations in the amino acids of its capsid protein play crucial roles in altering viral virulence, immunogenicity, host selection, and other abilities. In this study, the epidemiology of FPV was studied using 746 gastrointestinal swab samples derived from cats that presented gastrointestinal symptoms specifically, diarrhea or vomiting during the period spanning from 2018 to 2022. The overall prevalence of FPV-positive patients among these samples was determined to be 45.4%. Capsid (virion) protein 2 (VP2) gene of each FPV-positive sample was sequenced and amplified, yielding 65 VP2 sequences. Among them, six VP2 gene sequences were detected in the majority of the samples test positive for FPV, and these positive samples originated from a diverse range of geographical locations. These isolates were named FPV-6, FPV-10, FPV-15, FPV-251, FPV-271 and FPV-S2. Additionally, the substitution of Ala300Pro (A300P) in VP2 was detected for the first time in feline-derived FPV (FPV-251). FPV-251 isolate, with this substitution in VP2 protein, exhibited stable proliferative capacity in Madin-Darby canine kidney (MDCK) cells and A72 cells. FPV-271 was selected as the FPV control isolate due to its single amino acid difference from VP2 protein of FPV-251 at position 300 (FPV-271 has alanine, while FPV-251 has proline). After oral infection, both FPV-251 and FPV-271 isolates caused feline panleukopenia, which is characterized by clinical signs of enterocolitis. However, FPV-251 can infect dogs through the oral route and cause gastrointestinal (GI) symptoms with lesions in the intestine and mesenteric lymph nodes (MLNs) of infected dogs. This is the first report on the presence of an A300P substitution in VP2 protein of feline-derived FPV. Additionally, FPV isolate with a substitution of A300P at VP2 protein demonstrated efficient replication capabilities in canine cell lines and the ability to infect dogs.

Unprecedented selectivity for homologous lectin targets: differential targeting of the viral receptors L-SIGN and DC-SIGN.

DC-SIGN (CD209) and L-SIGN (CD209L) are two C-type lectin receptors (CLRs) that facilitate SARS-CoV-2 infections as viral co-receptors. SARS-CoV-2 manipulates both DC-SIGN and L-SIGN for enhanced infection, leading to interest in developing receptor antagonists. Despite their structural similarity (82% sequence identity), they function differently. DC-SIGN, found in dendritic cells, shapes the immune response by recognizing pathogen-associated carbohydrate patterns. In contrast, L-SIGN, expressed in airway epithelial endothelial cells, is not directly involved in immunity. COVID-19's primary threat is the hyperactivation of the immune system, potentially reinforced if DC-SIGN engages with exogenous ligands. Therefore, L-SIGN, co-localized with ACE2-expressing cells in the respiratory tract, is a more suitable target for anti-adhesion therapy. However, designing a selective ligand for L-SIGN is challenging due to the high sequence identity of the Carbohydrate Recognition Domains (CRDs) of the two lectins. We here present Man84, a mannose ring modified with a methylene guanidine triazole at position 2. It binds L-SIGN with a K D of 12.7μM ± 1 μM (ITC) and is the first known L-SIGN selective ligand, showing 50-fold selectivity over DC-SIGN (SPR). The X-ray structure of the L-SIGN CRD/Man84 complex reveals the guanidinium group's role in achieving steric and electrostatic complementarity with L-SIGN. This allows us to trace the source of selectivity to a single amino acid difference between the two CRDs. NMR analysis confirms the binding mode in solution, highlighting Man84's conformational selection upon complex formation. Dimeric versions of Man84 achieve additional selectivity and avidity in the low nanomolar range. These compounds selectively inhibit L-SIGN dependent trans-infection by SARS-CoV-2 and Ebola virus. Man84 and its dimeric constructs display the best affinity and avidity reported to date for low-valency glycomimetics targeting CLRs. They are promising tools for competing with SARS-CoV-2 anchoring in the respiratory tract and have potential for other medical applications.

Function-structure approach reveals novel insights on the interplay of Immunoglobulin G 1 proteoforms and Fc gamma receptor IIa allotypes.

Human Fc gamma receptor IIa (FcγRIIa) or CD32a has two major allotypes with a single amino acid difference at position 131 (histidine or arginine). Differences in FcγRIIa allotypes are known to impact immunological responses such as the clinical outcome of therapeutic monoclonal antibodies (mAbs). FcγRIIa is involved in antibody-dependent cellular phagocytosis (ADCP), which is an important contributor to the mechanism-of-action of mAbs by driving phagocytic clearance of cancer cells. Hence, understanding the impact of individual mAb proteoforms on the binding to FcγRIIa, and its different allotypes, is crucial for defining meaningful critical quality attributes (CQAs). Here, we report a function-structure based approach guided by novel FcγRIIa affinity chromatography-mass spectrometry (AC-MS) assays to assess individual IgG1 proteoforms. This allowed to unravel allotype-specific differences of IgG1 proteoforms on FcγRIIa binding. FcγRIIa AC-MS confirmed and refined structure-function relationships of IgG1 glycoform interactions. For example, the positive impact of afucosylation was higher than galactosylation for FcγRIIa Arg compared to FcγRIIa His. Moreover, we observed FcγRIIa allotype-opposing and IgG1 proteoform integrity-dependent differences in the binding response of stress-induced IgG1 proteoforms comprising asparagine 325 deamidation. The FcγRIIa-allotype dependent binding differences resolved by AC-MS were in line with functional ADCP-surrogate bioassay models. The molecular basis of the observed allotype specificity and proteoform selectivity upon asparagine 325 deamidation was elucidated using molecular dynamics. The observed differences were attributed to the contributions of an inter-molecular salt bridge between IgG1 and FcγRIIa Arg and the contribution of an intra-molecular hydrophobic pocket in IgG1. Our work highlights the unprecedented structural and functional resolution of AC-MS approaches along with predictive biological significance of observed affinity differences within relevant cell-based methods. This makes FcγRIIa AC-MS an invaluable tool to streamline the CQA assessment of therapeutic mAbs.

Characterization of tigurilysin, a novel human CD59-specific cholesterol-dependent cytolysin, reveals a role for host specificity in augmenting toxin activity.

Cholesterol-dependent cytolysins (CDCs) are a large family of pore-forming toxins, produced by numerous Gram-positive pathogens. CDCs depend on host membrane cholesterol for pore formation; some CDCs also require surface-associated human CD59 (hCD59) for binding, conferring specificity for human cells. We purified a recombinant version of a putative CDC encoded in the genome of Streptococcus oralis subsp. tigurinus, tigurilysin (TGY), and used CRISPR/Cas9 to construct hCD59 knockout (KO) HeLa and JEG-3 cell lines. Cell viability assays with TGY on wild-type and hCD59 KO cells showed that TGY is a hCD59-dependent CDC. Two variants of TGY exist among S. oralis subsp. tigurinus genomes, only one of which is functional. We discovered that a single amino acid change between these two TGY variants determines its activity. Flow cytometry and oligomerization Western blots revealed that the single amino acid difference between the two TGY isoforms disrupts host cell binding and oligomerization. Furthermore, experiments with hCD59 KO cells and cholesterol-depleted cells demonstrated that TGY is fully dependent on both hCD59 and cholesterol for activity, unlike other known hCD59-dependent CDCs. Using full-length CDCs and toxin constructs differing only in the binding domain, we determined that having hCD59 dependence leads to increased lysis efficiency, conferring a potential advantage to organisms producing hCD59-dependent CDCs.

DEVELOPMENT OF A SELECTIVE CD16A-BASED NK CELL ENGAGER UTILIZING ANTIBODIES TARGETING A SINGLE AMINO ACID VARIATION

Abstract Background and Significance Natural killer (NK) cells play a vital role in the human innate immune system and are being explored as a promising approach for cancer immunotherapy. Of particular interest are NK cell engagers that can target and activate NK cells to attack cancer cells. In this study, we developed novel NK cell engagers by targeting the NK cell activating receptor CD16a using antibodies that selectively distinguish between CD16a on NK cells and CD16b on granulocytes, which are highly homologous to each other. Methods and Results To generate antibodies with high developability, we employed a rational design approach to construct large yeast display libraries of human antibodies. This approach was based on the analysis of a deep sequencing dataset of human antibodies from over 500 individuals, which allowed us to determine the natural amino acid usage patterns of human antibody CDRs and mimic human antibody repertoires. Through screening these libraries, we discovered two classes of antibody clones that selectively recognize CD16a without cross-reactivity to CD16b. Epitope mapping revealed that a single amino acid difference confers over 10,000-fold selectivity for one class of antibody clones, while for the other class a second unique epitope on CD16a was identified. To evaluate the activity of these antibody clones, we produced bispecific antibody clones with one arm targeting CD16a and the other arm targeting a tumor-associated antigen (TAA). Our results demonstrated potent tumor cell-dependent activation of NK cells and effective killing of tumor cells. Several of these antibodies had greatly enhanced resistance to human IgG inhibition in killing target cells. Significantly, our anti-CD16a antibody clones exhibited superior performance compared to leading reference anti-CD16a clones in two distinct NK cell engager formats. This included higher affinity for CD16a, higher thermostability, and more potent killing activity both in the absence and presence of 10 mg/mL human IgGs as competitors. Conclusion Our findings indicate that anti-CD16a antibody-based NK cell engagers have significant potential for cancer immunotherapies.

A Porcine DNMT1 Variant: Molecular Cloning and Generation of Specific Polyclonal Antibody.

DNA methyltransferase 1 (DNMT1), the first-identified DNA methyltransferase in mammals, has been well studied in the control of embryo development and somatic homeostasis in mice and humans. Accumulating reports have demonstrated that DNMT1 plays an important role in the regulation of differentiation and the activation of immune cells. However, little is known about the effects of porcine DNMT1 on such functional regulation, especially the regulation of the biological functions of immune cells. In this study, we report the cloning of DNMT1 (4833 bp in length) from porcine alveolar macrophages (PAMs). According to the sequence of the cloned DNMT1 gene, the deduced protein sequence contains a total of 1611 amino acids with a 2 amino acid insertion, a 1 amino acid deletion, and 12 single amino acid mutations in comparison to the reported DNMT1 protein. A polyclonal antibody based on a synthetic peptide was generated to study the expression of the porcine DNMT1. The polyclonal antibody only recognized the cloned porcine DNMT1 and not the previously reported protein due to a single amino acid difference in the antigenic peptide region. However, the polyclonal antibody recognized the endogenous DNMT1 in several porcine cells (PAM, PK15, ST, and PIEC) and the cells of other species (HEK-293T, Marc-145, MDBK, and MDCK cells). Moreover, our results demonstrated that all the detected tissues of piglet express DNMT1, which is the same as that in porcine alveolar macrophages. In summary, we have identified a porcine DNMT1 variant with sequence and expression analyses.

In-depth size and charge variants characterization of monoclonal antibody with native mass spectrometry

Although the reversed-phase liquid chromatography (RPLC) is the most used separation front for mass spectrometry, many other separation modes are critical for enabling characterization of the protein therapeutics. Specifically, chromatographic separations under native conditions, such as those based on size exclusion chromatography (SEC) and ion-exchange chromatography (IEX), are used for characterizing important biophysical properties of protein variants in drug substance and drug product. Because most native state separation modes use non-volatile buffers with high salt concentration, optical detection has been traditionally used. However, there is an increasing need to understand and identify the optical underlying peaks by mass spectrometry for structure elucidation. For size variant separation by SEC, the native MS helps to understand the nature of the high molecular weight species, as well as clipping sites for low molecular weight fragments. For charge variant separation by IEX, native MS can reveal the post-translational modifications or other important factors contributing to charge heterogeneity at the intact level. Here, we demonstrate the power of native MS by direct coupling of SEC and IEX eluent to a time-of-flight mass spectrometer to characterize bevacizumab and NISTmAb. Our studies exemplify the effectiveness of native SEC-MS for characterizing bevacizumab's high molecular weight species at less than 0.3% (based on SEC/UV peak area%) and analyzing the fragment pathway with single amino acid difference for its low molecular weight species at less than 0.05%. Good IEX charge variant separation was obtained with consistent UV and MS profiles. The identity of separated acidic and basic variants were elucidated by native MS at intact level. We successfully differentiated several charge variants including glycoform variants that have not been reported before. In addition, native MS allowed identification of higher molecular weight species as late eluted variants. Overall, the SEC and IEX separation combined with high resolution and high sensitivity native MS, which is significantly different from the traditional RPLC-MS workflows, can be an effective tool that offers valuable insights for us to understand protein therapeutics at native state.

A Spike-Control Approach that Evaluates High Resolution Mass Spectrometry-Based Sequence Variant Analytical Method Performance for Therapeutic Proteins.

An amino acid sequence variant (SV) is defined as an unintended amino acid substitution in protein drug products. SVs contribute to product heterogeneity and can potentially impact product quality, safety, immunogenicity, and efficacy. The analysis of biotherapeutics for SVs is important throughout the product life cycle including clone selection, development of nutrient feed strategies, commercial manufacturing process, and post-approval changes to monitor product quality. The proposed analytical procedure for SVs consists of both qualitative (identification of SVs) and quantitative (quantitation of identified SVs) components. The complexities of SV analysis and the variety of current procedures highlight the need for a systematic approach for assessing the capability of these methodologies to reliably identify and quantitate SVs in biotherapeutics. We described here a "spike-control" approach for evaluating SV analytical procedure. The concept was adopted from quality control samples routinely used in analytical procedure validation. One FDA approved monoclonal antibody (mAb) was spiked with accurate amounts of highly homologous mAb to create mAb samples containing low yet accurate levels of "artificial" SVs. Spike-control samples were denatured, reduced, alkylated, digested and then analyzed by high resolution Orbitrap mass spectrometry. In silico analysis revealed four single amino acid differences between the two mAbs that could be used to represent SVs in the spike-control samples. All four "artificial" SVs were reliably identified by the current workflow. Analytical range (0.01% to 2%), accuracy and precision of identified SVs have also been evaluated. Overall, spike-control sample(s) helped to demonstrate that the SV analytical procedure (i.e., sample preparation, LC separation, mass spectrometry determinations and bioinformatic software) was fit for purpose and suitable for the identification and quantitation of SVs at a pre-determined threshold.

The Interaction of the F-Like Plasmid-Encoded TraN Isoforms with Their Cognate Outer Membrane Receptors.

Horizontal gene transfer via conjugation plays a major role in bacterial evolution. In F-like plasmids, efficient DNA transfer is mediated by close association between donor and recipient bacteria. This process, known as mating pair stabilization (MPS), is mediated by interactions between the plasmid-encoded outer membrane (OM) protein TraN in the donor and chromosomally-encoded OM proteins in the recipient. We have recently reported the existence of 7 TraN sequence types, which are grouped into 4 structural types, that we named TraNα, TraNβ, TraNγ, and TraNδ. Moreover, we have shown specific pairing between TraNα and OmpW, TraNβ and OmpK36 of Klebsiella pneumoniae, TraNγ and OmpA, and TraNδ and OmpF. In this study, we found that, although structurally similar, TraNα encoded by the Salmonella enterica pSLT plasmid (TraNα2) binds OmpW in both Escherichia coli and Citrobacter rodentium, while TraNα encoded by the R100-1 plasmid (TraNα1) only binds OmpW in E. coli. AlphaFold2 predictions suggested that this specificity is mediated by a single amino acid difference in loop 3 of OmpW, which we confirmed experimentally. Moreover, we show that single amino acids insertions into loop 3 of OmpK36 affect TraNβ-mediated conjugation efficiency of the K. pneumoniae resistance plasmid pKpQIL. Lastly, we report that TraNβ can also mediate MPS by binding OmpK35, making it the first TraN variant that can bind more than one OM protein in the recipient. Together, these data show that subtle sequence differences in the OM receptors can impact TraN-mediated conjugation efficiency. IMPORTANCE Conjugation plays a central role in the spread of antimicrobial resistance genes among bacterial pathogens. Efficient conjugation is mediated by formation of mating pairs via a pilus, followed by mating pair stabilization (MPS), mediated by tight interactions between the plasmid-encoded outer membrane protein (OMP) TraN in the donor (of which there are 7 sequence types grouped into the 4 structural isoforms α, β, γ, and δ), and an OMP receptor in the recipient (OmpW, OmpK36, OmpA, and OmpF, respectively). In this study, we found that subtle differences in OmpW and OmpK36 have significant consequences on conjugation efficiency and specificity, highlighting the existence of selective pressure affecting plasmid-host compatibility and the flow of horizontal gene transfer in bacteria.

A set of closely related methyltransferases for site-specific tailoring of anthraquinone pigments

Modification of the polyketide anthraquinone AQ-256 in the entomopathogenic Photorhabdusluminescens involves several O-methylations, but the biosynthetic gene cluster antA-I lacks corresponding tailoring enzymes. We here describe the identification of five putative, highly homologous O-methyltransferases encoded in the genome of P.luminescens. Activity assays invitro and deletion experiments invivo revealed that three of them account for anthraquinone tailoring by producing three monomethylated and two dimethylated species of AQ-256. X-ray structures of all five enzymes indicate high structural and mechanistic similarity. As confirmed by structure-based mutagenesis, a conserved histidine at the active site likely functions as a general base for substrate deprotonation and subsequent methyl transfer in all enzymes. Eight complex structures with AQ-256 as well as mono- and dimethylated derivatives confirm the substrate specificity patterns found invitro and visualize how single amino acid differences in the active-site pockets impact substrate orientation and govern site-specific methylation.

Identification of the molecular determinants of antagonist potency in the allosteric binding pocket of human P2X4.

P2X receptors are a family of ATP-gated cation channels comprising seven subtypes in mammals, which play key roles in nerve transmission, pain sensation and inflammation. The P2X4 receptor in particular has attracted significant interest from pharmaceutical companies due to its physiological roles in neuropathic pain and modulation of vascular tone. A number of potent small-molecule P2X4 receptor antagonists have been developed, including the allosteric P2X4 receptor antagonist BX430, which is approximately 30-fold more potent at human P2X4 compared with the rat isoform. A single amino-acid difference between human and rat P2X4 (I312T), located in an allosteric pocket, has previously been identified as critical for BX430 sensitivity, implying that BX430 binds in this pocket. Using a combination of mutagenesis, functional assay in mammalian cells and in silico docking we confirmed these findings. Induced-fit docking, permitting the sidechains of the amino-acids of P2X4 to move, showed that BX430 could access a deeper portion of the allosteric pocket, and that the sidechain of Lys-298 was important for shaping the cavity. We then performed blind docking of 12 additional P2X4 antagonists into the receptor extracellular domain, finding that many of these compounds favored the same pocket as BX430 from their calculated binding energies. Induced-fit docking of these compounds in the allosteric pocket enabled us to show that antagonists with high potency (IC50 ≤ 100nM) bind deep in the allosteric pocket, disrupting a network of interacting amino acids including Asp-85, Ala-87, Asp-88, and Ala-297, which are vital for transmitting the conformational change following ATP binding to channel gating. Our work confirms the importance of Ile-312 for BX430 sensitivity, demonstrates that the allosteric pocket where BX430 binds is a plausible binding pocket for a series of P2X4 antagonists, and suggests a mode of action for these allosteric antagonists involving disruption of a key structural motif required for the conformational change induced in P2X4 when ATP binds.

A single amino acid difference between archaeal and human type 2 methionine aminopeptidases differentiates their affinity towards ovalicin

In almost all living cells, methionine aminopeptidase (MetAP) co-translationally cleaves the initiator methionine in at least 70% of the newly synthesized polypeptides. MetAPs are typically classified into Type 1 and Type 2. While prokaryotes and archaea contain only either Type 1 or Type 2 MetAPs respectively, eukaryotes contain both types of enzymes. Almost all MetAPs published till date cleave only methionine from the amino terminus of the substrate peptides. Earlier experiments on crude Type 2a MetAP isolated from Pyrococcus furiosus (PfuMetAP2a) cosmid protein library was shown to cleave leucine in addition to methionine. Authors in that study have ruled out the PfuMetAP2a activity against leucine substrates and assumed it to be a background reaction contributed by other contaminating proteases. In the current paper, using the pure recombinant enzyme, we report that indeed activity against leucine is directly carried out by the PfuMetAP2a. In addition, the natural product ovalicin which is a specific covalent inhibitor of Type 2 MetAPs does not show efficient inhibition against the PfuMetAP2a. Bioinformatic analysis suggested that a glycine in eukaryotic MetAP2s (G222 in human MetAP2b) and asparagine (N53 in PfuMetAP2a) in archaeal MetAP2s positioned at the analogous position. N53 side chain forms a hydrogen bond with a conserved histidine (H62) at the entrance of the active site and alters its orientation to accommodate the ovalicin. This slight orientational difference of the H62, reduces affinity of the ovalicin by 300,000-fold when compared with the HsMetAP2b inhibition. This difference in the activity is partly reduced in the case of N53G mutation of the PfuMetAP2a.

Unusual “Asian-origin” 2c to 2b point mutant canine parvovirus (Parvoviridae) and canine astrovirus (Astroviridae) co-infection detected in vaccinated dogs with an outbreak of severe haemorrhagic gastroenteritis with high mortality rate in Hungary

In this study, the aetiological background of an outbreak of severe haemorrhagic gastroenteritis (HGE) in a colony of purebred Jack Russell Terriers vaccinated against CPV-2 in Hungary was investigated. Canine parvovirus 2 (CPV-2, Parvoviridae) and canine astrovirus (CaAstV, Astroviridae) co-infection was identified by viral metagenomics and next-generation sequencing (VM-NGS) methods from a rectal swab of an affected 7-week-old puppy. The complete coding sequence of CPV-2 strain FR1/CPV2-2021-HUN (ON733252) and the complete genome of CaAstV strain FR1/CaAstV-2021-HUN (ON733251) were determined by VM-NGS and PCR methods. Results of sequence and phylogenetic analyses showed that CPV-2 strain FR1/CPV2-2021-HUN was different from the applied vaccine strains and previously identified strains from Hungary but showed high sequence identity (> 99.8%) and close phylogenetic relationship to recently described “Asian-origin” CPV-2c strains from Italy. But, based on the single amino acid difference on position 426 of VP2 (Glu/Asp) between the study strain and the closest relatives, FR1/CPV2-2021-HUN belonged to the 2b antigenic type rather than 2c. The CaAstV strain FR1/CaAstV-2021-HUN showed close relationship with a CaAstV strain identified previously from a diarrhoeic dog in Hungary. Both viruses were continuously detectable by PCR in additional enteric samples, and the CPV-2 could also be detected in several (n = 32) tissue samples from 9 affected deceased puppies. Further comparative studies are necessary to confirm the role of the point mutation causing the change in the antigenic type of this “Asian-origin” CPV-2 and/or the role of CaAstV co-infection in the development and/or severity of (haemorrhagic) gastroenteritis among dogs vaccinated against CPV-2.

Impact of Micropolymorphism Outside the Peptide Binding Groove in the Clinically Relevant Allele HLA-C*14 on T Cell Responses in HIV-1 Infection.

ABSTRACTThere is increasing evidence for the importance of human leukocyte antigen C (HLA-C)-restricted CD8+ T cells in HIV-1 control, but these responses are relatively poorly investigated. The number of HLA-C-restricted HIV-1 epitopes identified is much smaller than those of HLA-A-restricted or HLA-B-restricted ones. Here, we utilized a mass spectrometry-based approach to identify HIV-1 peptides presented by HLA-C*14:03 protective and HLA-C*14:02 nonprotective alleles. We identified 25 8- to 11-mer HLA-I-bound HIV-1 peptides from HIV-1-infected HLA-C*14:02+/14:03+ cells. Analysis of T cell responses to these peptides identified novel 6 T cell epitopes targeted in HIV-1-infected HLA-C*14:02+/14:03+ subjects. Analyses using HLA stabilization assays demonstrated that all 6 epitope peptides exhibited higher binding to and greater cell surface stabilization of HLA-C*14:02 than HLA-C*14:03. T cell response magnitudes were typically higher in HLA-C*14:02+ than HLA-C*14:03+ individuals, with responses to the Pol KM9 and Nef epitopes being significantly higher. The results show that HLA-C*14:02 can elicit stronger T cell responses to HIV-1 than HLA-C*14:03 and suggest that the single amino acid difference between these HLA-C14 subtypes at position 21, outside the peptide-binding groove, indirectly influences the stability of peptide-HLA-C*14 complexes and induction/expansion of HIV-specific T cells. Taken together with a previous finding that KIR2DL2+ NK cells recognized HLA-C*14:03+ HIV-1-infected cells more than HLA-C*14:02+ ones, the present study indicates that these HLA-C*14 subtypes differentially impact HIV-1 control by T cells and NK cells.IMPORTANCE Some human leukocyte antigen (HLA) class I alleles are associated with good clinical outcomes in HIV-1 infection and are called protective HLA alleles. Identification of T cell epitopes restricted by protective HLA alleles can give important insight into virus-immune system interactions and inform design of immune-based prophylactic/therapeutic strategies. Although epitopes restricted by many protective HLA-A/B alleles have been identified, protective HLA-C alleles are relatively understudied. Here, we identified 6 novel T cell epitopes presented by both HLA-C*14:02 (no association with protection) and HLA-C*14:03 (protective) using a mass spectrometry-based immunopeptidome profiling approach. We found that these peptides bound to and stabilized HLA-C*14:02 better than HLA-C*14:03 and observed differences in induction/expansion of epitope-specific T cell responses in HIV-infected HLA-C*14:02+ versus HLA-C*14:03+ individuals. These results enhance understanding of how the microstructural difference at position 21 between these HLA-C*14 subtypes may influence cellular immune responses involved in viral control in HIV-1 infection.

​Cloning and Sequence Analysis of Transferrin Binding Protein Gene A of Field and Vaccine Strains of Pasteurella multocida B:2

Background: Pasteurella multocida is an important bacterial pathogen that causes many major diseases of which haemorrhagic septiciemia (HS) in cattle and buffalo is responsible for catastrophic epizootics in India and South Asia. In India, the disease haemorrhagic septiciemia is considered as the most dreaded bacterial disease. Various host- and pathogen- specific determinants are responsible for disease outcome. Various bacterial virulence genes (tbpA, pfhA, toxA, hgbB, hgbA, nanH, nanB, sodA, sodC, oma87 and ptfA) have been proposed to play a key role in this interaction. Methods: The present study was done to compare the gene and deduced amino acid sequence of transferrin binding protein gene (tbpA) gene of field isolates and vaccine strain of P. multocida B: 2. Result: It was observed that tbpA gene of field and vaccine strains have similar nucleotide sequence except at positions 574 and 620. The sequence of tbpA gene was used for prediction of matured TbpA protein characteristics. The deduced amino acid sequences of 242 amino acids revealed 99% homology with TbpA of P. multocida and with a variety of other TonB-dependent receptor proteins, indicating that it belongs to the family of outer membrane receptors. Deduced amino acid sequence was found to be similar in field and vaccine strains except at 207th amino acid. In field isolates Leucine was there while in vaccine strain Phenyl alanine was found. These both amino acids are hydrophobic in nature so no change in physico-chemical property of TbpA is expected. From this study it is concluded that single amino acid difference between field isolate and vaccine strain might not cause change in its binding and physico-chemical property.

Differences in a Single Extracellular Residue Underlie Adhesive Functions of Two Zebrafish Aqp0s.

Aquaporin 0 (AQP0) is the most abundant lens membrane protein, and loss of function in human and animal models leads to cataract formation. AQP0 has several functions in the lens including water transport and adhesion. Since lens optics rely on strict tissue architecture achieved by compact cell-to-cell adhesion between lens fiber cells, understanding how AQP0 contributes to adhesion would shed light on normal lens physiology and pathophysiology. We show in an in vitro adhesion assay that one of two closely related zebrafish Aqp0s, Aqp0b, has strong auto-adhesive properties while Aqp0a does not. The difference appears to be largely due to a single amino acid difference at residue 110 in the extracellular C-loop, which is T in Aqp0a and N in Aqp0b. Similarly, P110 is the key residue required for adhesion in mammalian AQP0, highlighting the importance of residue 110 in AQP0 cell-to-cell adhesion in vertebrate lenses as well as the divergence of adhesive and water permeability functions in zebrafish duplicates.

Beyond Volume Exclusion-Nanopore-Based Protein Sequencing

The amino acid sequence and structure of proteins largely determine the intermolecular interaction, protein functions and dynamic process. Nanopore technology has been developing as a powerful single-molecule approach, which has been widely used in biomolecule sensing and achieved DNA sequencing, now aiming at protein sequencing. Compared to the four kinds bases of DNA on nanopore sequencing, the twenty natural amino acids own non-uniform charges and small volumes. Hence, the discrimination of all amino acids is one of the main challenges in protein sequencing. Based on volume exclusion sensing mechanism, most efforts have been taken to explore the nanopore which owns a narrow sensing point with short length to match a single amino acid. However, apart from the fast translocation speed, such a sensing point actually has its limitation theoretically, which could not detect volume difference below 0.003 nm3. Hence, new sensing mechanism needs develop for accurate identification of single amino acid based on confined effect interaction. The long (∼ 10 nm) and narrow (∼ 1.0 nm) aerolysin nanopore could identify single nucleotide and single amino acid differences with long duration, indicating an entirely new sensing mechanism. Although the contribution of the tripeptide volume to the current was only about 0.8%, through reasonably construct mutant aerolysin nanopore sensing interface by modifying and regulating the channels interaction such as hydrogen bonds, hydrophilic and hydrophobic interactions, electrostatic interactions, van der Waals force, etc., the event duration is prolonged to 2.41ms in N226Q mutant nanopore and the small volume tripeptide could be recognized with about 33% ionic blockage. This new mechanism based nanopore holds great promise for the advancement of nanopore based protein sequencing.