Conserved Disulfide Bonds Research Articles

New Three-Finger Protein from Starfish Asteria rubens Shares Structure and Pharmacology with Human Brain Neuromodulator Lynx2.

Three-finger proteins (TFPs) are small proteins with characteristic three-finger β-structural fold stabilized by the system of conserved disulfide bonds. These proteins have been found in organisms from different taxonomic groups and perform various important regulatory functions or act as components of snake venoms. Recently, four TFPs (Lystars 1–4) with unknown function were identified in the coelomic fluid proteome of starfish A. rubens. Here we analyzed the genomes of A. rubens and A. planci starfishes and predicted additional five and six proteins containing three-finger domains, respectively. One of them, named Lystar5, is expressed in A. rubens coelomocytes and has sequence homology to the human brain neuromodulator Lynx2. The three-finger structure of Lystar5 close to the structure of Lynx2 was confirmed by NMR. Similar to Lynx2, Lystar5 negatively modulated α4β2 nicotinic acetylcholine receptors (nAChRs) expressed in X. laevis oocytes. Incubation with Lystar5 decreased the expression of acetylcholine esterase and α4 and α7 nAChR subunits in the hippocampal neurons. In summary, for the first time we reported modulator of the cholinergic system in starfish.

Mutations within scavenger receptor cysteine-rich (SRCR) protein domain 5 of porcine CD163 involved in infection with porcine reproductive and respiratory syndrome virus (PRRS).

CD163, a macrophage-specific membrane scavenger receptor, serves as a cellular entry receptor for porcine reproductive and respiratory syndrome virus (PRRSV). The removal of scavenger receptor cysteine-rich (SRCR) domain 5 (SRCR5) of CD163 is sufficient to make transfected cells or genetically modified pigs resistant to PRRSV-1 and PRRSV-2 genotypes, and substitution of SRCR5 with SRCR8 from human CD163-like protein (hCD163L1) confers resistance to PRRSV-1 but not PRRSV-2 isolates. However, the specific regions within the SRCR5 polypeptide involved in PRRSV infection remain largely unknown. In this report, we performed mutational studies in order to identify which regions or amino acid sequences in the SRCR5 domain are critical for PRRSV infection. The approach used in this study was to make proline-arginine (PR) insertions along the SRCR5 polypeptide. Constructs were transfected into HEK293T cells, and then evaluated for infection with PRRSV-2 or PRRSV-1. For PRRSV-2, four PR insertions located after amino acids 8 (PR-9), 47 (PR-48), 54 (PR-55), and 99 (PR-100) had the greatest impact on infection. For PRRSV-1, insertions after amino acids 57 (PR-58) and 99 (PR-100) were critical. Computer simulations based on the crystal structure of SRCR5 showed that the mutations that affected infection localized to a similar region on the surface of the 3-D structure. Specifically, we found two surface patches that are essential for PRRSV infection. PR-58 and PR-55, which were separated by only three amino acids, had reciprocal effects on PRRSV-1 and PRRSV-2. Substitution of Glu-58 with Lys-58 reduced PRRSV-1 infection without affecting PRRSV-2, which partially explains the resistance to PRRSV-1 caused by the SRCR5 replacement with the homolog human SRCR8 previously observed. Finally, resistance to infection was observed following the disruption of any of the four conserved disulfide bonds within SRCR5. In summary, the results confirm that there are distinct differences between PRRSV-1 and PRRSV-2 on recognition of CD163; however, all mutations that affect infection locate on a similar region on the same face of SRCR5.

Mechano-Redox Control of Integrins in Thromboinflammation.

Significance: How mechanical forces and biochemical cues are coupled remains a miracle for many biological processes. Integrins, well-known adhesion receptors, sense changes in mechanical forces and reduction-oxidation reactions (redox) in their environment to mediate their adhesive function. The coupling of mechanical and redox function is a new area of investigation. Disturbance of normal mechanical forces and the redox balance occurs in thromboinflammatory conditions; atherosclerotic plaques create changes to the mechanical forces in the circulation. Diabetes induces redox changes in the circulation by the production of reactive oxygen species and vascular inflammation. Recent Advances: Integrins sense changes in the blood flow shear stress at the level of focal adhesions and respond to flow and traction forces by increased signaling. Talin, the integrin-actin linker, is a traction force sensor and adaptor. Oxidation and reduction of integrin disulfide bonds regulate their adhesion. A conserved disulfide bond in integrin αlpha IIb beta 3 (αIIbβ3) is directly reduced by the thiol oxidoreductase endoplasmic reticulum protein 5 (ERp5) under shear stress. Critical Issues: The coordination of mechano-redox events between the extracellular and intracellular compartments is an active area of investigation. Another fundamental issue is to determine the spatiotemporal arrangement of key regulators of integrins' mechanical and redox interactions. How thromboinflammatory conditions lead to mechanoredox uncoupling is relatively unexplored. Future Directions: Integrated approaches, involving disulfide bond biochemistry, microfluidic assays, and dynamic force spectroscopy, will aid in showing that cell adhesion constitutes a crossroad of mechano- and redox biology, within the same molecule, the integrin. Antioxid. Redox Signal. 37, 1072-1093.

Crystal structure of a human MUC16 SEA domain reveals insight into the nature of the CA125 tumor marker

MUC16 is a membrane bound glycoprotein involved in the progression and metastasis of pancreatic and ovarian cancer. The protein is shed into the serum and the resulting cancer antigen 125 (CA125) can be detected by immunoassays. The CA125 epitope is used for monitoring ovarian cancer treatment progression, and has emerged as a potential target for antibody mediated immunotherapy. The extracellular tandem repeat domain of the protein is composed of repeating segments of heavily glycosylated sequence intermixed with homologous SEA (Sperm protein, Enterokinase and Agrin) domains. Here we report the purification and the first X-ray structure of a human MUC16 SEA domain. The structure was solved by molecular replacement using a Rosetta generated structure as a search model. The SEA domain reacted with three different MUC16 therapeutic antibodies, confirming that the CA125 epitope is localized to the SEA domain. The structure revealed a canonical ferredoxin-like fold, and contained a conserved disulfide bond. Analysis of the relative solvent accessibility of side chains within the SEA domain clarified the assignment of N-linked and O-linked glycosylation sites within the domain. A model of the glycosylated SEA domain revealed two major accessible faces, which likely represent the binding sites of CA125 specific antibodies. The results presented here will serve to accelerate future work to understand the functional role of MUC16 SEA domains and antibody recognition of the CA125 epitope.

Disulfide Bonds Play a Critical Role in the Structure and Function of the Receptor-binding Domain of the SARS-CoV-2 Spike Antigen

The current coronavirus pandemic is exerting a tremendously detrimental impact on global health. The Spike proteins of coronaviruses, responsible for cell receptor binding and viral internalization, possess multiple and frequently conserved disulfide bonds raising the question about their role in these proteins. Here, we present a detailed structural and functional investigation of the disulfide bonds of the SARS-CoV-2 Spike receptor-binding domain (RBD). Molecular dynamics simulations of the RBD predict increased flexibility of the surface loops when the four disulfide bonds of the domain are reduced. This flexibility is particularly prominent for the disulfide bond-containing surface loop (residues 456–490) that participates in the formation of the interaction surface with the Spike cell receptor ACE2. In vitro, disulfide bond reducing agents affect the RBD secondary structure, lower its melting temperature from 52 °C to 36–39 °C and decrease its binding affinity to ACE2 by two orders of magnitude at 37 °C. Consistent with these in vitro findings, the reducing agents tris(2-carboxyethyl)phosphine (TCEP) and dithiothreitol (DTT) were able to inhibit viral replication at low millimolar levels in cell-based assays. Our research demonstrates the mechanism by which the disulfide bonds contribute to the molecular structure of the RBD of the Spike protein, allowing the RBD to execute its viral function.

Four alternative splicing transcripts of intracellular copper/zinc superoxide dismutase 1 in Oxya chinensis

In this study, we obtained four alternative splicing transcripts of intracellular copper/zinc superoxide dismutase 1 (icCuZnSOD1) in Oxya chinensis. OcicCuZnSOD1a has all common characteristics of CuZnSOD family and is a canonical CuZnSOD. OcicCuZnSOD1b is missing a Zn binding site. OcicCuZnSOD1c lacks Zn ion and is a Cu-only SOD. OcicCuZnSOD1d is missing a CuZnSOD conserved sequence and lacks the E-loop, a conserved disulfide bond, and an active site arginine. OcicCuZnSOD1a was the most heat-resistant and OcicCuZnSOD1c was the most unstable at high temperatures above 55 °C. They were stable at a wide pH range, especially in alkaline conditions. The four variants expressed at the throughout developmental stages and had various tissue expression patterns. OcicCuZnSOD1a and OcicCuZnSOD1d were significantly induced by 8.79 mM CuCl2 and OcicCuZnSOD1b was significantly up-regulated by 14.67 mM CuCl2. OcicCuZnSOD1a was significantly inhibited by 19.13 mM ZnSO4 while OcicCuZnSOD1d were significantly induced by 22.61 mM ZnSO4. Disc diffusion assay showed that the four isoforms of OcicCuZnSOD1 made the killing zones smaller surrounding the CdCl2-soaked filter discs. However, the reduction ratios of OcicCuZnSOD1a were the highest. These results implied that the four transcripts played roles in defense against CdCl2-induced oxidative stress while OcicCuZnSOD1a had stronger antioxidant capacity.

Effects of removing a highly conserved disulfide bond in ubiquitin-associated domain of human HOIP on biochemical characteristics

Disulfide bond formed between the cysteine pairs plays a key role in maintaining the integrity of the protein structure and function. The ubiquitin-associated (UBA) domain of human HOIP contains three cysteine residues, Cys504, Cys551, and Cys572. Disulfide bonds formed by Cys504 and Cys551 residues are highly conserved, but the effect of disulfide bonds on the biochemical characteristics of UBA has not been elucidated. In addition, due to the presence of isolated Cys572, inactive inclusion bodies may be formed during protein expression or trigger protein aggregation during protein purification. In this study, the co-expression of SUMO fusion protein combined with SUMO protease (ULP enzyme) in Escherichia coli was successfully applied to improve the soluble expression of UBA domain. Introduced three mutants (UBAC551A, UBAC572A and UBAC551,572A) determined the effects of disulfide bonds on the biochemical characteristics of UBA. Circular dichroism and analytical size exclusion chromatography results showed that the target proteins obtained by co-expression could be folded correctly and had biological activity. Both thermal-induced and urea-induced results demonstrated that the elimination of disulfide bonds would significantly reduce the stability of UBA. Fluorescence spectroscopy result showed that the elimination of disulfide bonds slightly increases the binding affinity of UBA to ligands. In summary, soluble, stable and active UBA domain and its mutants were prepared by co-expression system, which will further contribute to the structural and functional research of UBA.

PINIR: a comprehensive information resource for Pin-II type protease inhibitors

BackgroundSerine protease inhibitors belonging to the Potato type-II Inhibitor family Protease Inhibitors (Pin-II type PIs) are essential plant defense molecules. They are characterized by multiple inhibitory repeat domains, conserved disulfide bond pattern, and a tripeptide reactive center loop. These features of Pin-II type PIs make them potential molecules for protein engineering and designing inhibitors for agricultural and therapeutic applications. However, the diversity in these PIs remains unexplored due to the lack of annotated protein sequences and their functional attributes in the available databases.ResultsWe have developed a database, PINIR (Pin-II type PIs Information Resource), by systematic collection and manual annotation of 415 Pin-II type PI protein sequences. For each PI, the number and position for signature sequences are specified: 695 domains, 75 linkers, 63 reactive center loops, and 10 disulfide bond patterns are identified and mapped. Database analysis revealed novel subcategories of PIs, species-correlated occurrence of inhibitory domains, reactive center loops, and disulfide bond patterns. By analyzing linker regions, we predict that alternative processing at linker regions could generate PI variants in the Solanaceae family.ConclusionPINIR (https://pinir.ncl.res.in) provides a web interface for browsing and analyzing the protein sequences of Pin-II type PIs. Information about signature sequences, spatio-temporal expression, biochemical properties, gene sequences, and literature references are provided. Analysis of PINIR depicts conserved species-specific features of Pin-II type PI protein sequences. Diversity in the sequence of inhibitory domains and reactive loops directs potential applications to engineer Pin-II type PIs. The PINIR database will serve as a comprehensive information resource for further research into Pin-II type PIs.

Different pharmacological properties between scorpion toxin BmKcug2 and its degraded analogs highlight the diversity of K+ channel blockers from thermally processed scorpions

Novel degraded potassium channel-modulatory peptides were recently found in thermally processed scorpions, but their pharmacological properties remain unclear. Here, we identified a full-length scorpion toxin (i.e., BmKcug2) and its four truncated analogs (i.e., BmKcug2-P1, BmKcug2-P2, BmKcug2-P3 and BmKcug2-P4) with three conserved disulfide bonds in processed scorpion medicinal material by mass spectrometry. The pharmacological experiments revealed that the recombinant BmKcug2 and BmKcug2-P1 could selectively inhibit the human Kv1.2 and human Kv1.3 potassium channels, while the other three analogs showed a much weaker inhibitory effect on potassium channels. BmKcug2 inhibited hKv1.2 and hKv1.3 channels, with IC50 values of 45.6 ± 5.8 nM and 215.2 ± 39.7 nM, respectively, and BmKcug2-P1 inhibited hKv1.2 and hKv1.3, with IC50 values of 89.9 ± 9.6 nM and 1142.4 ± 64.5 nM, respectively. The chromatographic analysis and pharmacological properties of BmKcug2 and BmKcug2-P1 boiled in water for different times further strongly supported their good thermal stability. Structural and functional dissection indicated that one amino acid, i.e., Tyr36, determined the differential affinities of BmKcug2 and four BmKcug2 analogs. Altogether, this research investigated the different pharmacological properties of BmKcug2 and its truncated analogs, and the findings highlighted the diversity of K+ channel blockers from various scorpion species through thermal processing.

Unusual quaternary structure of a homodimeric synergistic-type toxin from mamba snake venom defines its molecular evolution.

Snake venoms are complex mixtures of enzymes and nonenzymatic proteins that have evolved to immobilize and kill prey animals or deter predators. Among them, three-finger toxins (3FTxs) belong to the largest superfamily of nonenzymatic proteins. They share a common structure of three β-stranded loops extending like fingers from a central core containing all four conserved disulfide bonds. Most 3FTxs are monomers and through subtle changes in their amino acid sequences, they interact with different receptors, ion channels and enzymes to exhibit a wide variety of biological effects. The 3FTxs have further expanded their pharmacological space through covalent or noncovalent dimerization. Synergistic-type toxins (SynTxs) isolated from the deadly mamba venoms, although nontoxic, have been known to enhance the toxicity of other venom proteins. However, the details of three-dimensional structure and molecular mechanism of activity of this unusual class of 3FTxs are unclear. We determined the first three-dimensional structure of a SynTx isolated from Dendroaspis jamesoni jamesoni (Jameson's mamba) venom. The SynTx forms a unique homodimer that is held together by an interchain disulfide bond. The dimeric interface is elaborate and encompasses loops II and III. In addition to the inter-subunit disulfide bond, the hydrogen bonds and hydrophobic interactions between the monomers contribute to the dimer formation. Besides, two sulfate ions that mediate interactions between the monomers. This unique quaternary structure is evolved through noncovalent homodimers such as κ-bungarotoxins. This novel dimerization further enhances the diversity in structure and function of 3FTxs.

Steered molecular dynamic simulations reveal Marfan syndrome mutations disrupt fibrillin-1 cbEGF domain mechanosensitive calcium binding

Marfan syndrome (MFS) is a highly variable genetic connective tissue disorder caused by mutations in the calcium binding extracellular matrix glycoprotein fibrillin-1. Patients with the most severe form of MFS (neonatal MFS; nMFS) tend to have mutations that cluster in an internal region of fibrillin-1 called the neonatal region. This region is predominantly composed of eight calcium-binding epidermal growth factor-like (cbEGF) domains, each of which binds one calcium ion and is stabilized by three highly conserved disulfide bonds. Crucially, calcium plays a fundamental role in stabilizing cbEGF domains. Perturbed calcium binding caused by cbEGF domain mutations is thus thought to be a central driver of MFS pathophysiology. Using steered molecular dynamics (SMD) simulations, we demonstrate that cbEGF domain calcium binding decreases under mechanical stress (i.e. cbEGF domains are mechanosensitive). We further demonstrate the disulfide bonds in cbEGF domains uniquely orchestrate protein unfolding by showing that MFS disulfide bond mutations markedly disrupt normal mechanosensitive calcium binding dynamics. These results point to a potential mechanosensitive mechanism for fibrillin-1 in regulating extracellular transforming growth factor beta (TGFB) bioavailability and microfibril integrity. Such mechanosensitive “smart” features may represent novel mechanisms for mechanical hemostasis regulation in extracellular matrix that are pathologically activated in MFS.

Differential Roles of Extracellular Histidine Residues of GPR68 for Proton-Sensing and Allosteric Modulation by Divalent Metal Ions.

GPR68, an orphan G-protein coupled receptor, senses protons, couples to multiple G-proteins, and is also activated or inhibited by divalent metal ions. It has seven extracellular histidine residues, although it is not clear how these histidine residues play a role in both proton-sensing and metal ion modulation. Here we demonstrate that divalent metal ions are allosteric modulators that can activate or inhibit proton activity in a concentration- and pH-dependent manner. We then show that single histidine mutants have differential and varying degrees of effects on proton-sensing and metal ion modulation. Some histidine residues play dual roles in proton-sensing and metal ion modulation, while others are important in one or the other but not both. Two extracellular disulfide bonds are predicted to constrain histidine residues to be spatially close to each other. Combining histidine mutations leads to reduced proton activity and resistance to metal ion modulation, while breaking the less conserved disulfide bond results in a more severe reduction in proton-sensing over metal modulation. The small-molecule positive allosteric modulators (PAMs) ogerin and lorazepam are not affected by these mutations and remain active at mutants with severely reduced proton activity or are resistant to metal ion modulation. These results suggest GPR68 possesses two independent allosteric modulation systems, one through interaction with divalent metal ions at the extracellular surface and another through small-molecule PAMs in the transmembrane domains. A new GPR68 model is developed to accommodate the findings which could serve as a template for further studies and ligand discovery by virtual ligand docking.

The role of disulfide bonds in a Solanum tuberosum saposin-like protein investigated using molecular dynamics.

The Solanum tuberosum plant specific insert (StPSI) has a defensive role in potato plants, with the requirements of acidic pH and anionic lipids. The StPSI contains a set of three highly conserved disulfide bonds that bridge the protein's helical domains. Removal of these bonds leads to enhanced membrane interactions. This work examined the effects of their sequential removal, both individually and in combination, using all-atom molecular dynamics to elucidate the role of disulfide linkages in maintaining overall protein tertiary structure. The tertiary structure was found to remain stable at both acidic (active) and neutral (inactive) pH despite the removal of disulfide linkages. The findings include how the dimer structure is stabilized and the impact on secondary structure on a residue-basis as a function of disulfide bond removal. The StPSI possesses an extensive network of inter-monomer hydrophobic interactions and intra-monomer hydrogen bonds, which is likely the key to the stability of the StPSI by stabilizing local secondary structure and the tertiary saposin-fold, leading to a robust association between monomers, regardless of the disulfide bond state. Removal of disulfide bonds did not significantly impact secondary structure, nor lead to quaternary structural changes. Instead, disulfide bond removal induces regions of amino acids with relatively higher or lower variation in secondary structure, relative to when all the disulfide bonds are intact. Although disulfide bonds are not required to preserve overall secondary structure, they may have an important role in maintaining a less plastic structure within plant cells in order to regulate membrane affinity or targeting.

Structural basis of SARS-CoV-2 spike protein induced by ACE2.

MotivationThe recent emergence of the novel SARS-coronavirus 2 (SARS-CoV-2) and its international spread pose a global health emergency. The spike (S) glycoprotein binds ACE2 and promotes SARS-CoV-2 entry into host cells. The trimeric S protein binds the receptor using the receptor-binding domain (RBD) causing conformational changes in S protein that allow priming by host cell proteases. Unraveling the dynamic structural features used by SARS-CoV-2 for entry might provide insights into viral transmission and reveal novel therapeutic targets. Using structures determined by X-ray crystallography and cryo-EM, we performed structural analysis and atomic comparisons of the different conformational states adopted by the SARS-CoV-2-RBD.ResultsHere, we determined the key structural components induced by the receptor and characterized their intramolecular interactions. We show that κ-helix (polyproline-II) is a predominant structure in the binding interface and in facilitating the conversion to the active form of the S protein. We demonstrate a series of conversions between switch-like κ-helix and β-strand, and conformational variations in a set of short α-helices which affect the hinge region. These conformational changes lead to an alternating pattern in conserved disulfide bond configurations positioned at the hinge, indicating a possible disulfide exchange, an important allosteric switch implicated in viral entry of various viruses, including HIV and murine coronavirus. The structural information presented herein enables to inspect and understand the important dynamic features of SARS-CoV-2-RBD and propose a novel potential therapeutic strategy to block viral entry. Overall, this study provides guidance for the design and optimization of structure-based intervention strategies that target SARS-CoV-2.AvailabilityWe have implemented the proposed methods in an R package freely available at https://github.com/Grantlab/bio3dSupplementary informationbtaa744_Supplementary-dataClick here for additional data file.Supplementary data are available at Bioinformatics online.

Synthesis, Structure, and Activity of the Antifungal Plant Defensin PvD1.

Available treatments for invasive fungal infections have limitations, including toxicity and the emergence of resistant strains. Therefore, there is an urgent need for alternative solutions. Because of their unique mode of action and high selectivity, plant defensins (PDs) are worthy therapeutic candidates. Chemical synthesis remains a preferred method for the production of many peptide-based therapeutics. Given the relatively long sequence of PDs, as well as their complicated posttranslational modifications, the synthetic route can be considered challenging. Here, we describe a total synthesis of PvD1, the defensin from the common bean Phaseolus vulgaris. Analytical, structural, and functional characterization revealed that both natural and synthetic peptides fold into a canonical CSαβ motif stabilized by conserved disulfide bonds. Moreover, synthetic PvD1 retained the biological activity against four different Candida species and showed no toxicity in vivo. Adding the high resistance of synthetic PvD1 to proteolytic degradation, we claim that conditions are now met to consider PDs druggable biologicals.

Expression, purification, and glycosylation of epidermal growth factor-like repeat 27 from mouse NOTCH1

Notch receptors have large extracellular domains containing up to 36 tandem epidermal growth factor-like (EGF) repeats, which facilitate cell signaling by binding ligands on neighboring cells. Notch receptors play major roles in a variety of developmental processes by controlling cell fate decisions. Each EGF repeat consists of about 40 amino acids with 3 conserved disulfide bonds. Many of the EGF repeats are modified by O-linked fucose glycans, and more than half have calcium-binding sites, but the sequences of the EGF repeats vary giving distinct roles to each repeat. EGF repeat 27 (EGF27) from mouse NOTCH1 is modified with O-fucose and is 1 of 7 repeats that is differentially modified by specific Fringe enzymes, which are known to regulate NOTCH1 activation and ligand binding. To better understand the role of EGF27 in NOTCH1 function and regulation, the 3-dimensional structures of EGF27 and its glycoforms are being pursued. E. coli cells were used to produce EGF27 in sufficient quantities for nuclear magnetic resonance analysis. Previous attempts to express the repeat alone and refold the repeat under a steady redox environment were unsuccessful due to low yields and extensive mixed-disulfide bond cross-linking. A new strategy using a cleavable maltose binding protein fusion tag increased the solubility and yield of EGF27. With the fusion tag, EGF27 was refolded to produce the correct disulfide bond arrangement, which was verified enzymatically with the glycosyltransferases, Protein O-fucosyltransferase 1 (POFUT1) and Lunatic Fringe (LFNG).

Characterisation of a Novel A-Superfamily Conotoxin.

Conopeptides belonging to the A-superfamily from the venomous molluscs, Conus, are typically α-conotoxins. The α-conotoxins are of interest as therapeutic leads and pharmacological tools due to their selectivity and potency at nicotinic acetylcholine receptor (nAChR) subtypes. Structurally, the α-conotoxins have a consensus fold containing two conserved disulfide bonds that define the two-loop framework and brace a helical region. Here we report on a novel α-conotoxin Pl168, identified from the transcriptome of Conus planorbis, which has an unusual 4/8 loop framework. Unexpectedly, NMR determination of its three-dimensional structure reveals a new structural type of A-superfamily conotoxins with a different disulfide-stabilized fold, despite containing the conserved cysteine framework and disulfide connectivity of classical α-conotoxins. The peptide did not demonstrate activity on a range of nAChRs, or Ca2+ and Na+ channels suggesting that it might represent a new pharmacological class of conotoxins.

Recessive missense LAMP3 variant associated with defect in lamellar body biogenesis and fatal neonatal interstitial lung disease in dogs.

Neonatal interstitial lung diseases due to abnormal surfactant biogenesis are rare in humans and have never been reported as a spontaneous disorder in animals. We describe here a novel lung disorder in Airedale Terrier (AT) dogs with clinical symptoms and pathology similar to the most severe neonatal forms of human surfactant deficiency. Lethal hypoxic respiratory distress and failure occurred within the first days or weeks of life in the affected puppies. Transmission electron microscopy of the affected lungs revealed maturation arrest in the formation of lamellar bodies (LBs) in the alveolar epithelial type II (AECII) cells. The secretory organelles were small and contained fewer lamellae, often in combination with small vesicles surrounded by an occasionally disrupted common limiting membrane. A combined approach of genome-wide association study and whole exome sequencing identified a recessive variant, c.1159G>A, p.(E387K), in LAMP3, a limiting membrane protein of the cytoplasmic surfactant organelles in AECII cells. The substitution resides in the LAMP domain adjacent to a conserved disulfide bond. In summary, this study describes a novel interstitial lung disease in dogs, identifies a new candidate gene for human surfactant dysfunction and brings important insights into the essential role of LAMP3 in the process of the LB formation.

Identification and characterization of hirudin-HN, a new thrombin inhibitor, from the salivary glands of Hirudo nipponia.

Transcriptome sequencing data (6.5 Gb) of the salivary glands of the haematophagous leech Hirudo nipponia was obtained by using the BGIseq-500 platform. After identification and analysis, one transcript (Unigene5370) was annotated to hirudin HV3 from Hirudo medicinalis with an e-value of 1e-29 and was named hirudin-HN. This transcript was a new thrombin inhibitor gene belonging to the proteinase inhibitor I14 (hirudin) family. Hirudin-HN, with a 270-bp cDNA, encodes an 89-aa protein containing a 20-aa signal peptide. The mature hirudin-HN protein contains the typical structural characteristics of hirudin, e.g., three conserved disulfide bonds and the PKP and DFxxIP motifs. Proteins (Hir and M-Hir) were obtained via prokaryotic expression, and the mature hirudin-HN protein was shown to have anticoagulant activity and thrombin affinity by using the chromogenic substrate S2238 and surface plasmon resonance (SPR) interaction analysis, respectively. The N-terminal structure of the mature hirudin-HN protein was shown to be important for anticoagulant activity by comparing the activity and thrombin affinity of Hir and M-Hir. The abundances of Hirudin-HN mRNA and protein were higher in the salivary glands of starving animals than in those of feeding or fed leeches. These results provided a foundation for further study on the structure-function relationship of hirudin-HN with thrombin.

Preliminary analysis of PGRP-LC gene and structure characteristics in bumblebees

PGRP-LC is a significant pattern recognition receptor of the insect innate immune system that can recognize peptidoglycans and activate immune signaling pathways regulating the expression and release of antimicrobial peptides against infection. We for the first time analyzed the phylogenetic tree, purification and structure of bumblebee PGRP-LC. The results showed high conservation of bumblebee PGRP-LC among the 16 bumblebee species, and further phylogenetic analysis showed that the PGRP-LC phylogeny of different subgenera (Subterraneobombus, Megabombus, Melanobombus, Bombus) is consistent with that of the COI gene. Additionally, the phylogeny of PGRP-LCs among Bombus, Apis and the solitary bee Megachile rotundata coincides with the sociality evolution of bees. Moreover, bumblebee PGRP-LC (Bl-PGRP-LC) shares the Drosophila PGRP-LCx and PGRP-LCa topology, retaining conserved disulfide bonds and 80% binding residues involved in the interaction between TCT and PGRP-LCx. Therefore, Bl-PGRP-LC might share some similar binding characteristics with Drosophila PGRP-LCx. In addition, Bl-PGRP-LC has shorter β5 and β1 sheets, longer β2, β3, and β4 sheets and a shallow binding groove. To determine the characteristics of Bl-PGRP-LC, high-purity PGRP-LC inclusion bodies, soluble GST-tag Bl-PGRP-LC fusion protein and soluble pure Bl-PGRP-LC were obtained in vitro. The results will be helpful for further study of the function and structure of Bl-PGRP-LC.