Systemically administered wound-homing peptide accelerates wound healing by modulating syndecan-4 function.

CAR (CARSKNKDC) is a wound-homing peptide that recognises angiogenic neovessels. Here we discover that systemically administered CAR peptide has inherent ability to promote wound healing: wounds close and re-epithelialise faster in CAR-treated male mice. CAR promotes keratinocyte migration in vitro. The heparan sulfate proteoglycan syndecan-4 regulates cell migration and is crucial for wound healing. We report that syndecan-4 expression is restricted to epidermis and blood vessels in mice skin wounds. Syndecan-4 regulates binding and internalisation of CAR peptide and CAR-mediated cytoskeletal remodelling. CAR induces syndecan-4-dependent activation of the small GTPase ARF6, via the guanine nucleotide exchange factor cytohesin-2, and promotes syndecan-4-, ARF6- and Cytohesin-2-mediated keratinocyte migration. Finally, we show that genetic ablation of syndecan-4 in male mice eliminates CAR-induced wound re-epithelialisation following systemic administration. We propose that CAR peptide activates syndecan-4 functions to selectively promote re-epithelialisation. Thus, CAR peptide provides a therapeutic approach to enhance wound healing in mice; systemic, yet target organ- and cell-specific.

A reverse vaccinology approach on transmembrane carbonic anhydrases from Plasmodium species as vaccine candidates for malaria prevention.

BACKGROUND: Malaria is a significant parasitic infection, and human infection is mediated by mosquito (Anopheles) biting and subsequent transmission of protozoa (Plasmodium) to the blood. Carbonic anhydrases (CAs) are known to be highly expressed in the midgut and ectoperitrophic space of Anopheles gambiae. Transmembrane CAs (tmCAs) in Plasmodium may be potential vaccine candidates for the control and prevention of malaria. METHODS: In this study, two groups of transmembrane CAs, including α-CAs and one group of η-CAs were analysed by immunoinformatics and computational biology methods, such as predictions on transmembrane localization of CAs from Plasmodium spp., affinity and stability of different HLA classes, antigenicity of tmCA peptides, epitope and proteasomal cleavage of Plasmodium tmCAs, accessibility of Plasmodium tmCAs MHC-ligands, allergenicity of Plasmodium tmCAs, disulfide-bond of Plasmodium tmCAs, B cell epitopes of Plasmodium tmCAs, and Cell type-specific expression of Plasmodium CAs. RESULTS: Two groups of α-CAs and one group of η-CAs in Plasmodium spp. were identified to contain tmCA sequences, having high affinity towards MHCs, high stability, and strong antigenicity. All putative tmCAs were predicted to contain sequences for proteasomal cleavage in antigen presenting cells (APCs). CONCLUSIONS: The predicted results revealed that tmCAs from Plasmodium spp. can be potential targets for vaccination against malaria.

The production and biochemical characterization of α-carbonic anhydrase from Lactobacillus rhamnosus GG.

We report the production and biochemical characterization of an α-carbonic anhydrase (LrhCA) from gram-positive probiotic bacteria Lactobacillus rhamnosus GG. CAs form a family of metalloenzymes that catalyze hydration of CO2/interconversion between CO2 and water to bicarbonate ions and protons. They are divided into eight independent gene families (α, ß, γ, δ, ζ, η, θ, and ι). Interestingly, many pathogens have been identified with only ß- and/or γ-CAs, which can be targeted with CA-specific inhibitors (CAIs) acting as anti-pathogen drugs. Since it is important to study the potential off-target effects of CAIs for both the human body and its commensal bacteria, we took L. rhamnosus GG as our study subject. To date, only a single α-CA has been identified in L. rhamnosus GG, which was successfully produced and biochemically characterized. LrhCA showed moderate catalytic activity with the following kinetic parameters: kcat of 9.86 × 105 s-1 and kcat/KM of 1.41 × 107 s-1 M-1. Moderate inhibition was established with 11 of the 39 studied sulfonamides. The best inhibitors were 5-((4-aminophenyl)sulfonamido)-1,3,4-thiadiazole-2-sulfonamide, 4-(2-hydroxymethyl-4-nitrophenyl-sulfonamidoethyl)-benzenesulfonamide, and benzolamide with Ki values of 319 nM, 378 nM, and 387 nM, respectively. The other compounds showed weaker inhibitory effects. The Ki of acetazolamide, a classical CAI, was 733 nM. In vitro experiments with acetazolamide showed that it had no significant effect on cell growth in L. rhamnosus GG culture. Several sulfonamides, including acetazolamide, are in use as clinical drugs, making their inhibition data highly relevant to avoid any adverse off-target effects towards the human body and its probiotic organisms. KEY POINTS: • The α-carbonic anhydrase from Lactobacillus rhamnosus GG (LrhCA) is 24.3 kDa. • LrhCA has significant catalytic activity with a kcat of 9.9 × 105 s-1. • Acetazolamide resulted in a marginal inhibitory effect on cell growth.

Genotyping determination of Acanthamoeba strains: an original study and a systematic review in Iran.

This study aimed to detect the presence of Acanthamoeba spp. in different water resources of Zahedan, southeast of Iran, and also systematically reviewed all publications regarding Acanthamoeba in Iran (2005-2018). Fifty water samples were collected from different water resources in Zahedan. The positive samples were identified morphologically and subjected to polymerase chain reaction (PCR) using fragments of 18S rRNA. In the systematic review, data collection using particular terms was carried out using the following electronic databases including Science Direct, ISI Web of Science, MEDLINE, EBSCO, Scopus, and Google Scholar. A total of 17 (34%) samples were positive for Acanthamoeba spp., and nucleotide sequencing indicated that 15 samples (88.23%) belonged to the T4 genotype and the rest belonged to the T5 genotype. A total of 39 studies reported genotyping of Acanthamoeba spp. from various geographical areas of Iran and revealed that T4 (35 studies), T5 (19 studies), T3 (11 studies), T11 (8 studies), and T2 (6 studies) genotypes were the most prevalent in Iran. The T4 genotype of Acanthamoeba is a prevalent free-living amoeba and widely distributed not only in Zahedan but also in other provinces of Iran. Phylogenetic analysis reveals that A. castellanii and A. griffini predominantly colocalize with the T4 genotype.

Involvement of ß-Carbonic Anhydrase Genes in Bacterial Genomic Islands and Their Horizontal Transfer to Protists.

Genomic islands (GIs) are a type of mobile genetic element (MGE) that are present in bacterial chromosomes. They consist of a cluster of genes that produce proteins that contribute to a variety of functions, including, but not limited to, the regulation of cell metabolism, antimicrobial resistance, pathogenicity, virulence, and resistance to heavy metals. The genes carried in MGEs can be used as a trait reservoir in times of adversity. Transfer of genes using MGEs, occurring outside reproduction, is called horizontal gene transfer (HGT). Previous data have shown that numerous HGT events have occurred through endosymbiosis between prokaryotes and eukaryotes. ß-Carbonic anhydrase (ß-CA) enzymes play a critical role in the biochemical pathways of many prokaryotes and eukaryotes. We previously suggested the horizontal transfer of ß-CA genes from plasmids of some prokaryotic endosymbionts to their protozoan hosts. In this study, we set out to identify ß-CA genes that might have been transferred between prokaryotic and protist species through HGT in GIs. Therefore, we investigated prokaryotic chromosomes containing ß-CA-encoding GIs and utilized multiple bioinformatics tools to reveal the distinct movements of ß-CA genes among a wide variety of organisms. Our results identify the presence of ß-CA genes in GIs of several medically and industrially relevant bacterial species, and phylogenetic analyses reveal multiple cases of likely horizontal transfer of ß-CA genes from GIs of ancestral prokaryotes to protists.IMPORTANCE The evolutionary process is mediated by mobile genetic elements (MGEs), such as genomic islands (GIs). A gene or set of genes in the GIs is exchanged between and within various species through horizontal gene transfer (HGT). Based on the crucial role that GIs can play in bacterial survival and proliferation, they were introduced as environment- and pathogen-associated factors. Carbonic anhydrases (CAs) are involved in many critical biochemical pathways, such as the regulation of pH homeostasis and electrolyte transfer. Among the six evolutionary families of CAs, ß-CA gene sequences are present in many bacterial species, which can be horizontally transferred to protists during evolution. This study shows the involvement of bacterial ß-CA gene sequences in the GIs and suggests their horizontal transfer to protists during evolution.

Chromatin accessibility is associated with CRISPR-Cas9 efficiency in the zebrafish (Danio rerio).

CRISPR-Cas9 technology is routinely applied for targeted mutagenesis in model organisms and cell lines. Recent studies indicate that the prokaryotic CRISPR-Cas9 system is affected by eukaryotic chromatin structures. Here, we show that the likelihood of successful mutagenesis correlates with transcript levels during early development in zebrafish (Danio rerio) embryos. In an experimental setting, we found that guide RNAs differ in their onset of mutagenesis activity in vivo. Furthermore, some guide RNAs with high in vitro activity possessed poor mutagenesis activity in vivo, suggesting the presence of factors that limit the mutagenesis in vivo. Using open access datasets generated from early developmental stages of the zebrafish, and guide RNAs selected from the CRISPRz database, we provide further evidence for an association between gene expression during early development and the success of CRISPR-Cas9 mutagenesis in zebrafish embryos. In order to further inspect the effect of chromatin on CRISPR-Cas9 mutagenesis, we analysed the relationship of selected chromatin features on CRISPR-Cas9 mutagenesis efficiency using publicly available data from zebrafish embryos. We found a correlation between chromatin openness and the efficiency of CRISPR-Cas9 mutagenesis. These results indicate that CRISPR-Cas9 mutagenesis is influenced by chromatin accessibility in zebrafish embryos.

Interaction between ROR1 and MuSK activation complex in myogenic cells.

The ROR family of receptor tyrosine kinases, ROR1 and ROR2, is known to play an important role during skeletal muscle regeneration. ROR1 has a critical role in regulating satellite cell (SC) proliferation during muscle regeneration, and proinflammatory cytokines such as TNF-α and IL-1ß can induce expression of ROR1 in myogenic cells via NF-κB activation. While searching for ROR1-interacting proteins in myogenic cells, we identified MuSK as a ROR1-binding protein. MuSK interacts with and phosphorylates ROR1 at the cytoplasmic proline-rich domain. ROR1 also interacts with the MuSK activator Dok-7 independently of MuSK interaction. Collectively, our results identified ROR1 as a new interacting partner for MuSK and Dok-7, which may have an important role in myogenic cell signaling.

Identification and characterization of a novel zebrafish (Danio rerio) pentraxin-carbonic anhydrase.

BACKGROUND: Carbonic anhydrases (CAs) are ubiquitous, essential enzymes which catalyze the conversion of carbon dioxide and water to bicarbonate and H+ ions. Vertebrate genomes generally contain gene loci for 15-21 different CA isoforms, three of which are enzymatically inactive. CA VI is the only secretory protein of the enzymatically active isoforms. We discovered that non-mammalian CA VI contains a C-terminal pentraxin (PTX) domain, a novel combination for both CAs and PTXs. METHODS: We isolated and sequenced zebrafish (Danio rerio) CA VI cDNA, complete with the sequence coding for the PTX domain, and produced the recombinant CA VI-PTX protein. Enzymatic activity and kinetic parameters were measured with a stopped-flow instrument. Mass spectrometry, analytical gel filtration and dynamic light scattering were used for biophysical characterization. Sequence analyses and Bayesian phylogenetics were used in generating hypotheses of protein structure and CA VI gene evolution. A CA VI-PTX antiserum was produced, and the expression of CA VI protein was studied by immunohistochemistry. A knock-down zebrafish model was constructed, and larvae were observed up to five days post-fertilization (dpf). The expression of ca6 mRNA was quantitated by qRT-PCR in different developmental times in morphant and wild-type larvae and in different adult fish tissues. Finally, the swimming behavior of the morphant fish was compared to that of wild-type fish. RESULTS: The recombinant enzyme has a very high carbonate dehydratase activity. Sequencing confirms a 530-residue protein identical to one of the predicted proteins in the Ensembl database (ensembl.org). The protein is pentameric in solution, as studied by gel filtration and light scattering, presumably joined by the PTX domains. Mass spectrometry confirms the predicted signal peptide cleavage and disulfides, and N-glycosylation in two of the four observed glycosylation motifs. Molecular modeling of the pentamer is consistent with the modifications observed in mass spectrometry. Phylogenetics and sequence analyses provide a consistent hypothesis of the evolutionary history of domains associated with CA VI in mammals and non-mammals. Briefly, the evidence suggests that ancestral CA VI was a transmembrane protein, the exon coding for the cytoplasmic domain was replaced by one coding for PTX domain, and finally, in the therian lineage, the PTX-coding exon was lost. We knocked down CA VI expression in zebrafish embryos with antisense morpholino oligonucleotides, resulting in phenotype features of decreased buoyancy and swim bladder deflation in 4 dpf larvae. DISCUSSION: These findings provide novel insights into the evolution, structure, and function of this unique CA form.

Identification and inhibition of carbonic anhydrases from nematodes.

Carbonic anhydrases (CAs) are metalloenzymes, and classified into the evolutionarily distinct α, ß, γ, δ, ζ, and η classes. α-CAs are present in many living organisms. ß- and γ-CAs are expressed in most prokaryotes and eukaryotes, except for vertebrates. δ- and ζ-CAs are present in phytoplanktons, and η-CAs have been found in Plasmodium spp. Since the identification of α- and ß-CAs in Caenorhabditis elegans, the nematode CAs have been considered as an emerging target in research focused on antiparasitic CA inhibitors. Despite the presence of α-CAs in both helminths and vertebrates, structural studies have revealed different kinetic and inhibition results. Moreover, lack of ß-CAs in vertebrates makes this enzyme as an attractive target for inhibitory studies against helminthic infection. Some CA inhibitors, such as sulfonamides, have been evaluated against nematode CAs. This review article aims to present comprehensive information about the nematode CAs and their inhibitors as potential anthelminthic drugs.

Altered gene expression in the lower respiratory tract of Car6 (-/-) mice.

From birth, the respiratory tract mucosa is exposed to various chemical, physical, and microbiological stress factors. Efficient defense mechanisms and strictly regulated renewal systems in the mucosa are thus required. Carbonic anhydrase VI (CA VI) is the only secreted isoenzyme of the α-CA gene family. It is transported in high concentrations in saliva and milk into the alimentary tract where it contributes to optimal pH homeostasis. Earlier study of transcriptomic responses of Car6 (-/-) mice has shown changes in the response to oxidative stress and brown fat cell differentiation in the submandibular gland. It has been suggested that CA VI delivered to the mucosal surface of the bronchiolar epithelium is an essential factor in defense and renewal of the lining epithelium. In this study, the transcriptional effects of CA VI deficiency were investigated in both trachea and lung of Car6 (-/-) mice using a cDNA microarray analysis. Functional clustering of the results indicated significant changes of gene transcription in the lower airways. The altered biological processes included antigen transport by M-cells, potassium transport, muscle contraction, and thyroid hormone synthesis. Immunohistochemical staining confirmed the absence of CA VI in the submandibular gland of Car6 (-/-) mice. Immunostaining of the trachea and lung samples revealed no differences between the knockout and wild type groups nor were any morphological changes observed. The present findings can help us to recognize novel functions for CA VI-one of the major protein constituents of saliva and milk.

Horizontal transfer of ß-carbonic anhydrase genes from prokaryotes to protozoans, insects, and nematodes.

BACKGROUND: Horizontal gene transfer (HGT) is a movement of genetic information occurring outside of normal mating activities. It is especially common between prokaryotic endosymbionts and their protozoan, insect, and nematode hosts. Although beta carbonic anhydrase (ß-CA) plays a crucial role in metabolic functions of many living organisms, the origin of ß-CA genes in eukaryotic species remains unclear. METHODS: This study was conducted using phylogenetics, prediction of subcellular localization, and identification of ß-CA, transposase, integrase, and resolvase genes on the MGEs of bacteria. We also structurally analyzed ß-CAs from protozoans, insects, and nematodes and their putative prokaryotic common ancestors, by homology modelling. RESULTS: Our investigations of a number of target genomes revealed that genes coding for transposase, integrase, resolvase, and conjugation complex proteins have been integrated with ß-CA gene sequences on mobile genetic elements (MGEs) which have facilitated the mobility of ß-CA genes from bacteria to protozoan, insect, and nematode species. The prokaryotic origin of protozoan, insect, and nematode ß-CA enzymes is supported by phylogenetic analyses, prediction of subcellular localization, and homology modelling. CONCLUSION: MGEs form a complete set of enzymatic tools, which are relevant to HGT of ß-CA gene sequences from prokaryotes to protozoans, insects, and nematodes.

Ascaris lumbricoides ß carbonic anhydrase: a potential target enzyme for treatment of ascariasis.

BACKGROUND: A parasitic roundworm, Ascaris lumbricoides, is the causative agent of ascariasis, with approximately 760 million cases around the world. Helminthic infections occur with a high prevalence mostly in tropical and developing xcountries. Therefore, design of affordable broad-spectrum anti-helminthic agents against a variety of pathogens, including not only A. lumbricoides but also hookworms and whipworms, is desirable. Beta carbonic anhydrases (ß-CAs) are considered promising targets of novel anthelminthics because these enzymes are present in various parasites, while completely absent in vertebrates. METHODS: In this study, we identified an A. lumbricoides ß-CA (AIBCA) protein from protein sequence data using bioinformatics tools. We used computational biology resources and methods (including InterPro, CATH/Gene3D, KEGG, and METACYC) to analyze AlBCA and define potential roles of this enzyme in biological pathways. The AlBCA gene was cloned into pFastBac1, and recombinant AIBCA was produced in sf-9 insect cells. Kinetics of AlBCA were analyzed by a stopped-flow method. RESULTS: Multiple sequence alignment revealed that AIBCA contains the two sequence motifs, CXDXR and HXXC, typical for ß-CAs. Recombinant AIBCA showed significant CA catalytic activity with kcat of 6.0 × 10(5) s(-1) and kcat/KM of 4.3 × 10(7) M(-1) s(-1). The classical CA inhibitor, acetazolamide, showed an inhibition constant of 84.1 nM. Computational modeling suggests that the molecular architecture of AIBCA is highly similar to several other known ß-CA structures. Functional predictions suggest that AIBCA might play a role in bicarbonate-mediated metabolic pathways, such as gluconeogenesis and removal of metabolically produced cyanate. CONCLUSIONS: These results open new avenues to further investigate the precise functions of ß-CAs in parasites and suggest that novel ß-CA specific inhibitors should be developed and tested against helminthic diseases.

Inactivation of ca10a and ca10b Genes Leads to Abnormal Embryonic Development and Alters Movement Pattern in Zebrafish.

Carbonic anhydrase related proteins (CARPs) X and XI are highly conserved across species and are predominantly expressed in neural tissues. The biological role of these proteins is still an enigma. Ray-finned fish have lost the CA11 gene, but instead possess two co-orthologs of CA10. We analyzed the expression pattern of zebrafish ca10a and ca10b genes during embryonic development and in different adult tissues, and studied 61 CARP X/XI-like sequences to evaluate their phylogenetic relationship. Sequence analysis of zebrafish ca10a and ca10b reveals strongly predicted signal peptides, N-glycosylation sites, and a potential disulfide, all of which are conserved, suggesting that all of CARP X and XI are secretory proteins and potentially dimeric. RT-qPCR showed that zebrafish ca10a and ca10b genes are expressed in the brain and several other tissues throughout the development of zebrafish. Antisense morpholino mediated knockdown of ca10a and ca10b showed developmental delay with a high rate of mortality in larvae. Zebrafish morphants showed curved body, pericardial edema, and abnormalities in the head and eye, and there was increased apoptotic cell death in the brain region. Swim pattern showed abnormal movement in morphant zebrafish larvae compared to the wild type larvae. The developmental phenotypes of the ca10a and ca10b morphants were confirmed by inactivating these genes with the CRISPR/Cas9 system. In conclusion, we introduce a novel zebrafish model to investigate the mechanisms of CARP Xa and CARP Xb functions. Our data indicate that CARP Xa and CARP Xb have important roles in zebrafish development and suppression of ca10a and ca10b expression in zebrafish larvae leads to a movement disorder.

Analysis of evolution of carbonic anhydrases IV and XV reveals a rich history of gene duplications and a new group of isozymes.

Carbonic anhydrase (CA) isozymes CA IV and CA XV are anchored on the extracellular cell surface via glycosylphosphatidylinositol (GPI) linkage. Analysis of evolution of these isozymes in vertebrates reveals an additional group of GPI-linked CAs, CA XVII, which has been lost in mammals. Our work resolves nomenclature issues in GPI-linked fish CAs. Review of expression data brings forth previously unreported tissue and cancer types in which human CA IV is expressed. Analysis of collective glycosylation patterns of GPI-linked CAs suggests functionally important regions on the protein surface.