RNase T2 Family Research Articles

New Catalytic Residues and Catalytic Mechanism of the RNase T1 Family.

The ribonuclease T1 family, including RNase Po1 secreted by Pleurotus ostreatus, exhibits antitumor activity. Here, we resolved the Po1/guanosine-3'-monophosphate complex (3'GMP) structure at 1.75 Å. Structure comparison and fragment molecular orbital (FMO) calculation between the apo form and the Po1/3'GMP complex identified Phe38, Phe40, and Glu42 as the key binding residues. Two types of the RNase/3'GMP complex in RNasePo1 and RNase T1 were homologous to Po1, and FMO calculations elucidated that the biprotonated histidine on the β3 sheet (His36) on the β3 sheet and deprotonated Glu54 on the β4 sheet were advantageous to RNase activity. Moreover, tyrosine (Tyr34) on the β3 sheet was elucidated as a crucial catalytic residues. Mutation of Tyr34 with phenylalanine decreased RNase activity and diminished antitumor efficacy compared to that in the wild type. This suggests the importance of RNase activity in antitumor mechanisms.

Establishment of the improved colonization of Escherichia coli laboratory strain in the intestine mediated by single gene deletion

In light of the emerging importance of the gut microbiome in human health, there is a need to improve the colonization efficiency of therapeutic bacteria called probiotics. Despite their recognized potential, artificially administered bacteria exhibit poor colonization in the intestine, limiting their therapeutic efficacy. Addressing this challenge requires innovative strategies; however, reported examples are limited. In nature, including in the intestinal tract, bacteria live via biofilm formation. Recently, it has been reported that RNase I, a member of the RNase T2 family conserved among almost all species, including bacteria, inhibits biofilm formation in Escherichia coli. In this study, we focus on these results and investigate the relationship between high biofilm formation and intestinal attachment using a non-settling E. coli laboratory strain as a probiotic model. The intestinal colonization abilities were evaluated through a microfluidic device mimicking the intestinal tract and through oral administration to mice. The in vitro and in vivo experiments showed that the E. coli strain lacking RNase I exhibited remarkable stability in intestinal colonization. We investigated the observation of colonization using fluorescence in situ hybridization, and inoculated E. coli cells were aggregated with the gut microbiome in the cecum and colon. This study proposes a technique to improve the intestinal colonization of bacteria by simply manipulating a single gene disruption, and it is expected to contribute to future research on the colonization of useful bacteria.

Transmembrane protein TMEM230, regulator of metalloproteins and motor proteins in gliomas and gliosis.

Glial cells provide physical and chemical support and protection for neurons and for the extracellular compartments of neural tissue through secretion of soluble factors, insoluble scaffolds, and vesicles. Additionally, glial cells have regenerative capacity by remodeling their physical microenvironment and changing physiological properties of diverse cell types in their proximity. Various types of aberrant glial and macrophage cells are associated with human diseases, disorders, and malignancy. We previously demonstrated that transmembrane protein, TMEM230 has tissue revascularization and regenerating capacity by its ability to secrete pro-angiogenic factors and metalloproteinases, inducing endothelial cell sprouting and channel formation. In healthy normal neural tissue, TMEM230 is predominantly expressed in glial and marcophate cells, suggesting a prominent role in neural tissue homeostasis. TMEM230 regulation of the endomembrane system was supported by co-expression with RNASET2 (lysosome, mitochondria, and vesicles) and STEAP family members (Golgi complex). Intracellular trafficking and extracellular secretion of glial cellular components are associated with endocytosis, exocytosis and phagocytosis mediated by motor proteins. Trafficked components include metalloproteins, metalloproteinases, glycans, and glycoconjugate processing and digesting enzymes that function in phagosomes and vesicles to regulate normal neural tissue microenvironment, homeostasis, stress response, and repair following neural tissue injury or degeneration. Aberrantly high sustained levels TMEM230 promotes metalloprotein expression, trafficking and secretionwhich contribute to tumor associated infiltration and hypervascularization of high tumor grade gliomas. Following injury of the central nervous or peripheral systems, transcient regulated upregulation of TMEM230 promotes tissue wound healing, remodeling and revascularization by activating glial and macrophage generated microchannels/microtubules (referred to as vascular mimicry) and blood vessel sprouting and branching. Our results support that TMEM230 may act as a master regulator of motor protein mediated trafficking and compartmentalization of a large class of metalloproteins in gliomas and gliosis.

Identification of the Acidification Mechanism of the Optimal pH for RNase He1.

Ribonuclease (RNase) He1 is a small ribonuclease belonging to the RNase T1 family. Most of the RNase T1 family members are active at neutral pH, except for RNase Ms, U2, and He1, which function at an acidic pH. We crystallized and analyzed the structure of RNase He1 and elucidated how the acidic amino residues of the α1β3- (He1:26-33) and β67-loops (He1:87-95) affect their optimal pH. In He1, Ms, and U2, the hydrogen bonding network formed by the acidic amino acids in the β67-loop suggested that the differences in the acidification mechanism of the optimum pH specified the function of these RNases. We found that the amino acid sequence of the β67-loop was not conserved and contributed to acidification of the optimum pH in different ways. Mutations in the acidic residues in He1 promoted anti-tumor growth activity, which clarified the role of these acidic amino residues in the binding pocket. These findings will enable the identification of additional targets for modifying pH-mediated enzymatic activities.

Transcriptome Analysis of the Late-Acting Self-Incompatibility Associated with RNase T2 Family in Camellia oleifera.

The Camellia oil tree (Camellia oleifera Abel.) is an important nonwood forest species in China, and the majority of its cultivars are late-acting self-incompatibility (LSI) types. Although several studies have examined the mechanism of LSI, the process is quite complicated and unclear. In this study, pollen tube growth and fruit setting of two Camellia oil tree cultivars Huashuo (HS) and Huajin (HJ) were investigated after non and self-pollination, and transcriptomic analysis of the ovaries was performed 48 h after self-pollination to identify the potential genes implicated in the LSI of Camellia oil trees. The results showed that the fruit set of HS was significantly higher than that of HJ after self-pollination. Transcriptomic analysis revealed that plant hormone signal transduction, the phosphatidylinositol signaling system, ATP-binding cassette (ABC) transporters, reactive oxygen species (ROS) metabolism, and Ca2+ signaling were mainly contributed in the LSI of reaction of Camellia oil tree. Moreover, nine RNase T2 genes were identified from the transcriptome analysis, which also showed that CoRNase7 participated in the self-incompatibility reaction in HS. Based on phylogenetic analysis, CoRNase6 was closely related to S-RNase from coffee, and CoRNase7 and CoRNase8 were closely related to S-RNase from Camellia sinensis. The 9 RNase T2 genes successfully produced proteins in prokaryotes. Subcellular localization indicated that CoRNase1 and CoRNase5 were cytoplasmic proteins, while CoRNase7 was a plasma membrane protein. These results screened the main metabolic pathways closely related to LSI in Camellia oil tree, and SI signal transduction might be regulated by a large molecular regulatory network. The discovery of T2 RNases provided evidence that Camellia oil tree might be under RNase-based gametophytic self-incompatibility.

Genome-Wide Analysis of the RNase T2 Family and Identification of Interacting Proteins of Four ClS-RNase Genes in 'XiangShui' Lemon.

S-RNase plays vital roles in the process of self-incompatibility (SI) in Rutaceae plants. Data have shown that the rejection phenomenon during self-pollination is due to the degradation of pollen tube RNA by S-RNase. The cytoskeleton microfilaments of pollen tubes are destroyed, and other components cannot extend downwards from the stigma and, ultimately, cannot reach the ovary to complete fertilisation. In this study, four S-RNase gene sequences were identified from the ‘XiangShui’ lemon genome and ubiquitome. Sequence analysis revealed that the conserved RNase T2 domains within S-RNases in ‘XiangShui’ lemon are the same as those within other species. Expression pattern analysis revealed that S3-RNase and S4-RNase are specifically expressed in the pistils, and spatiotemporal expression analysis showed that the S3-RNase expression levels in the stigmas, styles and ovaries were significantly higher after self-pollination than after cross-pollination. Subcellular localisation analysis showed that the S1-RNase, S2-RNase, S3-RNase and S4-RNase were found to be expressed in the nucleus according to laser confocal microscopy. In addition, yeast two-hybrid (Y2H) assays showed that S3-RNase interacted with F-box, Bifunctional fucokinase/fucose pyrophosphorylase (FKGP), aspartic proteinase A1, RRP46, pectinesterase/pectinesterase inhibitor 51 (PME51), phospholipid:diacylglycerol acyltransferase 1 (PDAT1), gibberellin receptor GID1B, GDT1-like protein 4, putative invertase inhibitor, tRNA ligase, PAP15, PAE8, TIM14-2, PGIP1 and p24beta2. Moreover, S3-RNase interacted with TOPP4. Therefore, S3-RNase may play an important role in the SI of ‘XiangShui’ lemon.

The Origin and Evolution of RNase T2 Family and Gametophytic Self-incompatibility System in Plants.

Ribonuclease (RNase) T2 genes are found widely in both eukaryotes and prokaryotes, and genes from this family have been revealed to have various functions in plants. In particular, S-RNase is known to be the female determinant in the S-RNase-based gametophytic self-incompatibility (GSI) system. However, the origin and evolution of the RNase T2 gene family and GSI system are not well understood. In this study, 785 RNase T2 genes were identified in 81 sequenced plant genomes representing broad-scale diversity and divided into three subgroups (Class I, II, and III) based on phylogenetic and synteny network analysis. Class I was found to be of ancient origin and to emerge in green algae, Class II was shown to originate with the appearance of angiosperms, while Class III was discovered to be eudicot-specific. Each of the three major classes could be further classified into several subclasses of which some subclasses were found to be lineage-specific. Furthermore, duplication, deletion, or inactivation of the S/S-like-locus was revealed to be linked to repeated loss and gain of self-incompatibility in different species from distantly related plant families with GSI. Finally, the origin and evolutionary history of S-locus in Rosaceae species was unraveled with independent loss and gain of S-RNase occurred in different subfamilies of Rosaceae. Our findings provide insights into the origin and evolution of the RNase T2 family and the GSI system in plants.

Molecular characteristics of S-RNase alleles as the determinant of self-incompatibility in the style of Fragaria viridis

Strawberry (Fragaria spp.) is a member of the Rosoideae subfamily in the family Rosaceae. The self-incompatibility (SI) of some diploid species is a key agronomic trait that acts as a basic pollination barrier; however, the genetic mechanism underlying SI control in strawberry remains unclear. Two candidate S-RNases (Sa- and Sb-RNase) identified in the transcriptome of the styles of the self-incompatible Fragaria viridis 42 were confirmed to be SI determinants at the S locus following genotype identification and intraspecific hybridization using selfing progenies. Whole-genome collinearity and RNase T2 family analysis revealed that only an S locus exists in Fragaria; however, none of the compatible species contained S-RNase. Although the results of interspecific hybridization experiments showed that F. viridis (SI) styles could accept pollen from F. mandshurica (self-compatible), the reciprocal cross was incompatible. Sa and Sb-RNase contain large introns, and their noncoding sequences (promotors and introns) can be transcribed into long noncoding RNAs (lncRNAs). Overall, the genus Fragaria exhibits S-RNase-based gametophytic SI, and S-RNase loss occurs at the S locus of compatible germplasms. In addition, a type of SI-independent unilateral incompatibility exists between compatible and incompatible Fragaria species. Furthermore, the large introns and neighboring lncRNAs in S-RNase in Fragaria could offer clues about S-RNase expression strategies.

Phosphate-Starvation-Inducible S-Like RNase Genes in Rice Are Involved in Phosphate Source Recycling by RNA Decay.

The fine-tuning of inorganic phosphate (Pi) for enhanced use efficiency has long been a challenging subject in agriculture, particularly in regard to rice as a major crop plant. Among ribonucleases (RNases), the RNase T2 family is broadly distributed across kingdoms, but little has been known on its substrate specificity compared to RNase A and RNase T1 families. Class I and class II of the RNase T2 family are defined as the S-like RNase (RNS) family and have showed the connection to Pi recycling in Arabidopsis. In this study, we first carried out a phylogenetic analysis of eight rice and five Arabidopsis RNS genes and identified mono-specific class I and dicot-specific class I RNS genes, suggesting the possibility of functional diversity between class I RNS family members in monocot and dicot species through evolution. We then compared the in silico expression patterns of all RNS genes in rice and Arabidopsis under normal and Pi-deficient conditions and further confirmed the expression patterns of rice RNS genes via qRT-PCR analysis. Subsequently, we found that most of the OsRNS genes were differentially regulated under Pi-deficient treatment. Association of Pi recycling by RNase activity in rice was confirmed by measuring total RNA concentration and ribonuclease activity of shoot and root samples under Pi-sufficient or Pi-deficient treatment during 21 days. The total RNA concentrations were decreased by < 60% in shoots and < 80% in roots under Pi starvation, respectively, while ribonuclease activity increased correspondingly. We further elucidate the signaling pathway of Pi starvation through upregulation of the OsRNS genes. The 2-kb promoter region of all OsRNS genes with inducible expression patterns under Pi deficiency contains a high frequency of P1BS cis-acting regulatory element (CRE) known as the OsPHR2 binding site, suggesting that the OsRNS family is likely to be controlled by OsPHR2. Finally, the dynamic transcriptional regulation of OsRNS genes by overexpression of OsPHR2, ospho2 mutant, and overexpression of OsPT1 lines involved in Pi signaling pathway suggests the molecular basis of OsRNS family in Pi recycling via RNA decay under Pi starvation.

RNase T2 in Inflammation and Cancer: Immunological and Biological Views.

The RNase T2 family consists of evolutionarily conserved endonucleases that express in many different species, including animals, plants, protozoans, bacteria, and viruses. The main biological roles of these ribonucleases are cleaving or degrading RNA substrates. They preferentially cleave single-stranded RNA molecules between purine and uridine residues to generate two nucleotide fragments with 2'3'-cyclic phosphate adenosine/guanosine terminus and uridine residue, respectively. Accumulating studies have revealed that RNase T2 is critical for the pathophysiology of inflammation and cancer. In this review, we introduce the distribution, structure, and functions of RNase T2, its differential roles in inflammation and cancer, and the perspective for its research and related applications in medicine.

Comprehensive genomic analysis of the RNase T2 gene family in Rosaceae and expression analysis in Pyrus bretschneideri

RNase T2s exist widely in eukaryotes and prokaryotes, playing various physiological roles in plants. At present, the genomes of Chinese white pear and six other Rosaceae fruit crops have been sequenced. However, no comprehensive analysis has investigated the evolutionary characteristics of the RNase T2 family in Rosaceae species. Here, 93 RNase T2s were identified in seven Rosaceae species (Pyrus bretschneideri, Rubus occidentalis, Fragaria vesca, Malus domestica, Prunus persica, Prunus avium, and Prunus mume). On the basis of phylogenetic analysis and structural characteristics, the RNase T2 family can be divided into four classes (I, II, III, and IV). Class IV is a new class first identified among seven Rosaceae species. Intron–exon analysis further revealed a homologous intron in RNase T2s except for class IV. Although purifying selection was the major force acting on the RNase T2 family, some positively selected sites and protein regions were still found in class IV sequences by using PAML and sliding windows. Tandem and proximal gene duplications made a major contribution to the expansion of the PbrRNase T2 family. Expression analysis identified some PbrRNase T2 genes highly expressed in pear and several transcription factors that might regulate these genes. In conclusion, the study of the RNase T2 family in seven Rosaceae species will help us to better understand the molecular evolution of RNase T2 genes and facilitate further functional analysis in the future.

Ribonuclease T2 from Aspergillus fumigatus promotes T helper type 2 responses through M2 polarization of macrophages

Allergic bronchopulmonary aspergillosis (ABPA) is an allergic immunological response to Aspergillus fumigatus (Af) exposure, which induces a strong T helper 2 (Th2) response via mechanisms that have yet to be elucidated. The aim of the present study was to investigate the hypothesis that T2 ribonuclease from Af (Af RNASET2) induces M2-type macrophage polarization to produce a T helper 2 (Th2) immune response. Recombinant Af RNASET2 (rAf RNASET2) was expressed and purified in a prokaryotic pET system and BALB/c mice were immunized with rAf RNASET2 for in vivo analyses. Expression levels of M2 polarization factors were evaluated in RAW264.7 macrophages treated with rAf RNASET2 in vitro using flow cytometry, reverse transcription-quantitative PCR, and western blot analysis. The results predicted that the mature Af RNASET2 protein (382 amino acids; GenBank no. MN593022) contained two conserved amino acid sequence (CAS) domains, termed CAS-1 and CAS-2, which are also characteristic of the RNASET2 family proteins. The protein expression levels of the Th2-related cytokines interleukin (IL)-4, IL-10, and IL-13 were upregulated in mice immunized with rAf RNASET2. RAW264.7 macrophages treated with rAf RNASET2 showed increased mRNA expression levels of M2 factors [arginase 1, Il-10, and Il-13]; however, there was no difference in cells treated with rAf RNASET2 that had been inactivated with a ribonuclease inhibitor (RNasin). The protein expression levels of IL-10 in macrophage culture supernatant were also increased following stimulation with rAf RNASET2. In addition, rAf RNASET2 upregulated the expression of phosphorylated mitogen activated protein kinases (MAPKs) in RAW264.7 cells, whereas MAPK inhibitors attenuated rAf RNASET2-induced IL-10 expression in RAW264.7 cells. In conclusion, the present study reveals that high rAf RNASET2 activity is required for rAf RNASET2-induced M2 polarization of macrophages and suggests an important immune regulatory role for Af RNASET2 in ABPA pathogenesis.

X-Ray Crystallographic Structure of Hericium erinaceus Ribonuclease, RNase He1 in Complex with Zinc.

RNase He1 is a guanylic acid-specific ribonuclease of the RNase T1 family from Hericium erinaceus (Japanese name: Yamabushitake). Its RNA degrading activity is strongly inhibited by Zn2+, similar to other T1 family RNases. However, RNase He1 shows little inhibition of human tumor cell proliferation, unlike RNase Po1, another T1 family RNase from Pleurotus ostreatus (Japanese name: Hiratake). Here, we determined the three-dimensional X-ray crystal structure of RNase He1 in complex with Zn, which revealed that Zn binding most likely prevents substrate entry into the active site due to steric hindrance. This could explain why RNase He1 and other T1 family RNases are inhibited by Zn. The X-ray crystal structures revealed that RNase He1 and RNase Po1 are almost identical in their catalytic sites and in the cysteine residues involved in disulfide bonds that increase their stability. However, our comparison of the electrostatic potentials of their molecular surfaces revealed that RNase He1 is negative whereas RNase Po1 is positive; thus, RNase He1 may not be able to electrostatically bind to the plasma membrane, potentially explaining why it does not exhibit antitumor activity. Hence, we suggest that the cationic characteristics of RNase Po1 are critical to the anti-tumor properties of the protein.

Structural insights into nucleotide and protein sequence of Ageritin: a novel prototype of fungal ribotoxin.

Here, we report the amino acid sequence of Ageritin, the first ribotoxin-like protein from basidiomycetes (Agrocybe aegerita). This protein consists of 135 amino acid residues with a theoretical molecular mass of 14,801.80 Da (experimental mass 14,802.84 Da, [M + H+]+). Unlike both the classic ribotoxins and homologous RNases T1 family from ascomycetes, Ageritin has a single free cysteinyl residue and does not show homology with known RNases endowed with the specific enzymatic activity on the universally conserved Sarcin Ricin Loop. On the other hand, our 3D homology study shows that Ageritin has a structural core consisting of an antiparallel β-sheet and an adjacent long α-helix, typical of ribotoxins and RNase T1 family, although the sheet has an orthogonal arrangement with respect to them. Thus, Ageritin is the first prototype of novel ribotoxin-like protein family from fungi.

Cell growth and homeostasis are disrupted in arabidopsis rns2-2 mutants missing the main vacuolar RNase activity.

Enzymes belonging to the RNase T2 family are essential for normal rRNA turnover in eukaryotes. In Arabidopsis thaliana, this function is performed by RNS2. The null mutant rns2-2 has increased rRNA half-life and constitutive autophagy. The aim of this work was to determine the molecular changes that take place in the rns2-2 mutant that may lead to altered cellular homeostasis, manifested by the observed cellular phenotype. To determine the effect of defective rRNA turnover on cellular homeostasis, comparative transcriptome and metabolome analyses of 10-day-old wild-type and rns2-2 seedlings were used to identify molecular processes affected in the mutant. Bioinformatics analyses suggested additional phenotypes that were confirmed through direct plant size measurements and microscopy. Few genes were differentially expressed in the rns2-2 mutant, indicating that control of autophagy in this genotype is mainly achieved at the post-transcriptional level. Among differentially expressed genes, transcripts related to carbon flux processes, particularly the pentose phosphate pathway (PPP), were identified. Metabolite analyses confirmed changes in the levels of PPP intermediates. Genes related to cell wall loosening were also differentially expressed in the mutant, and a decrease in monosaccharide components of cell wall hemicellulose were found. As a potential effect of weaker cell walls, rns2-2 plants are larger than wild-type controls, due to larger cells and increased water content. Elevated levels of reactive oxygen species (ROS) were also measured in rns2-2, and the constitutive autophagy phenotype was blocked by preventing ROS production via NADPH oxidase. Lack of rRNA recycling in rns2-2 cells triggers a change in carbon flux, which is redirected through the PPP to produce ribose-5-phosphate for de novo nucleoside synthesis. rRNA or ribosome turnover is thus essential for cellular homeostasis, probably through maintenance of nucleoside levels as part of the salvage pathway.

Deficiencies in RNS2‐mediated ribosomal RNA turnover cause changes in the Pentose Phosphate Pathway flux and alter cell growth in Arabidopsis

Enzymes belonging to the RNase T2 family are essential for normal ribosomal RNA turnover in eukaryotes. In Arabidopsis thaliana this function is performed by RNS2. The null mutant rns2‐2 has increased rRNA half‐life and constitutive autophagy. To determine the effect of defective rRNA turnover on cellular homeostasis, we used comparative transcriptome and metabolome analyses of 10 day old wild‐type and rns2‐2 seedlings to identify molecular processes affected in the mutant. Bioinformatics analyses suggested additional phenotypes that were confirmed through direct plant size measurements and microscopy.Few genes were differentially expressed in the rns2‐2 mutant, indicating that control of autophagy in this genotype is mainly achieved at the posttranscriptional level. Among differentially expressed genes we identified transcripts related to carbon flux processes, particularly the pentose phosphate pathway, and metabolite analyses confirmed changes in the levels of PPP intermediates. We also found differentially expressed genes related to cell wall loosening and found a decrease in monosaccharide components of cell wall hemicellulose. As a potential effect of weaker cell walls, rns2‐2 plants are larger than wild‐type controls, due to larger cells and increased water content. We propose that lack of rRNA recycling in rns2‐2 cells triggers a change in carbon flux, which is redirected through the pentose phosphate pathway to produce ribose‐5‐P for de novo nucleoside synthesis. rRNA or ribosome turnover is thus essential to maintain nucleoside homeostasis in Arabidopsis.Support or Funding InformationThis work was supported by a grant from the United States National Science Foundation [MCB‐1051818] to G.C.M. and D.C.B.

Characterization of Schistosoma japonicum CP1412 protein as a novel member of the ribonuclease T2 molecule family with immune regulatory function

BackgroundSchistosome infection typically induces a polarized Th2 type host immune response. As egg antigen molecules play key roles in this immunoregulatory process, clarifying their functions in schistosomiasis would facilitate the development of vaccine and immunotherapeutic methods. Schistosoma japonicum (Sj) CP1412 (GenBank: AY57074.1) has been identified as a new member of the RNase T2 family with immune regulatory functions.MethodsThe expression plasmid Sj CP1412-pET28a was constructed and transformed into bacteria for production of recombinant Sj CP1412 protein (rSj CP1412) via IPTG induction. The RNase activity of Sj CP1412 was predicted by bioinformatic analysis and confirmed by digesting the yeast tRNA with rSj CP1412.C57BL/6j mice were immunized with rSj CP1412, and its immune regulatory effects in vivo and in vitro were investigated. Meanwhile, the relationship between the RNase activity of Sj CP1412 and its immune regulation was observed.ResultsSj CP1412 was confirmed as a novel RNase T2 family protein with RNase activity. Immunoblotting and RT-PCR analyses demonstrated Sj CP1412 as a protein exclusively secreted/excreted from eggs, but not cercariae and adult worms. Stimulating RAW264.7 macrophages with rSj CP1412 raised the expression of CD206, Arg-1 and IL-10, which are related to M2 type macrophage differentiation. Stimulating dendritic cells (DCs) with rSjCP1412 failed to induce their maturation, and the recombinant protein also inhibited LPS-stimulated DC maturation. Depletion of Sj CP1412 from soluble egg antigen (SEA) impaired the ability of SEA to induce M2 type polarization of RAW264.7 macrophages. Immunizing mice with rSj CP1412 induced high antibody titers, increased serum IL-4 and TGF-β levels and splenic CD4 + CD25 + Foxp3 + T cells, downregulated serum IFN-γ levels and alleviated the egg granuloma pathology of schistosome infection. In vitro stimulation by rSj CP1412 significantly increased CD4 + CD25 + Foxp3 + T cell numbers in splenocytes of healthy mice. The rSj CP1412 protein with RNase activity inactivated by DEPC failed to induce M2 surface marker CD206 expression in RAW264.7 macrophages.ConclusionsThe Sj CP1412 protein expressed specifically in S. japonicum eggs is a novel member of the RNase T2 family. Similar to Omega-1 of Schistosoma mansoni, the Sj CP1412 protein drives polarization of the host Th2 immune response, which is dependent on its RNase activity. These data provide new evidence towards understanding the immune regulatory role of RNase T2 family proteins during schistosome infection.

Amino acid sequence analysis and characterization of a ribonuclease from starfishAsterias amurensis

The aim of this study was to phylogenetically characterize the location of the RNase T2 enzyme in the starfish (Asterias amurensis). We isolated an RNase T2 ribonuclease (RNase Aa) from the ovaries of starfish and determined its amino acid sequence by protein chemistry and cloning cDNA encoding RNase Aa. The isolated protein had 231 amino acid residues, a predicted molecular mass of 25,906 Da, and an optimal pH of 5.0. RNase Aa preferentially released guanylic acid from the RNA. The catalytic sites of the RNase T2 family are conserved in RNase Aa; furthermore, the distribution of the cysteine residues in RNase Aa is similar to that in other animal and plant T2 RNases. RNase Aa is cleaved at two points: 21 residues from the N-terminus and 29 residues from the C-terminus; however, both fragments may remain attached to the protein via disulfide bridges, leading to the maintenance of its conformation, as suggested by circular dichroism spectrum analysis. The phylogenetic analysis revealed that starfish RNase Aa is evolutionarily an intermediate between protozoan and oyster RNases.

RNase T2 of Mortierella (phylum Zygomycota)

The gene encoding the RNase T2 family enzyme, RNMel36, was cloned from Mortierella elongata (Zygomycota, Mortierellales) by polymerase chain reaction. The deduced protein sequence contains a signal sequence, catalytic domain, S/T rich domain and an extended C-terminal domain. RNase T2 homologs were also cloned from M. alpina, M. parvispora, M. polycephala, and M. verticillata. Their deduced catalytic and the C-terminal domains are highly conserved, unlike that of their S/T rich domains. This is the first report of cloning, heterologous expression, enzyme activity analysis and phylogenetic evaluation of an RNase T2 enzyme from zygomycotan fungi containing a characteristic C-terminal extension.

Effect of the replacement of aspartic acid/glutamic acid residues with asparagine/glutamine residues in RNase He1 from Hericium erinaceus on inhibition of human leukemia cell line proliferation.

RNase He1 from Hericium erinaceus, a member of the RNase T1 family, has high identity with RNase Po1 from Pleurotus ostreatus with complete conservation of the catalytic sequence. However, the optimal pH for RNase He1 activity is lower than that of RNase Po1, and the enzyme shows little inhibition of human tumor cell proliferation. Hence, to investigate the potential antitumor activity of recombinant RNase He1 and to possibly enhance its optimum pH, we generated RNase He1 mutants by replacing 12 Asn/Gln residues with Asp/Glu residues; the amino acid sequence of RNase Po1 was taken as reference. These mutants were then expressed in Escherichia coli. Using site-directed mutagenesis, we successfully modified the optimal pH for enzyme activity and generated a recombinant RNase He1 that inhibited the proliferation of cells in the human leukemia cell line. These properties are extremely important in the production of anticancer biologics that are based on RNase activity.