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Bioinformatics analysis and characterization of a secretory cystatin from Thelohanellus kitauei.

ABSTRACT: Thelohanellus kitauei, is a member of obligate parasitic myxozoans, which causes intestinal giant-cystic disease of common carp (Cyprinus carpio) and has resulted in significant economic losses in carp farms. Cystatin secreted by parasites can regulate the immune response of host to facilitate parasite's survival. In this study, the secretory TK-cystatin gene, encoding a protein of 120 amino acid residues (13.65 kDa), was cloned from T. kitauei genome. Phylogenetic analysis showed that TK-cystatin gene is closely related to the cystatin-A from Hydra vulgaris. Multiple sequence alignment revealed that TK-cystatin had three conserved motifs: N-terminal G¹⁹G²⁰, Q⁷³VVAG⁷⁷, and C-terminal L¹⁰²P¹⁰³. Molecular docking between TK-cystatin and three cysteine proteases showed a lower binding energy (- 13 KJ/mol) with cathepsin L whereas a higher binding energy (- 8.6 KJ/mol) with cathepsin B. TK-cystatin gene was expressed in Escherichia coli. Activity assays revealed that TK-cystatin has stronger inhibitory activity on endopeptidases (papain and cathepsin L) and weaker inhibitory activity on exopeptidase (cathepsin B). TK-cystatin was stable under the condition of acidity or alkalinity or below 57 °C. This study laid a foundation for the design and development of the anti-T. kitauei vaccine in carp culture in the future.

SUBMITTER: Zhang F

PROVIDER: S-EPMC7311561 | biostudies-literature |

REPOSITORIES: biostudies-literature

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Project description:The cystatins are inhibitors of papain- and legumain-like cysteine proteinases, classified in MEROPS subfamilies I25A-I25C. This study shows that 84 % (42/50) of tick cystatins are putatively extracellular in subfamily I25B and the rest are putatively intracellular in subfamily I25A. On the neighbor joining phylogeny guide tree, subfamily I25A members cluster together, while subfamily I25B cystatins segregate among prostriata or metastriata ticks. Two Ixodes scapularis cystatins, AAY66864 and ISCW011771 that show 50-71 % amino acid identity to metastriata tick cystatins may be linked to pathways that are common to all ticks, while ISCW000447 100 % conserved in I. ricinus is important among prostriata ticks. Likewise metastriata tick cystatins, Dermacentor variabilis-ACF35512, Rhipicephalus microplus-ACX53850, A. americanum-AEO36092, R. sanguineus-ACX53922, D. variabilis-ACF35514, R. sanguineus-ACX54033 and A. maculatum-AEO35155 that show 73-86 % amino acid identity may be essential to metastriata tick physiology. RT-PCR expression analyses revealed that I. scapularis cystatins were constitutively expressed in the salivary glands, midguts and other tissues of unfed ticks and ticks that were fed for 24-120 h, except for ISCW017861 that are restricted to the 24 h feeding time point. On the basis of mRNA expression patterns, I. scapularis cystatins, ISCW017861, ISCW011771, ISCW002215 and ISCW0024528 that are highly expressed at 24 h are likely involved in regulating early stage tick feeding events such as tick attachment onto host skin and creation of the feeding lesion. Similarly, ISCW018602, ISCW018603 and ISCW000447 that show 2-3 fold transcript increase by 120 h of feeding are likely associated with blood meal up take, while those that maintain steady state expression levels (ISCW018600, ISCW018601 and ISCW018604) during feeding may not be associated with tick feeding regulation. We discuss our findings in the context of advancing our knowledge of tick molecular biology.

Project description:BackgroundMicroRNAs (miRNAs) are a family of approximately 22 nucleotide small RNA molecules which regulate gene expression by fully or partially binding to their complementary sequences in mRNAs or promoters. A large number of miRNAs and their expression patterns have been reported in human, mouse and rat. However, miRNAs and their expression patterns in live stock species such as beef cattle are not well studied.ResultsWe constructed and sequenced small-RNA libraries to yield a total of 13,541 small-RNA sequences from 11 bovine tissues including brain, subcutaneous fat, muscle, liver, kidney, spleen and thymus. In total, 228 miRNAs including 29 novel miRNA candidates were identified. Of the 199 miRNAs, 101 have been previously reported as bovine miRNAs and the other 98 are bovine orthologs of known miRNAs that have been identified in at least one other mammalian species. Of the 29 novel miRNA candidates, 17 appeared at this point in time to be bovine specific, while the remaining 12 had evidence of evolutionary conservation in other mammalian species. Five miRNAs (miR-23a, -23b, -99a, -125b and -126-5p) were very abundant across the 11 tissues, accounting for 44.3% of all small RNA sequences. The expression analysis of selected miRNAs using qRT-PCR also showed that miR-26a and -99a were highly expressed in all tissues, while miR-122 and miR-133a were predominantly expressed in liver and muscle, respectively.ConclusionThe miRNA expression patterns among 11 tissues from beef cattle revealed that most miRNAs were ubiquitously expressed in all tissues, while only a few miRNAs were tissue specific. Only 60% miRNAs in this study were found to display strand bias, suggesting that there are some key factors for mature miRNA selection other than internal stability. Most bovine miRNAs are highly conserved in other three mammalian species, indicating that these miRNAs may have a role in different species that are potential molecular markers for evolution.

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Bioinformatics analysis and characterization of a secretory cystatin from Thelohanellus kitauei.

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