Studies On The Localization And Function Of Chaperone Protein CCT Subunits δ And η Of Nosema Bombycis.

Microsporidia are obligate intracellular parasitic eukaryotes that can infect protists and mammals,including immunocompromised humans.The first discovered microsporidia was Nosema bombycis,which can infect silkworms and cause pebrine disease,causing huge economic losses to the sericulture industry.Molecular chaperones are a class of proteins that are responsible for the proper folding of a large number of polypeptides in prokaryotic and eukaryotic cells.Newly synthesized peptides are prone to non-specific interactions without the assist of chaperone proteins,forming toxic aggregates.Any misregulation of the protein folding process will result in the formation of misfolded proteins or toxic aggregates associated with various pathological diseases.Chaperonin-containing T-complex polypeptide-1(CCT)is a major chaperonin protein in eukaryotes,which prevents misfolding and aggregation and enables the protein effectively fold to its inherent functional conformation within a biologically relevant time period.CCT assist in the folding of actin,tubulin and many other cellular proteins in an ATP-dependent manner.Studying the function of chaperone CCT in N.bombycis is very important for further understanding the mechanism of proteins folding and to prevention and treatment of pebrine disease.In this paper,we cloned subunit δ and subunit η of chaperone CCT complex of Nosema bombycis,NbCCTδ and NbCCTη antibodies were prepared and colocalized with actin and β-tubulin,and the functions of NbCCTδ and NbCCTη were initially explored.NbCCTδ gene was cloned and the sequence similarity between with the NCBI database was 99.8 %.Sequence analysis results show that NbCCTδ gene is 1 497 bp and encodes 498 amino acids,of which 56.43 % are helical structure,22.49 % are random coil,13.86 % are extended fragment,and the rest are corners.NbCCTδ has no signal peptide,no transmembrane domain,seven phosphorylation sites,exist N-glycosylation sites and Oglycosylation sites.The multiple sequence alignment results showed that the N.bombycis CCTδ protein has higher sequence homology with other microsporidian NbCCTδ proteins,which are more than 40 %.The phylogenetic tree constructed based on the CCTδ amino acids sequence showed that N.bombycis and Nosema ceranae and Nosema apis clustered on the same branch,indicating that the three may have a close relationship.Construct NbCCTδ recombinant protein expression vector to express recombinant protein.NbCCTδ protein was used for antibody preparation after purification.The obtained antibody concentration was 1.04 mg/ml.Western blot results showed that NbCCTδ antibody could recognize the NbCCTδ protein in the total protein of N.bombycis.Indirect immunofluorescence results showed that the NbCCTδ protein was present in the mature spores.After germination of mature spores,CCTδ protein mainly exists in sporoplasm.NbCCTδ is present in the spore cytoplasm throughout the development of N.bombycis,and gradually accumulates toward the spore edge during the spore maturation,indicating that NbCCTδ is a cytoplasmic protein.The co-localization results of NbCCTδ with Nb-actin and Nb-β-tubulin proteins show that during the early development of N.bombycis,NbCCTδ co-localized with Nb-actin in the cytoplasm,and later all gathered toward the spore edge,and Nb-actin and NbCCTδ could completely overlap.At the same time,NbCCTδ is co-localization with Nb-β-tubulin in the cytoplasm,but Nb-β-tubulin is not evenly distributed in the cytoplasm during the development of N.bombycis.Nb-β-tubulin mainly gathers around the spore nucleus during the spore nucleus division of N.bombycis.After the nucleus division ends,Nb-β-tubulin gradually gathers at the edge of the spore.During the spore maturation process,NbCCTδ,Nbactin,and Nb-β-tubulin all gradually gathered toward the spore edge,which may be related to the formation and thickening of the spore wall.The above results indicate that Nb-actin and Nb-β-tubulin are involved in the formation of the skeleton of N.bombycis,and Nb-β-tubulin may also have a certain relationship with spore nuclear division.NbCCTδ is involved in the folding and assembly of Nb-actin and Nb-β-tubulin.NbCCTδ gene transcription characteristics analysis results show that the transcription level of NbCCTδ was very high at the early stage of the development of N.bombycis.After 54 h,the expression of NbCCTδ was significantly reduced,and the expression of NbCCTδ remained at a very low level afterwards,indicating NbCCTδ helps the folding of nascent proteins at the early stage of spore development.After RNA interference,the expression of NbCCTδ has been at a very low level,indicating that RNA interference NbCCTδ can inhibit the expression of NbCCTδ gene.At the same time,RNA interference changed the morphology of N.bombycis.It may be that the down-regulation of the NbCCTδ gene caused a significant decrease in NbCCTδ protein content,which in turn affected the folding and assembly of cytoskeletal proteins.Therefore,spores could not form their unique shapes.Bioinformatics predicts NbCCTη protein secondary structure of alpha-helix accounted for 56.09 %,extending segments accounted for 13.70 %,beta corner accounted for 7.39 %,random coil accounted for 22.83%.NbCCTη has no signal peptide,no transmembrane domain,has phosphorylation site,O-glycosylation site and N-glycosylation site.Multiple sequence alignment results show that the NbCCTη protein and Nosema ceranae CCTη protein homologous is 60.71 %,and the phylogenetic trees are also clustered on the same branch,indicating that the two have a close relationship.We cloned the NbCCTη gene,and the recombinant NbCCTη protein expressed by the constructed prokaryotic expression vector had a molecular weight of about 58 k D.After NbCCTη protein purification,specific antibody antibody NbCCTη was prepared.Indirect immunofluorescence results showed that NbCCTη can be localized in the cytoplasm during the entire development of N.bombycis,and converge in the spore edge during the spore mature.NbCCTη can be co-localized with Nb-actin and Nb-β-tubulin throughout the life cycle of N.bombycis,but Nb-β-tubulin is not always evenly distributed in the cytoplasm during spore development.Nb-β-tubulin is concentrated around the nucleus during nuclear division,indicating that Nb-β-tubulin may have a certain relationship with spore nucleus division.while the spores gradually mature,NbCCTη,Nb-actin,and Nb-β-tubulin all gradually converge toward the spore edge,may be related to the formation and thickening of the spore wall.This study shows that NbCCTη is mainly located in the cytoplasm of N.bombycis,exists throughout the life history of N.bombycis,and participates in the folding of Nb-actin and Nb-β-tubulin,and play an important role in maintaining the integrity of cytoskeleton and spore structure.NbCCTδ and NbCCTη exist throughout the life history of N.bombycis,are mainly located in the cytoplasm,which can help actin and tubulin folding.CCTδ and CCTη may also have functions other than the CCT complex.Studying the function of CCTδ and CCTη monomers is of great significance for a comprehensive understanding of the function of the CCT complex,which is helpful for in-depth understanding of the proliferation and development process of N.bombycis.It also has certain guiding significance for the prevention and control pebrine disease.