Trypanosoma brucei centrin5 is enriched in the flagellum and interacts with other centrins in a calcium‐dependent manner

Centrin is an evolutionarily conserved EF‐hand‐containing protein, which is present in eukaryotic organisms as diverse as algae, yeast, and humans. Centrins are associated with the microtubule‐organizing center and with centrosome‐related structures, such as basal bodies in flagellar and ciliated cells, and the spindle pole body in yeast. Five centrin genes have been identified in Trypanosoma brucei (T. brucei), a protozoan parasite that causes sleeping sickness in humans and nagana in cattle in sub‐Saharan Africa. In the present study, we identified that centrin5 of T. brucei (TbCentrin5) is localized throughout the cytosol and nucleus and enriched in the flagellum. We further identified that TbCentrin5 binds Ca2+ ions with a high affinity and constructed a model of TbCentrin5 bound by Ca2+ ions. Meanwhile, we observed that TbCentrin5 interacts with TbCentrin1, TbCentrin3, and TbCentrin4 and that the interactions are Ca2+‐dependent, suggesting that TbCentrin5 is able to form different complexes with other TbCentrins to participate in relevant cellular processes. Our study provides a foundation for better understanding of the biological roles of TbCentrin5.

Centrin is an evolutionarily conserved EF-hand-containing protein, which is present in eukaryotic organisms as diverse as algae, yeast, and humans. Centrins are associated with the microtubule-organizing center and with centrosome-related structures, such as basal bodies in flagellar and ciliated cells, and the spindle pole body in yeast. Five centrin genes have been identified in Trypanosoma brucei (T. brucei), a protozoan parasite that causes sleeping sickness in humans and nagana in cattle in sub-Saharan Africa. In the present study, we identified that centrin5 of T. brucei (TbCentrin5) is localized throughout the cytosol and nucleus and enriched in the flagellum. We further identified that TbCentrin5 binds Ca 2+ ions with a high affinity and constructed a model of TbCentrin5 bound by Ca 2+ ions. Meanwhile, we observed that TbCentrin5 interacts with TbCentrin1, TbCentrin3, and TbCentrin4 and that the interactions are Ca 2+ -dependent, suggesting that TbCentrin5 is able to form different complexes with other TbCentrins to participate in relevant cellular processes. Our study provides a foundation for better understanding of the biological roles of TbCentrin5.
Centrins are a member of the EF-hand calmodulin (CaM) superfamily, which are highly conserved in eukaryotic cells. Centrins were initially identified in unicellular green algae [1], as the essential components of basal bodies-associated and Ca 2+ -sensitive contracting fibers. Subsequently, homologous centrin proteins were observed in the microtubule-organizing center [2][3][4][5]. The functions of centrins were diverse in the cellular processes, including spindle pole body duplication [4], cellular morphogenesis [6], nucleotide excision repair [7], mRNA export [8], and protein degradation [9]. In green algae, the flagellar contraction is regulated by contractile fibers containing centrin and the contraction depending on the increase in intracellular concentration of Ca 2+ ions [1,10], suggesting that centrin is responsible for Ca 2+ -dependent cell motility. Target Sfi1 in yeast and its homologous protein (hSfi1) in humans were identified to interact centrins and responsible for SPB duplication [11,12]. In humans, two centrins (HsCentrin1 and HsCentrin2) with high sequence identity are involved in the centrosome/basal body segregation, ciliary beating, and mRNA and protein export [2,3,[13][14][15]. HsCentrin3 is involved in centrosome/basal body duplication [16,17].
The overall topology of centrins or centrin/target peptide complexes reported by NMR spectroscopy or X-ray crystallography [6,11,[18][19][20][21] is highly conserved and similar to CaM and troponin (TnC). The structure of centrin generally contains four EF-hands comprised of seven to eight a-helices. The structure of centrin can be divided into two independent domains, N-terminal domain (NTD) and C-terminal domain (CTD). Each domain contains a pair of EF-hands. The two domains are linked by a loop or a long a-helix and form a dumbbell structure. In all structures of centrins except Mus musculus Centrin1 (MmCentrin1) [19] and Chlamydomonas reinhardtii centrin (CrCentrin) [22], the two EF-hands of CTD instead of all the four EFhands can be saturated by Ca 2+ ions. The highly conserved CTD shows a higher affinity with Ca 2+ ions and other partners [11,23,24]. Meanwhile, NTD containing unconserved and unstructured 20-25 residues at the front shows a lower affinity with Ca 2+ ions and its partners [11,20]. Compared with CaM and TnC, the extended 20-25 residues in the NTD may have specialized biological function that is metal ion-dependent during self-assembly of centrin [25,26].
Here, we investigated the localization of TbCentrin5 in T. brucei and identified the interactions between TbCentrin5 and Ca 2+ ions. We further identified that TbCentrin5 is able to interact with other TbCentrins and the interactions are Ca 2+ -dependent. The work will provide a basis for better understanding of the biological functions of TbCentrin5.

Phylogenetic analyses of centrin protein sequences
The centrin protein sequences of T. brucei were obtained from TriTrypDB (https://tritrypdb.org/tritrypdb/). All other centrin protein sequences were obtained from NCBI protein database (https://www.ncbi.nlm.nih.gov/protein) and UniProt (https://www.uniprot.org/). Sequences were aligned using CLUSTALX [32] with default alignment parameters. The sequence trees were reconstructed with Neighbor-Joining algorithm using MEGA7 [33]. Bootstrap analysis (> 70, based on 500 replicates) provided a confidence measure for the detected relationships of branches in the phylogenetic tree.

In situ epitope tagging of endogenous proteins
For in situ tagging of TbCentrin5, the cDNA segment (nucleotide number 121-558) was cloned into pC-EYFP-NEO vector [35]. The recombinant vectors were linearized and electroporated into T. brucei procyclic forms from 427 strain. The transfection conditions were the same described as above. Successful transfectants were selected under 2.5 lgÁmL À1 G418. Expression of TbCentrin5-EYFP fusion protein was verified by western blotting.

Immunofluorescence microscopy
Cells stably expressing TbCentrin5-EYFP were harvested and washed twice with PBS. Resuspended cells were fixed with 4% paraformaldehyde and washed with PBS. Fixed cells were settled on the coverslip at room temperature for 30 min. The cells were stained with 4 0 ,6-diamidino-2phenylindole (DAPI) and examined with an inverted microscope (Model IX73; Olympus, Tokyo, Japan). Images were analyzed by IMAGEJ (NIH, Bethesda, MD, USA).

Protein expression and purification
Full-length gene encoding TbCentrin5 was amplified by PCR from the genomic DNA of T. brucei and cloned into a modifier vector pET-28a (+) (Novagen, Darmstadt, Germany), which provided a cut site of TEV protease at the N terminus to remove His-tag. The recombinant vector was transformed into Escherichia coli BL21 (DE3). The transformed cells were cultured in LB at 37°C until OD 600 reached 0.8 and induced by 0.5 mM IPTG at 16°C for 20 h. The cells were harvested and purified in lysis buffer containing 20 mM Tris and 500 mM NaCl at pH 7.0. The eluted protein was digested by TEV protease and further purified by gel filtration column Sephadex G-75 (GE Healthcare, Chicago, IL, USA).

Structure modeling
The 3D structure of TbCentrin5 or the complex of TbCen-trin5 and Ca 2+ ions was carried out using SWISS-MODEL based on homology modeling techniques [36]. The evolutionarily related protein structures were searched as templates based on the amino acid sequence of TbCentrin5. More than five hundred templates were searched and estimated by Global model quality estimate (GMQE) [37] and quaternary structure quality estimate (QSQE) [38]. Topranked templates were selected as templates for building model automatically. The model was finally evaluated and optimized by pairwise distance constraints that represented ensemble information from all template structures found.

Isothermal titration calorimetry
Isothermal titration calorimetry (ITC) measurements were performed on iTC200 (GE Healthcare) at 16°C to investigate the Ca 2+ -binding capacity and the interactions between TbCentrin5 and TbCentrin1/2/3/4. Samples of TbCentrin5 titrated with Ca 2+ ions were mixed with EGTA to remove potential Ca 2+ ions and then further purified by gel filtration column Sephadex G-75 (GE Healthcare). TbCentrin5 and calcium ions were equilibrated in the same buffer containing 20 mM Tris/HCl (pH 7.0) and 100 mM NaCl. 0.1 mM TbCen-trin5 in cell was titrated against 2.0 mM CaCl 2 from syringe. Two microliter of CaCl 2 was injected into 0.2 mL TbCen-trin5 at 120 s intervals. Samples of TbCentrin1/2/3/4 titrated with TbCentrin5 were purified and equilibrated as above expect mixed with EGTA or not. Fifty micromolar TbCen-trin1/2/3/4 in the cell was titrated against 0.5 mM TbCentrin5 from the syringe with the same sample volume and rotate speed. The data collected were analyzed by MICROCAL LLC ITC software (MicroCal, Malvern, UK).

Sedimentation assay
Sedimentation assay was carried out according to the procedures published previously [29]. In brief, the wild-type 29-13 cell line, the noninduced control cells, and TbCen-trin5 RNAi cells after tetracycline induction for 3 days were each suspended to~5910 6 cellsÁmL À1 in fresh medium. Each cell line was cultured in two cuvettes and incubated at 26°C and was measured by optical density (600 nm) every 2 h. To monitor the OD 600 , one cuvette was resuspended for monitoring cell growth while the other cuvette was not disturbed for monitoring sedimentation. The change in the OD 600 (DOD 600 ) was calculated by subtracting the OD 600 of the resuspended sample from that of the undisturbed sample. The experiment was repeated three times.

Phylogenetic analysis of TbCentrin5
To further understand the position of TbCentrin5 in evolution and its relationship with other centrins that have been identified in other organisms, a phylogenetic analysis was performed using a Neighbor-Joining method (Fig. 1A). Because the sequences of centrins are conserved, Neighbor-Joining method was selected. The presence of sequences of centrins from animal, fungi, and protist in this evolutionary tree indicates that those centrins come possibly from an ancestral protein in eukaryotic evolution. In the evolutionary tree, centrins from T. brucei are on different branches with centrins from higher animal, but closer to centrins from fungi and other protest, especially to centrins from Leishmania major (L. major) which belong to the same family of Trypanosomatidae as T. brucei. TbCen-trin5 and TbCentrin2 were on utterly different branches with other centrins but same as LmCentrin4 and LmCentrin3, suggesting that TbCentrin5 and TbCentrin2 are far from other centrins but closer to LmCentrin4 and LmCentrin3. The phylogenetic analysis indicated the position of TbCentrin5 and its relationship with other centrins, which is helpful for us to determine the location of Trypanosoma genera in the evolution.

TbCentrin5 is localized throughout the cytosol and nucleus and enriched in the flagellum
To determine the subcellular localization of TbCentrin5, TbLa was endogenously tagged with EYFP at the C terminus. The level of TbCentrin5-EYFP fusion protein was examined by western blot with GFP probe, which indicated the successful expression of TbCentrin5-EYFP in vivo (Fig. 1B). Fluorescence microscopy demonstrated that TbCentrin5-EYFP appeared to spread throughout the cell and was enriched in flagellum (Fig. 1C). Intriguingly, the localization of TbCentrin5 in nucleus changed through the cell cycle. In the early stage (1N1K) of the cell cycle, TbCentrin5 was slightly

Sequence analysis of TbCentrin5
The sequence of TbCentrin5 was aligned with other centrins from T. brucei, L. major and Homo sapiens using CLUSTALW2 and ESPRIPT 3.0 [32,39]. TbCentrin5 shares about 29%, 33%, 27%, 31%, 38%, and 45% sequence identity with TbCentrin1-4, HsCentrin1, and LmCentrin2, respectively (Fig. 2). The result of sequence alignment also verified the credibility of the evolutionary tree in Fig. 1A. Although the structures of centrins are conserved [6,11,[18][19][20][21], the sequences of centrins show diversity. Especially, the sequences of the first~25 residues at the N terminus show diverse length and very low similarity. In contrast with CaM and TnC, the extended~25 amino acids at the N  terminus of centrins display diversity in length and sequence, indicating that centrins may have more versatile functions than CaM and TnC.

Structure modeling of TbCentrin5
To better understand the functions of TbCentrin5, the model of TbCentrin5 was built using SWISS-MODEL [36], which builds model based on homology modeling techniques. Because the first 25 amino acids of TbCentrin5 show low conservation, the sequence of TbCentrin5 without the first 25 amino acids was placed for modeling. A total of 4015 templates were found from template library extracted from the PDB [40] to match the sequence of TbCen-trin5, and five templates were selected for modeling. The quality of model built by SWISS-MODEL is rapidly reduced when sequence identity is below 30% and is reliable when sequence identity is more than~40% [41,42]. Finally, MmCentrin1 (PDB: 5d 43), HsCentrin2 (PDB: 2ggm), SdCentrin (PDB: 3kf 9), and CrCentrin (PDB: 3qrx) with respective sequence identity of 43%, 42%, 41%, and 39% were selected to build model (Fig. 3). The scores of the model estimated by GMQE and QSQE were 0.64 and À1.23, respectively. In addition, the analysis of Ramachandran plots showed that 96% of the residues are in the most favored region (Fig. 4). The evaluation indicated that the model is of high quality and reliable. The structure model of TbCentrin5 is comprised of four EF-hands containing seven a-helices. Interestingly, a very short a-helix was formed in the loop of EF-hand IV because the loop is longer than that in general EF-hand. TbCentrin5 is divided into NTD and CTD by the long a4 and adopts a shape like a dumbbell.

Purification of TbCentrin5
Centrin5 of T. brucei was expressed and purified as described above. The elution volume of TbCentrin5 from Superdex G-75 column is approximate 66 mL, which corresponds to a molecular weight about 36 kDa (Fig. 5A). The calculated molecular weight of TbCentrin5 containing 186 amino acids is about 20 kDa. In addition, SDS/PAGE indicated no disulfide bond formation of the purified TbCentrin5 proteins, which suggested TbCentrin5 was expressed and purified as a homodimer.

TbCentrin5 binds to Ca 2+ ions with a high affinity
As an important second messenger, Ca 2+ ion is involved in many biological regulation processes [43]. ITC was performed to investigate the Ca 2+ -binding property of TbCentrin5 (Fig. 5B). The result showed that TbCentrin5 (pretreated with EDTA) binds to Ca 2+ ions with a high affinity (K d = 4.8 lM) in an exothermic mode and one molecule of TbCentrin5 is able to bind two Ca 2+ ions (N = 2).
The Ca 2+ -binding mode of TbCentrin5 is similar to that of TbCentrin4 [21], HsCentrin2 [20,44], and ScCdc31 [11] where one centrin molecule binds two Ca 2+ ions. In other Ca 2+ -binding modes, one centrin molecule binds more than two Ca 2+ ions [19,22]. Because of the higher binding affinity of CTD of centrins compared with NTD, CTD was preferentially saturated with Ca 2+ ions [11,20,23,24]. Therefore, the Ca 2+ -binding site of TbCentrin5 should be located in the CTD containing EF-hand III and EF-hand IV. The structure model of TbCentrin5 bound by Ca 2+ ions was built using SWISS-MODEL (Fig. 5C). As a Ca 2+ -binding motif, EFhand contains the specific amino acid sequences Based on an analysis of 118 structures of resolution of at least 2.0 Angstroms and R-factor no greater than 20%, a good quality model would be expected to have over 90% in the most favoured regions.    (Fig. 5D). The conformation of EF-hands may change due to the binding of Ca 2+ ions to EFhands, resulting in the change of the direction of Lys121, which may also provide a coordinated bond for Ca 2+ ions binding. The loop of EF-hand IV in TbCentrin5 is longer than its counterparts in other centrins (Fig. 2), resulting in the change of the location of the residues that provide coordinated bonds for Ca 2+ ions binding. The residues (Pro154, Asp162, Thr170, Glu175) in the loop of EF-hand IV may provide six coordinated bonds for Ca 2+ ions binding (Fig. 5E). The residues in the EF-hand III and EF-hand IV have the ability to provide sufficient coordinated bonds for Ca 2+ ions binding, indicating TbCentrin5 has a high Ca 2+ -binding affinity and forms a stable complex with Ca 2+ ions.

TbCentrin5 interacts with other centrins depending on Ca 2+ ions binding
In T. brucei, five centrin isoforms have been identified [27,28]. We have previously reported that TbCentrin4 interacts with TbCentrin1, TbCentrin2, and TbCen-trin5 [21]. To investigate the interactions between TbCentrin5 and other TbCentrins and further enrich the interaction network of centrins from T. brucei, GST-pull down assay and ITC were performed. GST-fused TbCentrin1-4 and TbCentrin5-HA were expressed and used in GST-pull down assay (Fig. 6A). The results showed that TbCentrin5 interacts with TbCentrin1, TbCentrin3, and TbCentrin4 but not TbCentrin2.
In the above experiments, EGTA was not added to remove the remaining Ca 2+ ions in TbCentrins. In the protein expression in LB culture medium and purification procedures, Ca 2+ ions may be copurified with TbCentrins. Therefore, it is necessary to ensure complete removal of Ca 2+ ions by treatment of EGTA to investigate the effect of Ca 2+ ions on the interactions between TbCentrin5 and other TbCentrins. ITC analysis of TbCentrin1, 2, 3, 4 titrated with TbCentrin5 was then performed without Ca 2+ ions. In the absence of Ca 2+ ions, TbCentrin5 is not able to interact with TbCentrin1, 2, 3, 4 (Fig. 6C). The results indicated that TbCentrin5 interacts with TbCentrin1, 3, 4 depending on Ca 2+ ions binding. Binding to Ca 2+ ions might induce the local conformational change of TbCentrin5, which results in the exposure of more hydrophobic region of TbCentrin5 to interact with other TbCentrins. Owing to these interactions, TbCen-trin5 is able to form different complexes with other TbCentrins depending on cellular Ca 2+ ions to participate in the relevant biological processes.

Depletion of TbCentrin5 does not compromise the cell motility
To investigate whether depletion of TbCentrin5 impacts cell growth, RNAi targeting on TbCentrin5 was performed in procyclic 29-13 cell line. Quantitative reverse transcription-PCR (RT-PCR) monitored that the mRNA level of TbCentrin5 in the RNAi cells decreased by~80% compared with that in the noninduced control cells after tetracycline induction for 2 days (Fig. 7A). The result demonstrated that depletion of TbCentrin5 does not inhibit the cell growth significantly (Fig. 7A).
TbCentrin5 has the same flagellum localization as TbCentrin3, and the effect of TbCentrin5 RNAi on cell growth is also similar to that of TbCentrin3 RNAi [29]. It was reported that knockdown of TbCentrin3 compromised the cell motility [29]. Therefore, the impact of knockdown of TbCentrin5 on the cell motility was also investigated. Sedimentation assay of cells after TbCentrin5 RNAi was performed. Monitored OD 600 values among wild-type 29-13 cell line, the noninduced control cells, and TbCentrin5 RNAi cells showed no significant distinction (Fig. 7B). Under light microscopy, cells after the deficiency of TbCentrin5 did not display any unusual phenotype such as spinning and tumbling or losing motility compared with the noninduced control cells. The results indicated that

Conclusion
In conclusion, we identified that TbCentrin5 is localized at the cytosol and nucleus and enriched in the flagellum. We further identified that TbCentrin5 binds Ca 2+ ions with a high affinity and built the model of TbCentrin5 bound by Ca 2+ ions. Besides, we demonstrated that TbCentrin5 interacts with TbCentrin1, TbCentrin3, and TbCentrin4 depending on Ca 2+ ions binding, suggesting TbCentrin5 might be able to form different complexes with other TbCentrins depending on cellular Ca 2+ ions to participate in the relevant biological processes. Our study will provide a basic information for better understanding the biological functions of TbCentrin5.