Survey of functional domains for cytostatic activity of TC14-3
Figure shows the alignment of TC14-1, TC14-2, and TC14-3 sequences. All 3 proteins are composed of 145 amino acids, of which 20 N-terminal amino acids are signal peptides. The remaining 125 amino acids constitute the mature protein. The CRD of TC14s consists of 2 α helices, 5 β strands, and 4 loops (Figure ) [19
]. The second α helix (α2) spanning positions 56-69 contributes to protein dimerization, and loop 3, loop 4, and β4 strand form a calcium pocket for galactose and fucose recognition (Figure ) [7
Two chimeric proteins containing complementary fragments from TC14-2 and TC14-3 were constructed (see Materials and methods). One of the chimeric proteins (TC14-221-60/TC14-361-145) consisted of N-terminal TC14-2 and C-terminal TC14-3. It reversibly blocked cell growth, similar to wild-type TC14-3 (Figure ). The other chimeric protein (TC14-321-60/TC14-261-145), like TC14-2, did not show such activity (Figure ), suggesting that the active site(s) for cell growth inhibition are located in the C-terminal region of TC14-3. In growth-arrested cells, the transcription of both cyclin A and cyclin B was suppressed (Figure ).
Figure 2 Effects of wild-type and chimeric proteins on in vitro cell growth (A-D) and gene expression (E-G). Cells were plated and cultured for 2 days in the growth medium containing 30 μg/ml proteins. (A)Wild-type TC14-3. (B) TC14-221-60/TC14-361-145 (more ...)
Next, we surveyed the polypeptide domains necessary for the cytostatic activity of TC14-3. Phe65 in the α2 helix, Glu106 in loop 3, and Asn109 in loop 4 were changed to Asp, Gly, and Gly, respectively. TC14-3F65D and TC14-3E106G completely lost cytostatic activity (Figure ), and TC14-3N109G exhibited lower activity (Table ), suggesting that α2 helix and loop 3 are important for cytostatic activity. However, because both Phe65 and Glu106 are conserved in both TC14-2 and TC14-3 (Figure ), these amino acids are insufficient to explain the unique cytostatic activity of TC14-3.
Figure 3 Quantitative data of cell growth inhibition of wild-type and mutant TC14s. Each histogram shows a mean ± standard deviation. (A)Effects of wild-type TC14-3, TC14-3F65D, and TC14-3E106G on cell growth. (B)Effects of wild-type TC14-2, TC14-2R69T (more ...)
Summary of the cytostatic activities of mutant TC14-3s.
Amino acids involved in TC14-3-specific protein dimerization and cytostatic activity
TC14-3 exhibited a relative electrophoretic mobility of 15 kDa (Figure , lane 1) on SDS-PAGE following heat denaturation, while under non-heated conditions, more than 99% of the total protein exhibited a relative mobility of 30 kDa (Figure , lane 2; Table ). In contrast, TC14-2 exhibited a single band of 18 kDa following heat denaturation (Figure , lane 3) and separated into 2 bands of 18 and 28 kDa under non-heated conditions (Figure , lane 4). The 28-kDa form of TC14-2 accounted for approximately 61% of the total amount of protein (Table ). The chimeric protein, TC14-221-60/TC14-361-145 exhibited an electrophoretic pattern similar to that of wild-type TC14-3 (Figure , lanes 5, 6). These results strongly suggest that wild-type TC14-3 may form more stable dimers than wild-type TC14-2.
Figure 4 Electrophoretic mobility of wild-type and mutant TC14s on SDS-PAGE. Odd lanes and even lanes show heat-denatured samples and non-heated samples, respectively. Lanes 1,2, wild TC14-3. Lanes 3,4, wild TC14-2. Lanes 5,6, TC14-2(21-60)/TC14-3(61-145). Lanes (more ...)
Relative amounts of monomeric and dimeric forms in wild-type TC14s and their mutant proteins.*
The mutant protein TC14-3F65D failed to dimerize (Figure , lanes 7, 8, Table ). At the extremity of the α2 helix (Figure ), Thr69 of TC14-3 was exchanged with Arg69 of TC14-2. Under heat denaturation, TC14-3T69R exhibited a major band of approximately 18 kDa instead of 15 kDa (Figure , lane 9), and under the non-heated condition, it yielded 2 bands of 18 and 28 kDa (Figure , lane 10, Table ), similar to wild-type TC14-2. On the other hand, heat-denatured TC14-2R69T exhibited a major band of 15 kDa (Figure , lane 11), similar to wild-type TC14-3. In contrast, the non-heated sample of TC14-2R69T yielded 2 bands of 15 and 28 kDa, intermediate between wild-type TC14-2 and TC14-3 (Figure , lane 12, Table ).
TC14-3T69R exhibited no cytostatic activity on cultured tunicate cells (Figure ). TC14-2R69T, on the other hand, acquired the cytostatic activity to some extent (Figure ). As a reference, the amino acid at position 70 was exchanged between TC14-2 and TC14-3. The cytostatic activity of the mutant proteins was unaffected (Table ).
These results indicate that the amino acid at position 69 can modulate multiple characteristics of TC14s, such as electrophoretic mobility, stability of protein dimers, and cytostatic activity.
Amino acids involved in TC14-3-specific Ca2+ binding and cytostatic activity
Figure shows the quantitative data of Ca2+ binding in wild-type and mutant TC14s. Wild-type TC14-2 bound to calcium at a molar ratio of 1:0.85, while the calcium binding ratio of wild-type TC14-3 was unexpectedly low (1:0.5) (Figure ). TC14-3E106G exhibited negligible Ca2+-binding activity (Figure ), and TC14-3N109G exhibited reduced calcium-binding efficiency (molar ratio, 0.4) (Figure ). As mentioned, TC14-3E106G lost the cytostatic activity near-completely, while TC14-3N109G exhibited weak cell growth inhibition.
Figure 5 Calcium-binding kinetics of wild-type and mutant TC14s. (A)Wild type TC14-2 and TC14-3. Note that the Ca2+-binding affinity of TC14-3 is lower than that of TC14-2. (B)TC14-3E106G, TC14-3N109G, and TC14-3K113S.N114E. TC14-3N109G showed lower Ca2+-binding (more ...)
We next focused on the amino acids at positions 113 and 114 in loop 4 of TC14s (Figure ). Although single mutations (TC14-3K113S or TC14-3N114E) did not improve calcium binding, the double mutation TC14-3K113S.N114E bound to calcium at a molar ratio of approximately 0.6 (Figure ), a value intermediate between wild-type TC14-3 and wild-type TC14-2 (Figure ).
Both TC14-3K113S and TC14-3N114E retained their growth-inhibitory activities on cultured cells (Figure ). On the other hand, the inhibitory activity was greatly diminished in the double mutant protein TC14-3K113S.N114E (Figure ). Mutations at C-terminal positions 136, 144, and 145 did not have any apparent influence on cell growth (Table ).
Only wild-type TC14-3 can induce PmEed
We examined whether TC14-3 influenced the gene expression of PmEed. Cultured cells of Polyandrocarpa were treated for 2 days with PBS, wild-type TC14-3, TC14-3T69R, or TC14-3E106G. PmEed cDNA could be amplified by RT-PCR only when wild-type TC14-3 was applied to cells (Figure ). The amount of PmEed continued to increase during PCR cycles (Figure ).
Figure 6 Quantification of in vitro and in vivo PmEed induction by wild-type and mutant TC14-3s. (A, B)Cultured cells. (A)PCR products of PmEed (upper) and Pmβ-actin (lower). Lane 1, control (PBS). Lane 2, wild-type TC14-3. Lane 3, TC14-3T69R. Lane 4, (more ...)
In intact animals, PmEed
was expressed abundantly from bud stages to juvenile zooid stages [see Additional file 1A, B, C, D
], but diminished conspicuously at adult zooid stages except the gonad [see Additional file 1A, E, F
] (More detailed results will be published elsewhere). In this study, adult zooids were cut into 3 pieces to facilitate TC14-3 infiltration, and treated with TC14-3 proteins for 2 days. Zooids of P. misakiensis
possess a high potential for regeneration [20
]. As expected, control zooid pieces treated with PBS could survive during the course of study. They did not exhibit any apparent signals for PmEed
in most tissues and organs except the gonad (Figure ), similar to intact adult zooids, indicating that the surgery by itself did not affect PmEed
expression. In contrast to the control, zooid pieces that had been treated with wild-type TC14-3 ubiquitously expressed PmEed
(Figure ), the expression pattern similar to buds. The strongest signal was detected in coelomic cells in the hemocoel (Figure ). The atrial, gastric, and perivisceral epithelia also expressed PmEed
(Figure ). The epidermis showed moderate expression of PmEed
, but muscle cells did not (Figure ).
Figure 7 In situ hybridization of PmEed in adult zooids treated with TC14-3. (A-C)Control, PBS. (A)Body wall of zooid. Bar, 100 μm. (B)Digestive tract. Bar, 50 μm. (C)Gonad. Bar, 50 μm. (D-I)Experiment, wild-type TC14-3. (D)Body wall of (more ...)
Results of RT-PCR showed that only wild-type TC14-3 could induce in vivo PmEed (Figure ). By semi-quantitative PCR, the PmEed products became visible at the 25th cycle (Figure ), and increased exponentially thereafter (Figure ). In the control, on the other hand, PmEed products became first visible at the 27th cycle (Figure ), and increased parallel to the experiment (Figure ). The result indicated that the amount of PmEed transcripts in wild-type TC14-3-treated animals was approximately 2-4-fold that of the control.
TC14-3 also induces mitochondrial respiratory gene
Our recent study showed that in P. misakiensis, PmEed and mitochondrial respiratory genes were both inactivated during zooidal senescence and reactivated remarkably during budding (Kawamura et al., submitted). We examined, therefore, whether wild-type TC14-3 could induce not only PmEed but also cytochrome c oxidase 1 (PmCOX1) in aged zooids. Results of in situ hybridization showed that in the control, signals were hardly detectable in the body wall, pharynx, and visceral organs (Figure ). In contrast, when TC14-3 was applied to zooids, a portion of epithelial cells and coelomic cells in the pharynx expressed PmCOX1 strongly (Figure ). The endostyle, digestive tract, and surrounding coelomic cells did not emit signals (Figure ). The increasing curves of PCR products indicated that TC14-3-treated samples had larger amount of PmCOX1 transcripts than untreated controls, although the difference was not so high (Figure ).
Figure 8 In vivo PmCOX1 induction in adult zooids by TC14-3. (A-F)In situ hybridization. (A-C)Control treated with PBS. (D-F)Experiment treated with wild-type TC14-3. (A)Body wall. Bar, 100 μm. (B)Pharynx. Bar, 50 μm. (C)Visceral organs. Bar, 100 (more ...)
Trimethylation of histone H3 by TC14-3
Anti-H3K27me3 antibody stained the in vivo nuclei of epithelial cells and coelomic cells in buds (Figure ). Nuclei of epidermal cells stained weakly (Figure ), whereas those of the atrial epithelium, multipotent epithelial cells in P. misakiensis, stained heavily (Figure ). In the hemocoel, many coelomic cells emitted strong signals (Figure ), but differentiated cells such as morula cells did not have apparent signals in the nucleus (Figure black arrowheads, 9D white arrowheads).
Figure 9 In vivo and in vitro immunostaining of trimethylated histone H3 in P. misakiensis. (A-D)Growing buds stained with anti-H3K27me3 antibody. (B, D)DAPI staining after immunostaining. (A, B)Bars, 50 μm. (C, D)Bars, 25 μm. Black and white arrowheads (more ...)
Cultured cells untreated with TC14-3 were not stained with anti-H3K27me3 antibody (Figure ). Cells treated with mutant protein (TC14-3E106G) were stained weakly (Figure ), whereas wild-type TC14-3-treated cells were stained heavily with the antibody (Figure ). Western blotting of in vitro cultured cells showed that anti-histone H3 antibody stained a single band of approximately 17 kDa (Figure , lane 1). Anti-H3K27me3 antibody, on the other hand, did not stain any bands when cells were not treated or treated with TC14-3E106G (Figure , lanes 2, 3), but stained a single band of 17 kDa when cultured cells were treated with wild-type TC14-3 (Figure , lane 4). We could not find in vivo differences in histone trimethylation between TC14-3-treated and untreated samples (not shown).
The gene expression of PmEzh2
, a Polyandrocarpa
homolog of Histone H3K27 methyltransferase, was examined. Adult zooid fragments treated with wild-type TC14-3 showed the same strength of signals as those of untreated zooids [see Additional file 2
lanes 1, 2). Cultured cells in the growth medium without TC14-3 showed a weak signal of PmEzh2
PCR products at 30th
cycle [see Additional file 2
lane 3]. When cells were treated in vitro with wild-type or mutant TC14-3s, the signals were approximately the same as those of the control [see Additional file 2
lanes 4-7). These results indicate that wild-type TC14-3 can induce H3K27me3 without affecting PmEzh2
Recovery from TC14-3-induced growth arrest by PmEed knockdown
We examined the effect of PmEed RNAi on cell growth arrest by wild-type TC14-3. Double-stranded RNA of PmEed (dsRNAPmEed) was introduced into cultured cells by electroporation. In the positive control, blunt electroporation was performed in the absence of dsRNAPmEed, and the cells were allowed to grow for 3 days without TC14-3. Cells spread on the culture dish (Figure ). In the negative control, cells were treated with TC14-3 after the blunt electroporation. Cells formed many aggregates (Figure ). In dsRNAPmEed experiments, cells spread again in the presence of TC14-3 (Figure ). The cell number was approximately twice as many as that of the negative control (Figure ). The recovery value accounted for 65% compared to the positive control.
Figure 10 Recovery of cell growth by RNAi of PmEed from TC14-3-induced growth arrest. (A)In control 1 (positive control), after blunt electroporation cells were allowed to grow for 3 days in the absence of TC14-3. Cell spread normally. Bar, 100 μm. (B)In (more ...)