α2 helix and loop 3 are essential for the cytostatic activity of TC14-3
The results of the chimera experiments revealed that the amino acids at positions 61-145 in the C-terminal region of TC14-3 are responsible for cytostatic activity. The C-terminal region contains 1 α helix (α2), 4 β strands (β2-β5), and 4 loops (L1-L4) (see Figure 1F). In the α2 helix of TC14-1, hydrophobic amino acids (Ala61 and Phe65) play a key role in protein dimerization . Our study, using site-directed mutagenesis and SDS-PAGE of recombinant proteins, confirmed that in TC14-3, Phe65 of α2 helix is essential for protein dimerization and also critical for cytostatic activity.
TC14s are Ca2+-binding proteins . The ligands for calcium are the side-chain oxygen atoms of Glu106 (loop 3), Asn109 (loop 4), Asp127 (β4 strand), and Asp128 (β4 strand), as well as the main-chain carbonyl oxygen of Asp128 (see Figure 1F) . In TC14-3, Glu106 of loop 3 played a key role in Ca2+ binding, and the loss of Ca2+ binding was associated with the loss of cytostatic activity. Glu106 and Asn109 of TC14s correspond to Glu185 and Asn187 of mannose-binding protein A (MBP-A), respectively. In MBP-A, double mutations, Glu185Gln and Asn187Asp, alter the sugar substrate specificity from mannose to galactose .
In E-selectin, the sequence Trp-Ala-Pro-Gly-Glu-Pro (76-81) regulates carbohydrate-binding specificity . If Ala at position 77 is replaced with Ser, the sugar specificity of the mutant E-selectin changes from sialic acid to mannose. An exactly identical sequence exists in loop 3 of TC14-3 (see Figure 1F, positions 102-107). The corresponding sequence of TC14-2 was Trp-Ser-Pro-Asp-Glu-Pro. Both TC14-3A103S and TC14-3G105D retained strong cytostatic activity (see Table 1). It is, therefore, unlikely that the loop 3 is responsible for the difference between TC14-2 and TC14-3, although the loop 3 is essential for determining biological and biochemical features of TC14s.
Angiostatin and endostatin are specific, potent inhibitors of endothelial proliferation and angiogenesis [1, 2]. Endostatin is a 20-kDa C-terminal fragment of collagen XVIII. TC14-3 is similar to endostatin in several respects. The X-ray structure of murine endostatin is similar to that of C-type lectin . It lacks a characteristic Ca2+-binding site, but instead binds zinc at the N-terminus. This metal binding enables the dimerization of human endostatin . Similar to TC14-3, protein dimerization is essential for endostatin to carry out the antitumor activity .
Thr69 modulates TC14-3 dimerization
TC14-3 differed from TC14-2 in protein dimer stability. As Phe65 of α2 helix is conserved in both TC14-2 and TC14-3, we hypothesized that the differences in the biological and biochemical properties of TC14-2 and TC14-3 may consist in α2 helix neighboring Phe65.
The amino acids at position 69 of TC14-3 and TC14-2 are Thr and Arg, respectively. As Arg has a large side chain, it would interfere with the fitting and hydrophobic bonds at the α2 helix between juxtaposing proteins. As expected, TC14-3T69R changed the electrophoretic mobility and the stability of protein dimers, and lost the cytostatic activity. In contrast, TC14-2R69T could not form stable dimers comparable to that of wild-type TC14-3. This result suggests that additional as yet unidentified amino acids may contribute to the stability of protein dimers. However, it is undoubted that the amino acid at position 69 can modulate the biological and biochemical properties of TC14s.
Lys113 and Asn114 modulate Ca2+ binding of TC14-3
The cytostatic activities of TC14-3 depend on calcium-dependent galactose binding . Therefore, we initially expected that the affinity of TC14-3 for calcium may be higher than that of TC14-2. However, contrary to our expectation, the Ca2+-binding affinity of TC14-3 was apparently lower than that of TC14-2.
Lys113 and Asn114 are specific for TC14-3. They are located at the boundary between loop 4 and the β3 strand. When both these amino acids were replaced with those of TC14-2, the resultant TC14-3K113S.N114E exhibited an increase in Ca2+-binding affinity (> 0.6) and a decrease in cytostatic activity. As mentioned, TC14-3N109G had low Ca2+-binding affinity (0.4), and exhibited reduced cytostatic activity. Taken together, TC14-3 appears to have the highest cytostatic activity when the binding ratio of protein to Ca2+ is 1:0.5.
PmEed mediates cytostatic activity of TC14-3
In P. misakiensis, the atrial epithelium is a transdifferentiation-competent, multipotent tissue [5, 25]. It undergoes the terminal differentiation into the pharynx, gut, and brain when growing buds enter the developmental stage . TC14-3 is induced remarkably during budding, and it disappears from the morphogenesis domain where transdifferentiation takes place . This disappearance of TC14-3 may be caused by retinoic acid-inducible serine protease . TC14-3 can block in vitro cell growth and differentiation in Polyandrocarpa cell lines that have been established from explants of the atrial epithelium [6, 27]. Consequently, Matsumoto et al.  have argued that in P. misakiensis, TC14-3 serves as a negative regulator of terminal differentiation of multipotent cells.
In P. misakiensis, PmEed was developmentally regulated during budding cycle. The gene expression of PmEed was the highest at bud stages, gradually diminish during zooid growth, and was almost absent in somatic tissues and organs of adult zooids (Kawamura et al., submitted). This expression pattern was similar to that of TC14. In the present study, wild-type TC14-3 could induce PmEed in both cultured cells and adult zooid tissues, and interestingly, mutant proteins with abnormalities in protein dimerization or Ca2+ binding failed to induce PmEed.
Semi-quantitative PCR analysis of zooid pieces revealed that in the presence of TC14-3, the amount of PmEed transcripts was 2-4-fold higher than that of the control. This value seemed smaller than that expected from the results of in situ hybridization. This may be due to strong signals from the gonads in the control as well as the experiment. In fact, many gonads are embedded in the ventral body wall (see Figure 1A), and they particularly expressed PmEed in adult tissues in a TC14-3-independent manner. Therefore, the net induction of PmEed may be much larger, if the background value in the gonad could be subtracted from the total signal.
In P. misakiensis, dsRNA
rescued cultured cells from the growth-inhibitory effect of wild-type TC14-3. This result affords further evidence that PmEed is a downstream mediator of cytostatic TC14-3. In mammals, when Eed is deficient in ES cells, PcG target genes are de-repressed , leading to cell growth and differentiation. Therefore, PcG is thought to play roles in stem cell renewal and inhibition of cell differentiation in ES cells . Our results are consistent with these findings and notion in mammals.
Other genes regulated by TC14-3
A previous study has shown that in P. misakiensis, TC14-3 up-regulates α-integrin gene expression . In this study, wild-type TC14-3 suppressed the gene expression of both cyclin A and cyclin B. In Drosophila, PcG directly down-regulates cyclin A.
In P. misakiensis, mitochondrial respiratory complex genes are regulated in accordance with PmEed during budding life cycle (Kawamura et al., submitted). When wild-type TC14-3 was applied to zooid pieces of P. misakiensis, PmCOX1 gene was up-regulated. This gene regulation may also be related to PmEed. However, it should be noted that, unlike PmEed, the expression of PmCOX1 was not ubiquitous, but restricted around the pharynx. It is, therefore, possible that mitochondrial respiratory complex genes may be up-regulated via a route other than PmEed.
Epigenetic histone H3 trimethylation involved in cell growth and differentiation
Eed and Ezh2 are the components of PRC2 in PcG . Eed acts as Ezh2 activator, and Ezh2 catalyzes H3K27me3 in the so-called histone tail . Trimethylation of histone H3K27 recruits PRC1 to the chromatin. PRC1 possesses a discrete enzyme activity that modifies histone H2A, resulting in genome-wide, epigenetic gene repression . Polyandrocarpa histone H3 showed 100% sequence similarity to mammalian histone H3.3 (not shown). Rabbit anti-histone H3K27me3 antibody indeed stained nuclei of the atrial epithelium and coelomic cells in intact buds of P. misakiensis. Our in vitro studies indicated that wild-type TC14-3 could induce H3K27me3 in Polyandrocarpa cultured cells. It is notable that TC14-3 up-regulated the PmEed gene expression, but not PmEzh2. Therefore, epigenetic trimethylation of histone H3K27 should be ascribable exclusively to enhanced PmEed gene expression.
In contrast with the atrial epithelium and coelomic cells, nuclei of epidermal cells and coelomic morula cells were stained very weakly with anti-H3K27me3 antibody. The epidermis is a specialized tissue to synthesize and secrete tunic components. Morula cells are differentiated cells engaged in self-defense mechanisms. In the light of multipotency of the atrial epithelium [5, 25], it is probable that H3K27me3 is related to the block of terminal differentiation in budding tunicates. In ES cells, STAT3, Oct-3/4, and Sox2 induce Eed that influences H3K27me3 in the nucleus [29, 30]. These transcription factors are essential for stem cell maintenance. Although the atrial epithelium in tunicates is quite different from ES cells in origin and developmental potential, the basic mechanism for keeping the multipotent cell state appears to be shared by tunicate cells and mammalian ES cells.
Trithorax group also modifies histone H3 by trimethylation of Lys4. However, the result of histone methylation is quite different from the case of PcG, making chromatin loose and activating differentiation genes . In P. misakiensis, Lys4 trimethylation occurs in the process of transdifferentiation, which will be reported in the near future.