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Giardia Duodenalis 14-3-3 Protein Is Polyglycylated by a Tubulin Tyrosine Ligase-like Member and Deglycylated by Two Metallocarboxypeptidases

2010; Elsevier BV; Volume: 286; Issue: 6 Linguagem: Inglês

10.1074/jbc.m110.181511

ISSN

1083-351X

Autores

Marco Lalle, Serena Camerini, Serena Cecchetti, Claudia Blasetti Fantauzzi, Marco Crescenzi, Edoardo Pozio,

Tópico(s)

Galectins and Cancer Biology

Resumo

The flagellated protozoan Giardia duodenalis is a parasite of the upper part of the small intestine of mammals, including humans, and an interesting biological model. Giardia harbors a single 14-3-3 isoform, a multifunctional protein family, that is modified at the C terminus by polyglycylation, an unusual post-translational modification consisting of the covalent addition of one or multiple glycines on the γ-carboxyl groups of specific glutamic acids. Polyglycylation affects the intracellular localization of g14-3-3, as the shortening of the polyglycine chain is correlated with a partial relocalization of 14-3-3 inside the nuclei during encystation. In this work we demonstrate that the gTTLL3, a member of the tubulin tyrosine ligase-like family, is the enzyme responsible for the 14-3-3 polyglycylation. We also identify two metallopeptidases of the M20 family, here termed gDIP1 (giardial dipeptidase 1) and gDIP2, as enzymes able to shorten the g14-3-3 polyglycine tail both in vivo and in vitro. Finally, we show that the ectopic expression of gDIP2 alters the g14-3-3 localization and strongly hampers the cyst formation. In conclusion, we have identified a polyglycylase and two deglycylases that act in concert to modulate the stage-dependent glycylation status of the multifunctional regulatory g14-3-3 protein in G. duodenalis. The flagellated protozoan Giardia duodenalis is a parasite of the upper part of the small intestine of mammals, including humans, and an interesting biological model. Giardia harbors a single 14-3-3 isoform, a multifunctional protein family, that is modified at the C terminus by polyglycylation, an unusual post-translational modification consisting of the covalent addition of one or multiple glycines on the γ-carboxyl groups of specific glutamic acids. Polyglycylation affects the intracellular localization of g14-3-3, as the shortening of the polyglycine chain is correlated with a partial relocalization of 14-3-3 inside the nuclei during encystation. In this work we demonstrate that the gTTLL3, a member of the tubulin tyrosine ligase-like family, is the enzyme responsible for the 14-3-3 polyglycylation. We also identify two metallopeptidases of the M20 family, here termed gDIP1 (giardial dipeptidase 1) and gDIP2, as enzymes able to shorten the g14-3-3 polyglycine tail both in vivo and in vitro. Finally, we show that the ectopic expression of gDIP2 alters the g14-3-3 localization and strongly hampers the cyst formation. In conclusion, we have identified a polyglycylase and two deglycylases that act in concert to modulate the stage-dependent glycylation status of the multifunctional regulatory g14-3-3 protein in G. duodenalis. IntroductionThe flagellated and binucleated protozoan Giardia duodenalis (synonymous of Giardia lamblia and Giardia intestinalis) is an extracellular parasite of the upper part of the small intestine of mammals, including humans, where it causes giardiasis, an acute enteritis (1Thompson R.C. Int. J. Parasitol. 2000; 30: 1259-1267Crossref PubMed Scopus (356) Google Scholar). Besides its relevance for human and animal health, G. duodenalis is also a fascinating and simple eukaryotic organism with a minimalistic genomic and cellular organization that arouses a great interest as a biological model (2Morrison H.G. McArthur A.G. Gillin F.D. Aley S.B. Adam R.D. Olsen G.J. Best A.A. Cande W.Z. Chen F. Cipriano M.J. Davids B.J. Dawson S.C. Elmendorf H.G. Hehl A.B. Holder M.E. Huse S.M. Kim U.U. Lasek-Nesselquist E. Manning G. Nigam A. Nixon J.E. Palm D. Passamaneck N.E. Prabhu A. Reich C.I. Reiner D.S. Samuelson J. Svard S.G. Sogin M.L. Science. 2007; 317: 1921-1926Crossref PubMed Scopus (589) Google Scholar). In this perspective we have previously characterized the single giardial 14-3-3 (g14-3-3) isoform, a member of a small dimeric protein family ubiquitously conserved in eukaryotes (3Lalle M. Salzano A.M. Crescenzi M. Pozio E. J. Biol. Chem. 2006; 281: 5137-5148Abstract Full Text Full Text PDF PubMed Scopus (41) Google Scholar, 4Lalle M. Bavassano C. Fratini F. Cecchetti S. Boisguerin P. Crescenzi M. Pozio E. Int. J. Parasitol. 2010; 40: 201-213Crossref PubMed Scopus (16) Google Scholar). The 14-3-3s are able to bind a wide range of proteins containing consensus binding motifs usually phosphorylated on serine or threonine, thus, regulating multiple cellular processes, i.e. the metabolism, cell cycle progression, signal transduction pathways, cell growth, and differentiation (5Aitken A. Semin. Cancer. Biol. 2006; 16: 162-172Crossref PubMed Scopus (627) Google Scholar). We demonstrated that the g14-3-3 is modified in a peculiar fashion by the phosphorylation of Thr-214 and the polyglycylation of Glu-246 (3Lalle M. Salzano A.M. Crescenzi M. Pozio E. J. Biol. Chem. 2006; 281: 5137-5148Abstract Full Text Full Text PDF PubMed Scopus (41) Google Scholar, 4Lalle M. Bavassano C. Fratini F. Cecchetti S. Boisguerin P. Crescenzi M. Pozio E. Int. J. Parasitol. 2010; 40: 201-213Crossref PubMed Scopus (16) Google Scholar). The glycylation, first discovered at the C-terminal domain of α- and β-tubulin, is a post-translational modification consisting of the covalent addition of one or multiple glycines to the γ-carboxyl groups of specific glutamic acids of target proteins (6Bré M.H. Redeker V. Vinh J. Rossier J. Levilliers N. Mol. Biol. Cell. 1998; 9: 2655-2665Crossref PubMed Scopus (59) Google Scholar, 7Vinh J. Langridge J.I. Bré M.H. Levilliers N. Redeker V. Loyaux D. Rossier J. Biochemistry. 1999; 38: 3133-3139Crossref PubMed Scopus (44) Google Scholar). Recently, polyglycylation has been also reported for several proteins, including the mammalian nucleosome assembly proteins (8Ikegami K. Horigome D. Mukai M. Livnat I. MacGregor G.R. Setou M. FEBS Lett. 2008; 582: 1129-1134Crossref PubMed Scopus (39) Google Scholar, 9Ikegami K. Setou M. FEBS Lett. 2009; 583: 1957-1963Crossref PubMed Scopus (36) Google Scholar, 10Rogowski K. Juge F. van Dijk J. Wloga D. Strub J.M. Levilliers N. Thomas D. Bré M.H. Van Dorsselaer A. Gaertig J. Janke C. Cell. 2009; 137: 1076-1087Abstract Full Text Full Text PDF PubMed Scopus (98) Google Scholar). Whereas the phosphorylation of g14-3-3 is a constitutive post-translational modification, the polyglycylation of the protein is regulated during the G. duodenalis life cycle with a remarkable reduction in the length of the polyglycine chain during the early phase of the encystation process (3Lalle M. Salzano A.M. Crescenzi M. Pozio E. J. Biol. Chem. 2006; 281: 5137-5148Abstract Full Text Full Text PDF PubMed Scopus (41) Google Scholar, 4Lalle M. Bavassano C. Fratini F. Cecchetti S. Boisguerin P. Crescenzi M. Pozio E. Int. J. Parasitol. 2010; 40: 201-213Crossref PubMed Scopus (16) Google Scholar). Polyglycylation has been related to the intracellular localization of g14-3-3, as the shortening of the polyglycine chain is correlated with a partial relocalization of the protein inside the nuclei. In fact, in G. duodenalis parasites expressing the g14-3-3 mutant E246A, which cannot be polyglycylated, the protein localizes in the nuclei throughout the parasite life cycle, resulting in a faster differentiation of the trophozoite into the cyst stage once the process has been induced (3Lalle M. Salzano A.M. Crescenzi M. Pozio E. J. Biol. Chem. 2006; 281: 5137-5148Abstract Full Text Full Text PDF PubMed Scopus (41) Google Scholar, 4Lalle M. Bavassano C. Fratini F. Cecchetti S. Boisguerin P. Crescenzi M. Pozio E. Int. J. Parasitol. 2010; 40: 201-213Crossref PubMed Scopus (16) Google Scholar).Furthermore, the enzymes that catalyze the glycylation on tubulin and on other substrate proteins have been identified as members of the tubulin tyrosine ligase-like (TTLL) 2The abbreviations used are: TTLL, tubulin tyrosine ligase (TTL)-like; g-, giardial; Ab, antibody; pAb, polyclonal Ab; CWP, cyst wall protein. family, which also includes other amino acids ligases such as the tubulin tyrosine ligase (TTL) (11Ersfeld K. Wehland J. Plessmann U. Dodemont H. Gerke V. Weber K. J. Cell Biol. 1993; 120: 725-732Crossref PubMed Scopus (179) Google Scholar) and polyglutamylases, which add glutamic acid instead of glycines (12Janke C. Rogowski K. Wloga D. Regnard C. Kajava A.V. Strub J.M. Temurak N. van Dijk J. Boucher D. van Dorsselaer A. Suryavanshi S. Gaertig J. Eddé B. Science. 2005; 308: 1758-1762Crossref PubMed Scopus (227) Google Scholar, 13van Dijk J. Rogowski K. Miro J. Lacroix B. Eddé B. Janke C. Mol. Cell. 2007; 26: 437-448Abstract Full Text Full Text PDF PubMed Scopus (176) Google Scholar, 14Wloga D. Rogowski K. Sharma N. Van Dijk J. Janke C. Eddé B. Bré M.H. Levilliers N. Redeker V. Duan J. Gorovsky M.A. Jerka-Dziadosz M. Gaertig J. Eukaryot. Cell. 2008; 7: 1362-1372Crossref PubMed Scopus (74) Google Scholar). Glycylases can be classified as: primases, which add the first glycine, like TTLL3s of vertebrates and Tetrahymena thermophila and the mammalian TTLL8 (9Ikegami K. Setou M. FEBS Lett. 2009; 583: 1957-1963Crossref PubMed Scopus (36) Google Scholar, 10Rogowski K. Juge F. van Dijk J. Wloga D. Strub J.M. Levilliers N. Thomas D. Bré M.H. Van Dorsselaer A. Gaertig J. Janke C. Cell. 2009; 137: 1076-1087Abstract Full Text Full Text PDF PubMed Scopus (98) Google Scholar, 15Wloga D. Webster D.M. Rogowski K. Bré M.H. Levilliers N. Jerka-Dziadosz M. Janke C. Dougan S.T. Gaertig J. Dev. Cell. 2009; 16: 867-876Abstract Full Text Full Text PDF PubMed Scopus (106) Google Scholar); elongases, which are only able to elongate the polyglycine chain, like mammalian TTLL10, with the exception of the non-functional human TTLL10 (9Ikegami K. Setou M. FEBS Lett. 2009; 583: 1957-1963Crossref PubMed Scopus (36) Google Scholar, 10Rogowski K. Juge F. van Dijk J. Wloga D. Strub J.M. Levilliers N. Thomas D. Bré M.H. Van Dorsselaer A. Gaertig J. Janke C. Cell. 2009; 137: 1076-1087Abstract Full Text Full Text PDF PubMed Scopus (98) Google Scholar); bifunctional initiating/elongating enzymes, like Drosophila melanogaster dmTTLL3A and dmTTLL3B (10Rogowski K. Juge F. van Dijk J. Wloga D. Strub J.M. Levilliers N. Thomas D. Bré M.H. Van Dorsselaer A. Gaertig J. Janke C. Cell. 2009; 137: 1076-1087Abstract Full Text Full Text PDF PubMed Scopus (98) Google Scholar). However, the existence of enzymes responsible for the removal of glycines has only been indirectly demonstrated, but information about their identity is still missing (16Weber K. Schneider A. Westermann S. Müller N. Plessmann U. FEBS Lett. 1997; 419: 87-91Crossref PubMed Scopus (51) Google Scholar, 17Campanati L. Bré M.H. Levilliers N. de Souza W. Biol. Cell. 1999; 91: 499-506Crossref PubMed Scopus (16) Google Scholar).In this work we demonstrate that the giardial TTLL3 (gTTLL3), a member of the TTLL family, is the enzyme responsible for the 14-3-3 polyglycylation. We also identify two metallopeptidases of the M20 family, here termed gDIP1 and gDIP2, as enzymes able to shorten the g14-3-3 polyglycine tail both in vivo and in vitro. Finally, we show that the ectopic expression of gDIP2 alters the g14-3-3 localization and strongly hampers the cyst formation.DISCUSSIONIn this work we have shown that in G. duodenalis, despite the presence of several TTLL encoding genes, only the gTTLL3 protein displays an appreciable polyglycylation activity on g14-3-3. An in vitro glycylation assay using the AXO49 Ab, which recognizes polyglycine chains of at least four residues (3Lalle M. Salzano A.M. Crescenzi M. Pozio E. J. Biol. Chem. 2006; 281: 5137-5148Abstract Full Text Full Text PDF PubMed Scopus (41) Google Scholar, 34Bré M.H. Redeker V. Quibell M. Darmanaden-Delorme J. Bressac C. Cosson J. Huitorel P. Schmitter J.M. Rossler J. Johnson T. Adoutte A. Levilliers N. J. Cell Sci. 1996; 109: 727-738Crossref PubMed Google Scholar), together with the recombinant unmodified g14-3-3 suggests that gTTLL3 could be initiating/elongating enzymes that catalyze both the addition of the first glycine on the C terminus of g14-3-3 and the subsequent elongation of the polyglycine chain. Recent findings have demonstrated that in vertebrates polyglycylation of microtubules occurs by a two-step mechanism requiring the combined activities of the TTLL3/8 and TTLL10 enzymes (9Ikegami K. Setou M. FEBS Lett. 2009; 583: 1957-1963Crossref PubMed Scopus (36) Google Scholar, 10Rogowski K. Juge F. van Dijk J. Wloga D. Strub J.M. Levilliers N. Thomas D. Bré M.H. Van Dorsselaer A. Gaertig J. Janke C. Cell. 2009; 137: 1076-1087Abstract Full Text Full Text PDF PubMed Scopus (98) Google Scholar). A similar mechanism probably functions in the ciliate T. thermophila, where the TTLL3 homolog shows only glycine-ligase/initiase activity on tubulin (15Wloga D. Webster D.M. Rogowski K. Bré M.H. Levilliers N. Jerka-Dziadosz M. Janke C. Dougan S.T. Gaertig J. Dev. Cell. 2009; 16: 867-876Abstract Full Text Full Text PDF PubMed Scopus (106) Google Scholar). On the contrary, in organisms lacking TTLL10 homologs, bifunctional activities have been reported. In D. melanogaster, DmTTLL3A mono- and polyglycylates the α- and the β-tubulin, whereas DmTTLL3B mono- and polyglycylates non-tubulin proteins (10Rogowski K. Juge F. van Dijk J. Wloga D. Strub J.M. Levilliers N. Thomas D. Bré M.H. Van Dorsselaer A. Gaertig J. Janke C. Cell. 2009; 137: 1076-1087Abstract Full Text Full Text PDF PubMed Scopus (98) Google Scholar). Notably, mammalian TTLL10 is also able to act as a bifunctional enzyme on nucleosome assembly proteins (8Ikegami K. Horigome D. Mukai M. Livnat I. MacGregor G.R. Setou M. FEBS Lett. 2008; 582: 1129-1134Crossref PubMed Scopus (39) Google Scholar). The evidence that gTTLL3 is the only member of the giardial TTLL family clustering close to other mono- and polyglycylases and that G. duodenalis does not have a TTLL10 homolog protein (Ref. 20Redeker V. Levilliers N. Vinolo E. Rossier J. Jaillard D. Burnette D. Gaertig J. Bré M.H. J. Biol. Chem. 2005; 280: 596-606Abstract Full Text Full Text PDF PubMed Scopus (60) Google Scholar; supplemental Fig. S1) is in favor of a direct role of gTTLL3 not only in g14-3-3 but most probably also in the tubulin polyglycylation.As suggested by anti-polyGly immunoblotting and immunolocalizations, expression of the FLAG-gTTLL3 led also to an increase in polyglycylation levels of tubulin and microtubule structures. However, further studies are needed to clarify the activity of gTTLL3 on G. duodenalis tubulin as well as to define the function of the other gTTLLs, in particular gTTLL7, the only one that failed to be expressed.In T. thermophila, TTLL3 protein homologs localize primarily in cilia or basal bodies, with the exception of TTLL3Dp, which is present in the cell body (15Wloga D. Webster D.M. Rogowski K. Bré M.H. Levilliers N. Jerka-Dziadosz M. Janke C. Dougan S.T. Gaertig J. Dev. Cell. 2009; 16: 867-876Abstract Full Text Full Text PDF PubMed Scopus (106) Google Scholar). Here, we show that the FLAG-gTTLL3 protein partially co-localizes with g14-3-3 at all stages, in agreement with its polyglycylation activity toward g14-3-3, and it is not present in flagella or basal bodies. Intriguingly, in cysts the FLAG-gTTLL3 signal accumulates just underneath the cyst wall, most probably at the plasmalemma level. In agreement with gene expression data, this peculiar localization could suggest that physiologically the protein is constitutively expressed and stored during the dormant cyst stage to be eventually available during excystation. A similar conservative strategy is adopted by G. duodenalis during cyst formation; the ventral disc and flagella are broken into several large fragments, stored in the cytoplasm of the cyst, and apparently reassembled during excystation (35Palm D. Weiland M. McArthur A.G. Winiecka-Krusnell J. Cipriano M.J. Birkeland S.R. Pacocha S.E. Davids B. Gillin F. Linder E. Svärd S. Mol. Biochem. Parasitol. 2005; 141: 199-207Crossref PubMed Scopus (77) Google Scholar, 36Midlej V. Benchimol M. Parasitol. Int. 2009; 58: 72-80Crossref PubMed Scopus (30) Google Scholar).The production of FLAG-gTTLL3 protein did not affect the deglycylation of g14-3-3 during encystation, g14-3-3 localization, or the encystation timing. These data suggest that the developmental modulation of polyglycine chain length is exerted by deglycylating enzymes irrespective of the starting polyglycylation level. This hypothesis is supported by the almost constant expression of the gttll3 gene in the wild type G. duodenalis strain.To the best of our knowledge this is the first study reporting the identification of two metallopeptidases, gDIP1 and gDIP2, with a deglycylase activity. The reversible nature of polyglycylation and the existence of tubulin deglycylases were previously shown in cytoplasmic extracts of the ciliate Paramecium tetraurelia, but the enzymes were not identified (6Bré M.H. Redeker V. Vinh J. Rossier J. Levilliers N. Mol. Biol. Cell. 1998; 9: 2655-2665Crossref PubMed Scopus (59) Google Scholar). gDIP1 and gDIP2 belong to the MH clan, subfamily M20C. Metallopeptidases of the clan MH contains six families (M18, M20, M25, M28, M40, and M42) with enzymes having known activities such as carboxypeptidases or aminopeptidases. Members of the M20 metallohydrolase family, further divided into four subfamilies (A–D), require co-catalytic divalent metal ions, generally zinc, for their activity, and all include different exopeptidases, like the members of the clans MF and MG (37Rawlings N.D. Barrett A.J. Bateman A. Nucleic Acids Res. 2010; 38: D227-D233Crossref PubMed Scopus (713) Google Scholar). However, the M20 and M28 families are unique in attacking the C terminus of the substrate (37Rawlings N.D. Barrett A.J. Bateman A. Nucleic Acids Res. 2010; 38: D227-D233Crossref PubMed Scopus (713) Google Scholar). The glutamate carboxypeptidase G2 of Pseudomonas sp. (subfamily M20A) hydrolyzes the C-terminal glutamate moiety from folic acid and its analogues (38Minton N.P. Atkinson T. Sherwood R.F. J. Bacteriol. 1983; 156: 1222-1227Crossref PubMed Google Scholar), the tripeptidase T (M20B) of Lactococcus lacti hydrolyzes only tripeptides (39Mori S. Nirasawa S. Komba S. Kasumi T. Biochim. Biophys. Acta. 2005; 1748: 26-34Crossref PubMed Scopus (3) Google Scholar), the bacterial aminoacylhistidine dipeptidase performs hydrolysis of dipeptides (40Henrich B. Klein J.R. Handbook of Proteolytic Enzymes. 2nd Ed. Elsevier, London2004: 951-953Crossref Scopus (1) Google Scholar), and the thermostable carboxypeptidase Ss1 (M20D) of the archean Sulfolobus solfataricus is able to cleave N-blocked tripeptides (41Colombo S. Toietta G. Zecca L. Vanoni M. Tortora P. J. Bacteriol. 1995; 177: 5561-5566Crossref PubMed Google Scholar).The ectopic expression of FLAG-gDIP1 and FLAG-gDIP2 in G. duodenalis results in a remarkable reduction in the length of the polyglycine chain of g14-3-3, down to a size comparable with that observed in the g14-3-3 from wild type encysting parasites (3Lalle M. Salzano A.M. Crescenzi M. Pozio E. J. Biol. Chem. 2006; 281: 5137-5148Abstract Full Text Full Text PDF PubMed Scopus (41) Google Scholar). Nevertheless, a complete deglycylation of g14-3-3 was not observed at any stage in FLAG-gDIP1 or FLAG-gDIP2 transgenic parasite. A possible explanation for these observations might be related to the need for FLAG-gDIP1 and -2 activities for a cofactor protein, likely present in limiting amounts. Alternatively or in addition, the function of both enzymes might be partially counteracted by the activity of endogenous gTTLL3. The physiological, simultaneous expression of both the gttll3 and gdip1 genes in trophozoites and encysting parasites weighs in favor of the second hypothesis, again indicating that in G. duodenalis a fine balance exists between polyglycylation and deglycylation of g14-3-3. However, even though the partial deglycylation of g14-3-3 obtained in vitro with FLAG-tagged enzymes confirms the in vivo observations, the experimental conditions used in vitro might not allow the enzymes to express their full activities. Furthermore, the modest-to-absent inhibitory effect exerted by the metal chelator EDTA on the FLAG-gDIPs activities has also been reported for other metallopeptidases of different clans, such as human aminopeptidase N, Bacillus stearothermophilus aminopeptidase I, and Sabellastarte magnifica metallocarboxypeptidase (42Moser P. Roncari G. Zuber H. Int. J. Protein. Res. 1970; 2: 191-207Crossref PubMed Scopus (26) Google Scholar, 43Vanderheyden P.M. Demaegdt H. Swales J. Lenaerts P.J. De Backer J.P. Vogel L.K. Vauquelin G. Fundam. Clin. Pharmacol. 2006; 20: 613-619Crossref PubMed Scopus (11) Google Scholar, 44Alonso-del-Rivero M. Trejo S.A. Rodríguez de la Vega M. González Y. Bronsoms S. Canals F. Delfín J. Diaz J. Aviles F.X. Chávez M.A. FEBS J. 2009; 276: 4875-4890Crossref PubMed Scopus (13) Google Scholar). In these cases the absence of the inhibitory effect has been related to a strong binding of the metal to the enzyme.Previously, we have demonstrated that the E246A mutant of g14-3-3, which cannot be glycylated, localizes to the nuclei already at the trophozoite stage, resulting in a faster differentiation of trophozoites into cysts (4Lalle M. Bavassano C. Fratini F. Cecchetti S. Boisguerin P. Crescenzi M. Pozio E. Int. J. Parasitol. 2010; 40: 201-213Crossref PubMed Scopus (16) Google Scholar). Here, we show that despite the comparable shortening of g14-3-3 polyglycine chain attained by both metallopeptidases, the g14-3-3 localization in the trophozoites nuclei occurs only in FLAG-gDIP2 transfectants. Moreover, we observe that the expression of FLAG-gDIP2 strongly hampers cyst formation and affects CWP protein levels, whereas the forced expression of FLAG-gDIP1 results only in a limited reduction of encystation efficiency. Nevertheless, these results are not in conflict, as the g14-3-3 E246A point mutation only affects the g14-3-3 itself, whereas the expression of FLAG-gDIP1 and FLAG-gDIP2 might exert its effect on multiple targets, most likely including tubulin. Taken together, these data indicate that gDIP1 and gDIP2, besides their role in deglycylation of 14-3-3 and possibly tubulin, might carry out other functions, as also suggested by the different localization of the two proteins. Indeed, FLAG-gDIP2 labeling is evident in the median body, in the marginal groove, and in the ventrolateral flanges, whereas FLAG-gDIP1 localizes also in the nuclei. Moreover, the fact that gdip2 is down-regulated at the encystation, whereas gdip1 is almost constitutively expressed supports the possible involvement of these enzymes in different cellular processes. Deglycylation of nuclear proteins, such as transcriptional or chromatin remodeling factors, might be responsible for the defect in encystation most clearly observed in the FLAG-gDIP2 transgenic line. It has been proposed that glycylation by TTLL10 of nucleosome assembly protein 1 (NAP1) in the mouse might modulate NAP1-histone interactions, thus inducing transcriptional repression during spermiogenesis (8Ikegami K. Horigome D. Mukai M. Livnat I. MacGregor G.R. Setou M. FEBS Lett. 2008; 582: 1129-1134Crossref PubMed Scopus (39) Google Scholar). In D. melanogaster, depletion of DmTTLL3B protein results in several defects attributed to the potential lack of glycylation of its substrates, including chaperonins and motor proteins (10Rogowski K. Juge F. van Dijk J. Wloga D. Strub J.M. Levilliers N. Thomas D. Bré M.H. Van Dorsselaer A. Gaertig J. Janke C. Cell. 2009; 137: 1076-1087Abstract Full Text Full Text PDF PubMed Scopus (98) Google Scholar). Intriguingly, G. duodenalis motor proteins are localized in an area corresponding to the lateral crest of the trophozoite (45Narcisi E.M. Paulin J.J. Fechheimer M. J. Parasitol. 1994; 80: 468-473Crossref PubMed Scopus (14) Google Scholar). However, further studies are needed to verify the existence of other polyglycylation substrates and their possible involvement in the differentiation of G. duodenalis.Recently, Kimura et al. (46Kimura Y. Kurabe N. Ikegami K. Tsutsumi K. Konishi Y. Kaplan O.I. Kunitomo H. Iino Y. Blacque O.E. Setou M. J. Biol. Chem. 2010; 285: 22936-22941Abstract Full Text Full Text PDF PubMed Scopus (77) Google Scholar) published the identification in Caenorhabditis elegans and humans of tubulin deglutamylases as cytosolic carboxypeptidases of the MC clan, M14 family, subfamily M14D of metallopeptidases. G. duodenalis has four genes encoding proteins assigned to the M14 family but all classified as non-peptidase homologs (23Rawlings N.D. Barrett A.J. Handbook of Proteolytic Enzymes. 2nd Ed. Elsevier, London2004: 231-268Crossref Scopus (60) Google Scholar). Notably, metallopeptidases of the MC and the MH clans share no detectable sequence homologies (47Wouters M.A. Husain A. J. Mol. Biol. 2001; 314: 1191-1207Crossref PubMed Scopus (30) Google Scholar), thus, excluding the hypothesis of a common evolutionary origin. However, they show a common protein fold similar to peptidases in which two zinc atoms are essential for their activity (such as MH metallopeptidases), implying that metallopeptidase activity might be acquired in independent evolutionary events (37Rawlings N.D. Barrett A.J. Bateman A. Nucleic Acids Res. 2010; 38: D227-D233Crossref PubMed Scopus (713) Google Scholar). Because the identified deglutamylases (46Kimura Y. Kurabe N. Ikegami K. Tsutsumi K. Konishi Y. Kaplan O.I. Kunitomo H. Iino Y. Blacque O.E. Setou M. J. Biol. Chem. 2010; 285: 22936-22941Abstract Full Text Full Text PDF PubMed Scopus (77) Google Scholar) and deglycylases (this study) do not seem to belong to a single well conserved protein family, as in the case of the TTLLs, but share metallopeptidase properties, it is plausible that other metallopeptidases of different clans might exert similar functions in other organisms. Indeed, members of the metallopeptidase clans M14 and M28 and subfamilies M20A and M20D, but none of the M20C, have been identified in the partially annotated genomes of the ciliates T. thermophila and P. tetraurelia as well as in humans and D. melanogaster (23Rawlings N.D. Barrett A.J. Handbook of Proteolytic Enzymes. 2nd Ed. Elsevier, London2004: 231-268Crossref Scopus (60) Google Scholar). In conclusion, we have identified in G. duodenalis a polyglycylase and two deglycylases that act in concert to modulate the stage-dependent glycylation state of the multifunctional regulatory g14-3-3 protein. IntroductionThe flagellated and binucleated protozoan Giardia duodenalis (synonymous of Giardia lamblia and Giardia intestinalis) is an extracellular parasite of the upper part of the small intestine of mammals, including humans, where it causes giardiasis, an acute enteritis (1Thompson R.C. Int. J. Parasitol. 2000; 30: 1259-1267Crossref PubMed Scopus (356) Google Scholar). Besides its relevance for human and animal health, G. duodenalis is also a fascinating and simple eukaryotic organism with a minimalistic genomic and cellular organization that arouses a great interest as a biological model (2Morrison H.G. McArthur A.G. Gillin F.D. Aley S.B. Adam R.D. Olsen G.J. Best A.A. Cande W.Z. Chen F. Cipriano M.J. Davids B.J. Dawson S.C. Elmendorf H.G. Hehl A.B. Holder M.E. Huse S.M. Kim U.U. Lasek-Nesselquist E. Manning G. Nigam A. Nixon J.E. Palm D. Passamaneck N.E. Prabhu A. Reich C.I. Reiner D.S. Samuelson J. Svard S.G. Sogin M.L. Science. 2007; 317: 1921-1926Crossref PubMed Scopus (589) Google Scholar). In this perspective we have previously characterized the single giardial 14-3-3 (g14-3-3) isoform, a member of a small dimeric protein family ubiquitously conserved in eukaryotes (3Lalle M. Salzano A.M. Crescenzi M. Pozio E. J. Biol. Chem. 2006; 281: 5137-5148Abstract Full Text Full Text PDF PubMed Scopus (41) Google Scholar, 4Lalle M. Bavassano C. Fratini F. Cecchetti S. Boisguerin P. Crescenzi M. Pozio E. Int. J. Parasitol. 2010; 40: 201-213Crossref PubMed Scopus (16) Google Scholar). The 14-3-3s are able to bind a wide range of proteins containing consensus binding motifs usually phosphorylated on serine or threonine, thus, regulating multiple cellular processes, i.e. the metabolism, cell cycle progression, signal transduction pathways, cell growth, and differentiation (5Aitken A. Semin. Cancer. Biol. 2006; 16: 162-172Crossref PubMed Scopus (627) Google Scholar). We demonstrated that the g14-3-3 is modified in a peculiar fashion by the phosphorylation of Thr-214 and the polyglycylation of Glu-246 (3Lalle M. Salzano A.M. Crescenzi M. Pozio E. J. Biol. Chem. 2006; 281: 5137-5148Abstract Full Text Full Text PDF PubMed Scopus (41) Google Scholar, 4Lalle M. Bavassano C. Fratini F. Cecchetti S. Boisguerin P. Crescenzi M. Pozio E. Int. J. Parasitol. 2010; 40: 201-213Crossref PubMed Scopus (16) Google Scholar). The glycylation, first discovered at the C-terminal domain of α- and β-tubulin, is a post-translational modification consisting of the covalent addition of one or multiple glycines to the γ-carboxyl groups of specific glutamic acids of target proteins (6Bré M.H. Redeker V. Vinh J. Rossier J. Levilliers N. Mol. Biol. Cell. 1998; 9: 2655-2665Crossref PubMed Scopus (59) Google Scholar, 7Vinh J. Langridge J.I. Bré M.H. Levilliers N. Redeker V. Loyaux D. Rossier J. Biochemistry. 1999; 38: 3133-3139Crossref PubMed Scopus (44) Google Scholar). Recently, polyglycylation has been also reported for several proteins, including the mammalian nucleosome assembly proteins (8Ikegami K. Horigome D. Mukai M. Livnat I. MacGregor G.R. Setou M. FEBS Lett. 2008; 582: 1129-1134Crossref PubMed Scopus (39) Google Scholar, 9Ikegami K. Setou M. FEBS Lett. 2009; 583: 1957-1963Crossref PubMed Scopus (36) Google Scholar, 10Rogowski K. Juge F. van Dijk J. Wloga D. Strub J.M. Levilliers N. Thomas D. Bré M.H. Van Dorsselaer A. Gaertig J. Janke C. Cell. 2009; 137: 1076-1087Abstract Full Text Full Text PDF PubMed Scopus (98) Google Scholar). Whereas the phosphorylation of g14-3-3 is a constitutive post-translational modification, the polyglycylation of the protein is regulated during the G. duodenalis life cycle with a remarkable reduction in the length of the polyglycine chain during the early phase of the encystation process (3Lalle M. Salzano A.M. Crescenzi M. Pozio E. J. Biol. Chem. 2006; 281: 5137-5148Abstract Full Text Full Text PDF PubMed Scopus (41) Google Scholar, 4Lalle M. Bavassano C. Fratini F. Cecchetti S. Boisguerin P. Crescenzi M. Pozio E. Int. J. Parasitol. 2010; 40: 201-213Crossref PubMed Scopus (16) Google Scholar). Polyglycylation has been related to the intracellular localization of g14-3-3, as the shortening of the polyglycine chain is correlated with a partial relocalization of the protein inside the nuclei. In fact, in G. duodenalis parasites expressing the g14-3-3 mutant E246A, which cannot be polyglycylated, the protein localizes in the nuclei throughout the parasite life cycle, resulting in a faster differentiation of the trophozoite into the cyst stage once the process has been induced (3Lalle M. Salzano A.M. Crescenzi M. Pozio E. J. Biol. Chem. 2006; 281: 5137-5148Abstract Full Text Full Text PDF PubMed Scopus (41) Google Scholar, 4Lalle M. Bavassano C. Fratini F. Cecchetti S. Boisguerin P. Crescenzi M. Pozio E. Int. J. Parasitol. 2010; 40: 201-213Crossref PubMed Scopus (16) Google Scholar).Furthermore, the enzymes that catalyze the glycylation on tubulin and on other substrate proteins have been identified as members of the tubulin tyrosine ligase-like (TTLL) 2The abbreviations used are: TTLL, tubulin tyrosine ligase (TTL)-like; g-, giardial; Ab, antibody; pAb, polyclonal Ab; CWP, cyst wall protein. family, which also includes other amino acids ligases such as the tubulin tyrosine ligase (TTL) (11Ersfeld K. Wehland J. Plessmann U. Dodemont H. Gerke V. Weber K. J. Cell Biol. 1993; 120: 725-732Crossref PubMed Scopus (179) Google Scholar) and polyglutamylases, which add glutamic acid instead of glycines (12Janke C. Rogowski K. Wloga D. Regnard C. Kajava A.V. Strub J.M. Temurak N. van Dijk J. Boucher D. van Dorsselaer A. Suryavanshi S. Gaertig J. Eddé B. Science. 2005; 308: 1758-1762Crossref PubMed Scopus (227) Google Scholar, 13van Dijk J. Rogowski K. Miro J. Lacroix B. Eddé B. Janke C. Mol. Cell. 2007; 26: 437-448Abstract Full Text Full Text PDF PubMed Scopus (176) Google Scholar, 14Wloga D. Rogowski K. Sharma N. Van Dijk J. Janke C. Eddé B. Bré M.H. Levilliers N. Redeker V. Duan J. Gorovsky M.A. Jerka-Dziadosz M. Gaertig J. Eukaryot. Cell. 2008; 7: 1362-1372Crossref PubMed Scopus (74) Google Scholar). Glycylases can be classified as: primases, which add the first glycine, like TTLL3s of vertebrates and Tetrahymena thermophila and the mammalian TTLL8 (9Ikegami K. Setou M. FEBS Lett. 2009; 583: 1957-1963Crossref PubMed Scopus (36) Google Scholar, 10Rogowski K. Juge F. van Dijk J. Wloga D. Strub J.M. Levilliers N. Thomas D. Bré M.H. Van Dorsselaer A. Gaertig J. Janke C. Cell. 2009; 137: 1076-1087Abstract Full Text Full Text PDF PubMed Scopus (98) Google Scholar, 15Wloga D. Webster D.M. Rogowski K. Bré M.H. Levilliers N. Jerka-Dziadosz M. Janke C. Dougan S.T. Gaertig J. Dev. Cell. 2009; 16: 867-876Abstract Full Text Full Text PDF PubMed Scopus (106) Google Scholar); elongases, which are only able to elongate the polyglycine chain, like mammalian TTLL10, with the exception of the non-functional human TTLL10 (9Ikegami K. Setou M. FEBS Lett. 2009; 583: 1957-1963Crossref PubMed Scopus (36) Google Scholar, 10Rogowski K. Juge F. van Dijk J. Wloga D. Strub J.M. Levilliers N. Thomas D. Bré M.H. Van Dorsselaer A. Gaertig J. Janke C. Cell. 2009; 137: 1076-1087Abstract Full Text Full Text PDF PubMed Scopus (98) Google Scholar); bifunctional initiating/elongating enzymes, like Drosophila melanogaster dmTTLL3A and dmTTLL3B (10Rogowski K. Juge F. van Dijk J. Wloga D. Strub J.M. Levilliers N. Thomas D. Bré M.H. Van Dorsselaer A. Gaertig J. Janke C. Cell. 2009; 137: 1076-1087Abstract Full Text Full Text PDF PubMed Scopus (98) Google Scholar). However, the existence of enzymes responsible for the removal of glycines has only been indirectly demonstrated, but information about their identity is still missing (16Weber K. Schneider A. Westermann S. Müller N. Plessmann U. FEBS Lett. 1997; 419: 87-91Crossref PubMed Scopus (51) Google Scholar, 17Campanati L. Bré M.H. Levilliers N. de Souza W. Biol. Cell. 1999; 91: 499-506Crossref PubMed Scopus (16) Google Scholar).In this work we demonstrate that the giardial TTLL3 (gTTLL3), a member of the TTLL family, is the enzyme responsible for the 14-3-3 polyglycylation. We also identify two metallopeptidases of the M20 family, here termed gDIP1 and gDIP2, as enzymes able to shorten the g14-3-3 polyglycine tail both in vivo and in vitro. Finally, we show that the ectopic expression of gDIP2 alters the g14-3-3 localization and strongly hampers the cyst formation.

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