Wunen, a homolog of a lipid phosphate phosphatase (LPP), has a crucial function in the migration and survival of primordial germ cells (PGCs) during Drosophila embryogenesis. Past work has indicated that the LPP isoforms may show functional redundancy in certain systems, and that they have broad-range lipid phosphatase activities in vitro, with little apparent specificity between them. This study shows that there are marked differences in biochemical activity between fly Wun and mammalian LPPs, with Wun having a narrower activity range than has been reported for the mammalian LPPs. Furthermore, although it is active on a range of substrates in vitro, mouse Lpp1 has no activity on an endogenous Drosophila germ-cell-specific factor in vivo. Conversely, human LPP3 is active, resulting in aberrant migration and PGC death. These results show an absolute difference in bioactivity among LPP isoforms for the first time in a model organism and may point towards an underlying signalling system that is conserved between flies and humans (Burnett, 2003).
The human LPPs are grouped into three isoforms, 1, 2 and 3, and the human genes also have splice variants (Waggoner, 1999). Sequence alignments of Wun and Wun2 were analyzed with mammalian LPPs, and it was concluded that both proteins have the greatest homology with human LPP3 (Burnett, 2003).
Past work has indicated that the three known LPP isoforms can dephosphorylate a broad range of lipid phosphates, notably lysophosphatidic acid (LPA), phosphatidic acid (PA), ceramide-1-phosphate (C(1)P), diacylglycerol pyrophosphate (DGPP) and sphingosine-1-phosphate (S(1)P), with relatively little apparent specificity (Dillon, 1997; Jasinska, 1999; Kai, 1997; Roberts, 1998; Waggoner, 1996). Apart from Wun and Wun2, little is known about the biological functions of these proteins. Dri42 (differentially expressed in rat intestine 42), the rat homolog of LPP3, is upregulated during differentiation of the crypt cells in the small intestine (Barila, 1996), whereas the human splice-variant LPP1-1 is downregulated in human colon-tumor tissue (Leung, 1998). In 2000, Zhang reported a homozygous null mutation in murine Lpp2 that produced viable, fertile mice with no detectable phenotype. In addition, Lpp2 is expressed at lower levels than the Lpp1 and Lpp3 isoforms, leading to the proposal that Lpp2 functions redundantly with them (Zhang, 2000b). Starz-Gaiano (2001) reported that null mutations in either wun or wun2 also present no detectable phenotype, with embryonic development and PGC migration occurring normally. By contrast, removal of both genes results in highly perturbed PGC migration, with PGCs scattering widely on exiting the midgut at stage 10. This led to the suggestion that these LPPs function redundantly (Burnett, 2003).
Given the remarkable bioactivity of the potential lipid substrates for these enzymes, and the specificity of some of their receptors (Takuwa, 2002), it was curious that the separate isoforms present such broad-range activity in biochemical assays and seem to be functionally redundant in vivo. This study examines specificity in substrate recognition among the fly and mammalian isoforms in vitro for a range of known substrates, and in vivo by investigating their ability to dephosphorylate the PGC-specific guidance molecule that functions as a substrate for Wun. It is shown that there are marked differences in relative activity between immunopurified Wun and the mammalian isoforms in a biochemical phosphate-release assay, with Wun showing negligible activity for two of the substrates. Transgenic flies were created expressing mouse Lpp1 and human LPP3; although active on all of the tested substrates in vitro, mouse Lpp1 is completely inactive in the germ-cell migration bioassay. Conversely, overexpression of human LPP3 results in aberrant PGC migration and death, with a remarkably similar phenotype to Wun overexpression. This demonstrates a distinct difference in bioactivity between the isoforms for the first time, and may point towards an underlying signalling system that is conserved between flies and humans (Burnett, 2003).
Although detailed kinetic analyses was not performed, the fly LPP Wun and the mammalian LPP1 and LPP3 isoforms differ in their relative activities on lysophosphatidic acid (LPA), phosphatidic acid (PA), ceramide-1-phosphate (C(1)P) in a PiPer® phosphate-release assay. Although Wun can dephosphorylate LPA with a similar efficiency to mouse Lpp1, it shows negligible activity on both PA and C(1)P. This indicates that there are distinct differences in substrate preference between the fly and mammalian enzymes, with Wun showing a narrower activity range in this assay than was previously reported for the mammalian LPPs. Both mouse Lpp1 and human LPP3 are active on all three substrates. Mouse Lpp1 has a 1.7-fold higher activity on C(1)P than human LPP3 in this assay. Human LPP3, however, had a 1.6-fold higher activity than mouse Lpp1 on PA (Burnett, 2003).
Human LPP3 is highly active when ectopically expressed in Drosophila embryos, as assayed by the disruption of PGC migration and survival, which results in a phenotype similar to the ectopic expression of Wun or Wun2. That human LPP3 shows the same phenotype in a bioassay as Drosophila Wun suggests a conserved signalling pathway that regulates germ-cell migration and survival from flies to humans. Conversely, although active in the biochemical assay, mouse Lpp1 is completely inactive in vivo, and has no apparent effect on PGC migration or survival. This shows an absolute difference in functional bioactivity between the mammalian LPP isoforms. That the LPP isoforms present different functional outputs when assayed in vivo has a number of implications. It may be that the Lpp1 isoform simply cannot recognize or catalyse the dephosphorylation of the specific factor acted on by Wun and LPP3. This would demonstrate specificity in substrate choice between the isoforms, and may indicate that the germ-cell-specific factor is not PA, LPA or C(1)P, on which mouse Lpp1 is active in vitro, particularly as Wun shows negligible activity on PA and C(1)P in the same assay. Alternatively, there may be specific components of the pathway that are required for selection and recognition of the factor. It is possible that as yet unidentified conformational or structural differences in mouse Lpp1 preclude its association with these factors, thus inhibiting its enzymatic function in this system. Thus, LPA could be the factor, and the unnatural presentation and high concentration of LPA in the biochemical assay may override the specific selection mechanisms used to regulate activity in vivo, allowing mouse Lpp1 access to this otherwise inaccessible substrate. Primary sequence analyses have not identified any immediate candidates for residues conferring such a difference. It is speculated that the observed differences in substrate preference may be related to the proteins' structure, which are as yet unsolved. Barila has studied the properties of the internal sequences of Dri42 in its trafficking to the cell surface (Barila, 1996). The contributions of the termini to biological and biochemical properties are yet to be reported in any detail (Burnett, 2003).
In conclusion, evidence is presented of differences in relative activity between the mammalian and fly LPP isoforms on LPA, PA and C(1)P in a biochemical assay. These results indicate that the fly LPP Wun has a narrower activity range than the mammalian LPPs in this assay. Evidence is also presented to show that, although active in vitro, mouse Lpp1 cannot dephosphorylate the same endogenous germ-cell-specific factor as Wun in vivo, whereas human LPP3 seems to do so. This demonstrates that despite broad-range activity in Triton-micelle assays, the mammalian LPP isoforms do show distinct differences in bioactivity when assayed in a physiological context. It is expected that a combination of biochemical and biological data, such as those presented here, will in time help to identify the physiological substrate for Wun, which is, presumably, a stem-cell control factor (Burnett, 2003).
Lipid phosphate phosphatases (LPPs) are integral membrane proteins believed to dephosphorylate bioactive lipid messengers, thereby modifying or attenuating their activities. Wunen, a Drosophila LPP homolog, has been shown to play a pivotal role in primordial germ cell (PGC) migration and survival during embryogenesis. It has been hypothesised that LPPs may form oligomeric complexes, and may even function as hexamers. This possibility was explored to confirm whether LPPs can oligomerise, and if they do, whether oligomerisation is required for either in vitro or in vivo activity. Evidence is presented that Wunen dimerises and that these associations require the last thirty-five C-terminal amino-acids and depend upon the presence of an intact catalytic site. Expression of a truncated, monomeric form of Wunen in Drosophila embryos results in perturbation of germ cell migration and germ cell loss, as observed for full-length Wunen. Murine LPP-1 and human LPP-3 can also form associations, but do not form interactions with Wunen or each other. Furthermore, Wunen does not form dimers with its closely related counterpart Wunen-2. Finally, addition of a trimeric myc tag to the C-terminus of Wunen does not prevent dimerisation or in vitro activity, but does prevent activity in vivo. It is concluded that LPPs do form complexes, but these do not seem to be specifically required for activity either in vitro or in vivo. Since neither dimerisation nor the C-terminus seem to be involved in substrate recognition, they may instead confer structural or functional stability through dimerisation. The results indicate that the associations seen are highly specific and occur only between monomers of the same protein (Burnett, 2004).
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