Lack of phosphomannomutase 2 affects Xenopuslaevis morphogenesis and the non-canonical Wnt5a/ Ror2 signalling
Nastassja Himmelreich1, Lilian T. Kaufmann2, Herbert Steinbeisser†2, Christian Körner†1, Christian Thiel1
Supplemental data
Identification of Pmm2 in Xenopuslaevis—As in a multitude of organisms ranging from human to yeast, also in Xenopuslaevis two genes are known to encode the evolutionarily related enzymes phosphomannomutase 1 (Pmm1; NM_001094315.1) and phosphomannomutase 2 (Pmm2; NM_001092472.1). In human and mouse both proteins are organ specific expressed and display PMM activity in functional assays but having different kinetics and a distinct substrate specificity, indicating that PMM1 and PMM2 may have distinct functions. While PMM2 deficiency is associated with CDG, the role of PMM1 in glycoprotein biosynthesis remains unsolved (Pirard et al 1999; Heykants et al 2001; Cromphout et al 2006). On protein level the frog Pmm1 and Pmm2 share a 68% identity which corresponds to the 65% identity between the human PMM1 and PMM2 proteins. Curiously, comparison of the human PMM2 or mouse Pmm2 with the Xenopus Pmm2 revealed an identity of 66%, while the Xenopus Pmm1 sequence was more than 74% identical to PMM2 of both mammalian species (analysed by MegAline, DNAStar, Lasergene Software; Supplemental Figure a). Since also the protein lengths and the protein composition of the frog Pmm1 correlates with human and mouse PMM2 better than the frog Pmm2 does (analysed by Protean, DNAStar, Lasergene Software; Supplemental Figure b), we assumed that the annotation of the Xenopus Pmm1 and Pmm2 sequences in NCBI was interchanged.
To conclusively verify the identity of both Xenopusphosphomannomutases, we cloned the coding regions of pmm1 and pmm2 in the mammalian expression vector pCI-neo, stably transfected the plasmids in COS-7 cells and measured the enzymatic activity in cytosolic extracts derived from the respective sample in a coupled photometric assay. Empty vector transfected COS-7 cells (activity set to 100% +/- 1.4%) and human control fibroblasts (96.9% +/-1.2%) show similar Pmm2 activity, indicating a comparable endogenous phosphomannomutase expression level. Notably, overexpression of NM_001092472.1 resulted in an only 2-fold higher (207.5% +/- 59.2%) activity, while overexpression of NM_001094315.1 led to a more than 4-fold higher conversion (411.6% +/- 97.7%) of NADP+ than the controls (Supplemental Figure c). Moreover, an antibody against the human PMM2 protein was not able to detect the protein derived from the sequence NM_001092472.1 but rather cross reacted with the protein obtained from NM_001094315.1 (Supplemental Figure d). Taken together, we conclude that NM_001094315.1 is truly the Xenopuslaevisorthologue of the human PMM2, while NM_001092472.1 most likely is the Xenopuslaevisorthologue of human PMM1. Therefore, further experiments were conducted on the basis of NM_001094315.1 as the Xenopus Pmm2.
Pmm2 is ubiquitous expressed during development—After identifying the Xenopus Pmm2, we investigated its developmental expression pattern by RT-PCR analysis of uninjected embryos ranging from stage NF stages 1 to 46. Before midblastula transition and onset of zygotic transcription, pmm2 is maternally expressed and transcript level remains constant (stages 1-9). Accordingly pmm2 transcription increases from early gastrula (stage 10) on throughout neurulation (stage 13-18) until tail bud stage (stage 20-30). From tail bud to tadpole (stages 42-46) pmm2 transcription is slightly declining again. This demonstrates that Pmm2 is continuously expressed throughout frog embryogenesis which further indicates its significant role during development (Supplemental Figure e).
Supplemental data figurelegend
Supplemental Figure 1: Identification of Pmm2 in Xenopuslaevis. a, Comparison of protein identity and divergence of human, mouse and Xenopuslaevisphosphomannomutases 1 and 2. Xenopuslaevis protein sequences of Pmm1 and Pmm2 were derived from NM_00194315.1 and NM_001092472.1. b, Kyte-Doolittle analysis of human and mouse phosphomannomutase 2 with Xenopuslaevisphosphomannomutase 1 and 2. The comparison of the hydrophilicity plots indicates that the frog Pmm1 is more related to human and mouse PMM2 than frog Pmm2. The circles point to divergent regions. c, Measurement of phosphomannomutase 2 activity in COS-7 cells. COS-7 cells expressing the open reading frames of NM_00194315.1 and NM_001092472.1 were analysed for their respective phosphomannomutase 2 activity. In contrast to mock transfected cells (pCI-neo) and human fibroblasts (human fibroblasts), activity of cells expressing the Xenopusphosphomannomutases (NM_00194315.1 and NM_001092472.1) were significantly higher. Expression of NM_00194315.1 showed the highest activity (n=3; **p≤0.005; ***p≤0.001). d, Western blot analysis for Pmm2 in COS-7 cells. COS-7 cells expressing the open reading frames of NM_00194315.1 and NM_001092472.1 were analysed by Western blot with an antibody against human PMM2 (top). In contrast to cells expressing the phosphomannomutase derived from NM_001092472.1 the antibody only cross-reacted with the phosphomannomutase of sequence NM_00194315.1. Even by doubling the amount of protein isolated from NM_001092472.1 expressing cells (see ß-actin; bottom) no signal was detected. e, Expression of pmm2 during early Xenopuslaevisembryogenesis. Expression of pmm2 was analysed by RT-PCR of stages NF 1-42 in comparison to the reference gene ornithine decarboxylase (odc). Beside the ubiquitous expression throughout the stages, pmm2 level is up-regulated through gastrulation, neurulation, tail bud phase and organogenesis (NF stages 10-30).
Citations
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