van Rosmalen JW and Martens GJ (2007), Mutagenesis studies in transgenic Xenopus inter...

XB-ART-35797

Dev Neurobiol 2007 May 01;676:715-27. doi: 10.1002/dneu.20330.

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Mutagenesis studies in transgenic Xenopus intermediate pituitary cells reveal structural elements necessary for correct prion protein biosynthesis.

van Rosmalen JW , Martens GJ .

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The cellular prion protein (PrP(C)) is generally accepted to be involved in the development of prion diseases, but its physiological role is still under debate. To obtain more insight into PrP(C) functioning, we here used stable Xenopus transgenesis in combination with the proopiomelanocortin (POMC) gene promoter to express mutated forms of Xenopus PrP(C) fused to the C-terminus of the green fluorescent protein (GFP) specifically in the neuroendocrine Xenopus intermediate pituitary melanotrope cells. Similar to GFP-PrP(C), the newly synthesized GFP-PrP(C)K81A mutant protein was stepwise mono- and di-N-glycosylated to 48- and 51-kDa forms, respectively, and eventually complex glycosylated to yield a 55-kDa mature form. Unlike GFP-PrP(C), the mature GFP-PrP(C)K81A mutant protein was not cleaved, demonstrating the endoproteolytic processing of Xenopus PrP(C) at lysine residue 81. Surprisingly, removal of the glycosylphosphatidylinositol (GPI) anchor signal sequence or insertion of an octarepeat still allowed N-linked glycosylation, but the GFP-PrP(C)DeltaGPI and GFP-PrP(C)octa mutant proteins were not complex glycosylated and not cleaved, indicating that the GPI/octa mutants did not reach the mid-Golgi compartment of the secretory pathway. The transgene expression of the mutant proteins did not affect the ultrastructure of the melanotrope cells nor POMC biosynthesis and processing, or POMC-derived peptide secretion. Together, our findings reveal the evolutionary conservation of the site of metabolic cleavage and the importance of the presence of the GPI anchor and the absence of the octarepeat in Xenopus PrP(C) for its correct biosynthesis.

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Species referenced: Xenopus laevis
Genes referenced: c4bpa gnpda1 gpi myh3 pomc prnp sod1

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	Figure 1 Intermediate pituitary-specific fluorescence and transgene expression in Xenopus transgenic for GFP-PrPC, GFP-PrPCK81A, GFP-PrPCDGPI, GFP-PrPCocta, orGFP-GPI. A. Schematic representation of the linear injection fragments pPOMC-GFP-PrP, pPOMC-GFP-PrPK81A, pPOMCGFP- PrPDGPI, pPOMC-GFP-PrPocta, and pPOMC-GFP-GPI containing the Xenopus POMC gene A promoter fragment (pPOMC), and the GFP-PrP, GFP-PrPK81A, GFP-PrPDGPI, GFP-PrPocta, and GFP-GPI fusion protein-coding sequence, which were used to generate transgenic Xenopus lines 102, 140, 160, 170, and 150, respectively; SS, signal peptide sequence; octa, human octarepeat; *, location of the mutation of the metabolic cleavage site; GPI, glycosylphosphatidylinositol signal sequence. B. Intermediate pituitary-specific fluorescence in a living Xenopus F1 embryo (stage 40, staging according to Nieuwkoop and Faber, 1967; left panel) and in a black-adapted 6-month-old frog transgenic for GFP-PrPCDGPI (ventrocaudal view; right panel) as a representive example of the various transgenic Xenopus F1 and F2 lines used in this study. In adult Xenopus, the brain was lifted to reveal intense fluorescence in the intermediate lobe (IL), but not in the anterior lobe (AL) of the pituitary. Arrows indicate the localizations of the fluorescent intermediate pituitaries expressing the fusion product; the positions of the eye (E), nose (N), and gut (G) are also indicated in the tadpole. Bars equal 0.5 mm. C. Tissue lysates of neurointermediate lobes (NILs) and ALs from wild-type (wt) animals and animals transgenic for the GFP-PrPC (102), GFP-PrPCK81A (140), GFP-PrPCDGPI (160), GFP-PrPCocta (170), or GFP-GPI (150) fusion protein were subjected to SDS-PAGE. Western blot analysis was performed using an anti-GFP antibody (a-GFP). [Color figure can be viewed in the online issue, which is available at www.interscience.wiley.com.]
	Figure 2 Biosynthesis of the mutated forms of the GFP-PrPC protein in transgenic Xenopus intermediate pituitary cells. A. In lane 1, 5% of a total neurointermediate lobe (NIL) lysate (total) from wild-type (wt) Xenopus was directly loaded on the gel. A–F. Wt NILs and NILs transgenic for GFP-PrPC (102), GFP-PrPCK81A (140), GFP-PrPCDGPI (160), GFP-PrPCocta (170), or GFP-GPI (150) were pulse labeled with [35S]-Met/Cys for 30 min (P30), or 30-min pulse labeled and chase incubated for 180 min (P30C180), and subsequently the proteins were extracted. B–F. The extracted proteins were incubated in the presence (þ) or absence () of peptide N-glycosidase F (PNgase F) or phosphatidylinositol-specific phospholipase C (PIPLC). In all cases, newly synthesized proteins extracted from the lobes or secreted into the incubation medium were immunoprecipitated using an anti-GFP antibody, and the immunoprecipitates were resolved by SDS-PAGE on a 15% gel, and the radiolabeled proteins were visualized by autoradiography. Asterisks in A indicate major NIL proteins (proPC2 and POMC) nonspecifically interacting with the anti-GFP antibody.
	Figure 3 Biosynthesis and processing of newly synthesized POMC in wild-type (wt) Xenopus intermediate pituitary cells and cells transgenic for the GFP-PrPC (102), GFP-GPI (150), GFPPrP CK81A (140), GFP-PrPCDGPI (160), or GFP-PrPCocta (170) fusion protein. A. Neurointermediate lobes (NILs) from wt and transgenic animals were pulse labeled with [35S]-Met/Cys for 30 min and subsequently chased for 180 min. Newly synthesized proteins extracted from the lobes (5%) or secreted into the incubation medium (20%) were resolved by SDS-PAGE on 15% gels and visualized by autoradiography. B. The amounts of newly synthesized 37-kDa POMC and the 18-kDa POMC-derived product were quantified by densitometric scanning and are presented in arbitrary units (AU), relative to the amounts of newly synthesized actin. Shown are the means 6 SEM (n ¼ 3). Significant differences are indicated by (p < 0.01) or *(p < 0.001). In all cases, animals were adapted to a black background for >8 weeks (long-term adaptation).
	Figure 4 Electron microscopy on wild-type Xenopus melanotrope cells and cells transgenic for the GFP-PrPC, GFP-PrPCK81A, GFP-PrPCDGPI, GFP-PrPCocta, or GFP-GPI protein. Electron micrographs of wild-type (wt; A), GFP-PrPC- (102; B), GFP-PrPCK81A- (140; C), GFP-PrPCDGPI- (160; D), GFP-PrPCocta- (170; E), and GFP-GPI-transgenic (150; F) melanotrope cells. Animals were adapted to a black background for >8 weeks. Melanotrope cells from transgenic line 102 contain pleiomorph electron-dense crinosomes with capricious extensions, indicative of the process of crinophagy (B). Cr, crinosome; ER, endoplasmic reticulum; N, nucleus; Sg, secretory granules. Bars equal 1 lm.