In this manuscript we investigated whether Dpl reveals the same properties as PrPc regarding self-interaction and binding to the 37-kDa/67-kDa laminin receptor employing the yeast two- hybrid system (for review see (Vidal and Legrain, 1999)). We PCR-amplified on cDNA level the mature form of Doppel termed Dpl27-154 employing oligodeoxy ribonucleotides flanking the Dpl-sequence introducing an EcoRI (5‘) and a SalI (3‘) restriction site from cDNA generated from cultivated HeLa cells by RT-PCR. The PCR-product was cloned into the vector pSH2-1 via EcoRI and SalI restriction sites resulting in pSH2-1-Dpl27-154. The Dpl27-154 encoding cDNA was excised from pSH2-1-via EcoRI (5‘) and SalI (3‘) and subcloned into pJG4-5 restricted with EcoRI and XhoI resulting in pJG4-5-Dpl27-154. All plasmids were confirmed by didesoxy sequencing. The construction of pSH2-1-GST, pSH-2- 1-GST::huPrP23-230 and pJG4-5-LRP44-295 was described previously (Rieger et al., 1997). The different bait and prey plasmids and the reporter plasmid pSH18-34 (lacZ) were co- transformed into EGY48 yeast cells and transformants were tested employing the b- galactosidase assay.
The Dpl27-154 protein failed to interact with GST::huPrP23-230 (Fig. 1 row 2). GST in prey position did not interact with Dpl (Fig. 1 row 1) demonstrating that there is no unspecific effect of the GST part of the GST::huPrP23-230 fusion protein. In contrast to GST::huPrP23- 230 (Fig.1 row5) Dpl27-154 failed to interact with LRP44-295, the cellular receptor for PrP
(Fig. 1 row 3). In contrast to PrP which interact with each other (Hundt et al., submitted), Dpl27-154 did not interact with itself in the yeast two-hybrid system (Fig.1 row 4). To test whether Dpl 27-154 is expressed in the yeast two-hybrid system we co-transformed yeast cells EGY48 with pSH2-1-Dpl27-154 (bait-protein) and pJG4-5-Dpl27-154 (prey protein). Western-blot analysis with a polyclonal anti-Dpl antibody (kindly provided by H. M. Schätzl, Munich) revealed that the two fusion proteins were properly expressed (Fig. 2). The observed molecular weights of 21 kDa and 36 kDa for the bait and prey proteins match very well with calculated molecular weights of 21 kDa (lexA-DNA-binding domain: 7 kDa and Dpl27-154: 14 kDa) and 36 kDa (acidic activation domain B42: 22 kDa and Dpl27-154: 14 kDa). Therefore we can exclude that the lack of interaction of Dpl with PrP, LRP and itself is a consequence of non-expressed bait- and prey proteins.
Discussion
The incapability of Dpl to dimerize with itself was recently proposed by modelling the potential interaction interface (Warwicker, 2000). According to the model PrP dimerization might take place via a b-hairpin structure located between aa 119 and 128, a non-polar region lacking in the Dpl protein. We have further evidence that the major interaction domain for the PrP/PrP interaction is the octarepeat region (Hundt et al., submitted) which is highly conserved in different mammalian species (Schätzl et al., 1995). Besides the capability of the octarepeat region to bind HSPGs and therefore mediating the interaction between PrP and the 37-kDa/67-kDa laminin receptor (Hundt et al., 2001) one feature represents the binding of copper (Aronoff-Spencer et al., 2000) and the possibility to make a copper-mediated protein- protein interactions. In Alzheimer’s disease, which represents a neurologic disease comparable to TSEs copper-induced aggregation is observed (Atwood et al., 1998). An analogous induction of the aggregation of the prion protein which occurs during the disease process might be conceivable. The octarepeat region is missing within the Dpl protein and therefore metal-induced dimerization is unlikely. In a yeast two-hybrid analysis we were able to detect a further interaction domain for the PrP/PrP interaction which is located in the core region of the prion protein between aa 90-230 (Hundt et al., submitted). This core region shows similarities to the structured region of Dpl which was analysed by NMR (Mo et al., 2001). Dimerization via this region is conceivable analogous to PrP. In contrast to PrP, Dpl is able to form two disulfide bridges confirmed by several methods (Mo et al., 2001). This may
confer the structure of Dpl a more rigid behaviour which probably prevent the attachment of a second Dpl molecule forming a dimer. The formation of two disulfide bridges in the Doppel protein might as well not allow domain swapping which contribute to dimer formation of PrP observed in the investigation of the crystal structure of PrP (Knaus et al., 2001).
The different structural features of PrP and Dpl might also be the reason for the absence of an interaction of Dpl with the 37-kDa/67-kDa laminin receptor, the receptor for cellular PrP (Gauczynski et al., 2001b). We can not exclude that additional factors which are not present in the nucleus of the yeast cells might mediate the Dpl-LRP interaction. For PrP HSPGs arbitrate the interaction with LRP/LR detected in cell binding assays (Hundt et al., 2001). Whether HSPGs or other factors have an influence on the binding of Doppel to LRP has to be further investigated.
Dpl fails to interact with PrP. This might be explained with the different expression patterns of PrP and Dpl in vivo. PrP is expressed mainly in neuronal tissues and in the brain, which is the most relevant location for the development of TSEs. In contrast, the main expression locus for Dpl represents the testis (Silverman et al., 2000) and the heart of wild-type mice (Moore et al., 1999). Dpl was not detectable in the brain of wild-type mice. Mice however, lacking the prion gene express Dpl in the brain (Moore et al., 1999). Therefore, the failure of interaction of PrP and Dpl in the yeast two-hybrid system is not astonishing. Whether Dpl can fulfill the functions of PrP in PrP0/0 cells remains still unclear. The transport of copper and SOD activity proposed functions of the prion protein might not be adopted by Dpl due to the lack of the copper-binding octarepeat region. The role of Dpl in signal transduction necessary for cell survival postulated by Shmerling (Shmerling et al., 1998) has also to be further investigated.
Table 1: Comparison of Dpl and PrP