Wnt signaling involves multiple pathways and mediates embryonic induction, generation of cell polarity, specification of cell fate (Cadigan & Nusse (1997) Genes Dev. 11(24):3286-305; Peifer & Polakis (2000) Science 287(5458):1606-9), as well as being closely linked to tumorigenesis (Peifer & Polakis (2000) supra). Wnt signaling is also regulated by several types of endogenous antagonists (Kawano & Kypta (2003) J. Cell Sci. 116 (Pt 13):2627-34), where Dickkopf (Dkk) is probably the most notable Wnt antagonist inhibiting the canonical Wnt signaling pathway. The initiation of canonical Wnt/β-catenin signaling pathway requires the binding of secreted Wnt proteins to receptor Frizzled (Fz) proteins (Bhanot, et al. (1996) Nature 382(6588):225-30) and coreceptor LDL receptor-related protein 5 or 6 (LRP5/6) (Mao, et al. (2001) Mol. Cell. 7(4):801-9; Mao, et al. (2001) Nature 411(6835):321-5; Pinson, et al. (2000) Nature 407(6803):535-8; Tamai, et al. (2000) Nature 407(6803):530-5). To block the canonical Wnt signaling pathway, Dkk binds to LRP5/6 and another single transmembrane receptor Kremen simultaneously (Semënov, et al. (2001) Curr. Biol. 11(12):951-61; Mao & Niehrs (2003) Gene 302(1-2):179-83; Mao, et al. (2001) supra). The ternary DKK-Kremen-LRP5/6 complex not only prevents Wnt from interacting with LRP5/6, but also promotes the rapid internalization and removal of LRP5/6 from plasma membrane, further inhibiting the canonical Wnt signaling (Mao & Niehrs (2003) supra).
Four members of the Dkk family have been identified in mammals (Krupnik, et al. (1999) Gene 238(2):301-13). Dkk1, the most extensively studied member, was originally cloned as a molecule that is able to induce secondary axes with a complete head when its mRNA is injected into Xenopus embryos together with a dominant-negative mutant of the BMP-2/4 receptor (Glinka, et al. (1998) Nature 391(6665):357-62). The characteristic developmental function of Dkk1 is its head-inducing activity in vertebrate embryos (Glinka, et al. (1998) supra), a process that has been postulated to involve inhibition of Wnt signaling (Glinka, et al. (1997) Nature 389(6650):517-9).
Members of the Dkk family are composed of two characteristic cysteine-rich domains (CRDs) separated by a variable-length spacer region, each domain containing 10 conserved cysteines (Krupnik, et al. (1999) supra). Both domains remain well conserved among all four members; in particular, Dkk1 and Dkk2 share 50% identity in their N-terminal cysteine-rich region amino acid sequences and 70% identity in their C-terminal regions. Among the four Dkk members, Dkk1 and Dkk4 appear indistinguishable in terms of Wnt antagonist activity, whereas Dkk3 does not appear to modulate Wnt signaling (Krupnik, et al. (1999) Gene). However, Dkk2 is more complicated, since it functions as a Wnt activator or a Wnt inhibitor in a cell-context dependent way. On the other hand, previous studies have demonstrated that the C-terminal cysteine-rich domains of Dkk1 and Dkk2 behave similarly to one another in Wnt signaling: in isolation they are both necessary and sufficient for physically associating with LRP5/6 and inhibiting canonical Wnt signaling (Brott & Sokol (2002) Mol. Cell. Biol. 22(17):6100-10; Li, et al. (2002) J. Biol. Chem. 277(8):5977-81). By contrast, the N-terminal cysteine-rich domain of Dkk1/2 appears to play a regulatory role in these interactions, and likely responsible for the different activities of the intact Dkk1 and Dkk2 proteins (Brott & Sokol (2002) supra).
Despite the important roles of Dkk in regulating Wnt signaling, the particular molecular mechanism that results from Dkk interaction with LRP5/6 is not completely understood. Much of the information on the functions of the various Dkk genes has been derived from studies with cloned versions of Dkk, including the entire Dkk gene as well as portions that comprise only a single CRD domain of Dkk. Thus, the properties of the CRDs have been investigated by isolation of the individual domains and testing their effects upon wnt activity (Li, et al. (2002) supra) as well as more elaborate experiments where fusion proteins were created with the N-terminal CRD of Dkk1 fused to the carboxy-terminal CRD of Dkk2 and vice versa (Brott & Sokol (2002) supra).
In most cases, expression of the Dkk proteins has been limited to eukaryotic expression vectors, while in the case of Dkk1, prokaryotic vectors have also been used (Gregory, et al. (2003) J. Biol. Chem. 278:28067-28078; U.S. Patent Application 20080038775). In general, expression of eukaryotic genes in eukaryotic host cells insures the likelihood of the correct folding as well as allowing postsynthetic modifications such as glycosylation or protease cleavages. On the other hand, yields of target proteins are limited, since a large number of different proteins are expressed in the eukaryotic host cells and there are expenses associated with media and growth. In contrast, media for prokaryotic expression is very inexpensive and high yields of proteins can be achieved. The limiting factor in prokaryotic systems is that folding of proteins can be problematic both immediately after synthesis of the proteins as well as in steps that may be carried out at a later stage. The latter effect is due to the expression of the proteins commonly being found in the form of what are called “inclusion bodies”, insoluble masses of proteins that require essentially denaturing conditions to render them into soluble functional form.
Thus, although the ultimate yield of functional proteins may be high in prokaryotic systems, the specific activity may be much lower since a large number of inactive forms may be present as well. In some cases, the presence of the inactive forms is irrelevant since they may simply act as passive carriers, but other studies, such as crystallographic studies or binding experiments, depend upon the availability of purified highly active forms. Further problems are also specifically seen with the prokaryotically-derived Dkk proteins. For instance, yields of Dkk1 have been reported as being relatively low, with more than 60% of the product containing intermolecular cross-links between two different Dkk molecules (Gregory, et al. (2003) supra), thereby limiting proper intramolecular disulfide bond formation and altering the ultimate configuration of the Dkk protein. Treatment of inclusion bodies with either Guanidine Chloride or Urea (two standard solubilization methods for inclusion bodies) has been suggested; however, the resulting preparations were devoid of activity in both co-immuno-precipitation assays and TCF reporter assays (see U.S. Patent Application 20080038775). As such, there is a need in the art for an inexpensive source of Dkk, which is both soluble and active, for use in screening assays and in the analysis of Dkk and its domains.
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USPTO Backgrounds