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Gene
1999 Oct 01;2382:301-13. doi: 10.1016/s0378-1119(99)00365-0.
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Functional and structural diversity of the human Dickkopf gene family.
Krupnik VE, Sharp JD, Jiang C, Robison K, Chickering TW, Amaravadi L, Brown DE, Guyot D, Mays G, Leiby K, Chang B, Duong T, Goodearl AD, Gearing DP, Sokol SY, McCarthy SA.
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Wnt proteins influence many aspects of embryonic development, and their activity is regulated by several secreted antagonists, including the Xenopus Dickkopf-1 (xDkk-1) protein. xDkk-1 inhibits Wnt activities in Xenopus embryos and may play a role in induction of head structures. Here, we characterize a family of human Dkk-related genes composed of Dkk-1, Dkk-2, Dkk-3, and Dkk-4, together with a unique Dkk-3 related protein termed Soggy (Sgy). hDkks 1-4 contain two distinct cysteine-rich domains in which the positions of 10 cysteine residues are highly conserved between family members. Sgy is a novel secreted protein related to Dkk-3 but which lacks the cysteine-rich domains. Members of the Dkk-related family display unique patterns of mRNA expression in human and mouse tissues, and are secreted when expressed in 293T cells. Furthermore, secreted hDkk-2 and hDkk-4 undergo proteolytic processing which results in cleavage of the second cysteine-rich domain from the full-length protein. Members of the human Dkk-related family differ not only in their structures and expression patterns, but also in their abilities to inhibit Wnt signaling. hDkk-1 and hDkk-4, but not hDkk-2, hDkk-3 or Sgy, suppress Wnt-induced secondary axis induction in Xenopus embryos. hDkk-1 and hDkk-4 do not block axis induction triggered either by Xenopus Dishevelled (Xdsh) or Xenopus Frizzled-8 (Xfz8), both of which function to transduce signals from Wnt ligands. Thus, hDkks 1 and 4 may inhibit Wnt activity by a mechanism upstream of Frizzled. Our findings highlight the structural and functional heterogeneity of human Dkk-related proteins.
Fig. 1.
The human Dkk family. (A) Schematic illustration of the human Dkk/Sgy family showing signal peptides (darkened boxes), Cysteine-rich domains (Cys-1 and Cys-2) and putative sites of N-linked glycosylation in the human Dkks (branches). (B) Percent identities between full-length protein sequences for hDkks 1–4, mDkk-1 (AF030433), mDkk-3, cDkk-3 (D26311) and xDkk-1 (AF030434). Figures were generated using the Smith–Waterman algorithm as implemented in the program Bestfit of the GCG package, with gap penalties of 8 for opening and 1 for extending. All 20 cysteines were aligned in all but five of the 28 comparisons, in which the final cysteine was mis-matched. (C) Multiple sequence alignment of different Dkk proteins. Alignments were performed with the ClustalW algorithm as implemented in the GCG program PILEUP. Predicted signal peptides (Nielsen et al., 1997) are underlined, putative N-glycosylation sites are indicated by a thick bar, Cys-1 by an open box, Cys-2 by a shaded box. The proteolytic cleavage site within hDkk-4 is indicated by an arrow. (D) Multiple sequence alignment of hDkks with human colipase (J02883). Cys-2 of each hDkk is shown and indicated by the shaded box. The human colipase sequence is shown missing eight amino acids from the N-terminus of the mature peptide. The alignment was made using gap penalties of 12 for opening and 2 for extending. A minor adjustment was necessary, since PILEUP inserts a single gap in hDkk-1 and hDkk-2 between GS at position 56–57, even with gap opening penalty of 15. hDkk-1 is shown from amino acid 181. (E) Multiple sequence alignment of Dkk-3 and Soggy proteins. Signal peptides and N-glycosylation sites are indicated as in (C). Cys-1 (open box) and Cys-2 (shaded box) within Dkk-3 are shown for reference. Asterisks indicate amino-acid identities between all four proteins. Soggy protein sequences terminate at #.
Fig. 6.
Effect of hDkks and Sgy on Wnt-induced axis duplication in Xenopus embryos. Effect of hDkks and hSgy on Xwnt8-induced secondary axis formation in Xenopus embryos. Four or eight cell stage embryos were injected subequatorially into a ventralblastomere with mRNAs for: (A) Xwnt8, (B) Xwnt8+hDkk-1, (C) Xwnt8+hDkk-2, (D) Xwnt8+hDkk-3, (E) Xwnt8+hSgy, (F) Xwnt8+hDkk-4. Four representative embryos from multiple injections are shown at stage 35–37 in each panel.
Fig. 7.
Effect of human Dkk proteins on Wnt, Dsh and Fz-induced axis duplication. Four- or eight-cell stage embryos were injected subequatorially into a ventral blastomere with mRNAs for Xwnt-8, Xwnt-2b, Xwnt-3a, Xfz8 or Xdsh in the presence or absence of mRNAs for either hDkk-1, hDkk-3, hDkk-4 or hSgy as indicated. Embryos were allowed to develop until stage 33–37, then scored for secondary axis formation. Complete axes included eyes and cement glands; partial axes included partial neural tubes and hindbrain, but lacked fore/midbrain structures. n=number of embryos in the experiment; open bars, % of embryos with complete secondary axes; filled bars, % of embryos with partial secondary axes.
