| Summary: | 碩士 === 國立清華大學 === 生物資訊與結構生物研究所 === 95 === Members of the highly conserved Wnt secreted protein family encode critical mediators of cell-cell signaling events. Wnt gene family influence many aspects of embryonic development, and are required for adult tissue maintenance. Previous studies have also indicated that perturbation of the Wnt signaling is associated with human degenerative diseases and cancers. Wnt activity is regulated by several secreted proteins, including the conserved Dickkopf (DKK) protein family. DKK-related family members also play a role in embryonic development and cancers.
In previous clinical studies, we have identified a SNP in DKK2 exon_01_+791 G>T. This nucleotide change leads to Gly to Val substitution at amino acid position 29. DKK2 is a secretory glycoprotein which modulates canonical Wnt signaling levels (beta-catenin). Therefore, we hypothesized this amino acid change could influence the secretion of DKK2.
In order to explore this SNP function, we established in vitro system of Flp-In T-REx-293 cell line containing inducible DKK2 expression plasmids (pcDNA5/TO/FRT-DKK2-flag: wild type, mutant type, and vector-only).These plasmids were generated by specific Flp-mediated recombination at a specific genome site. The copy number was checked by Southern blot. Then, we compared DKK2 protein level in cell lysate and medium to demonstrate the influence of the SNP. On the other hand, canonical Wnt3a signaling (TCF/ beta-catenin) reporter assay was performed to demonstrate the difference between wild type and mutant type of DKK2-signaling.
Immunoblots revealed multiple forms of DKK2 in cell lysate and medium. In medium the amount of large-size DKK2(34 kD) seemed higher in wild type but small-size of DKK2(13.5 kD) seemed more in mutant type.On the other hand, in cell the amount of large-size form (33.5 kD) was higher in mutant type and small-size form (30.5 kD) was more in wild type after 24hr induction. According to previous studies, this size variation may be a result of post-translation modification or proteolytic processing. In fact, we discovered that these size variations are due to glycosylation by glycodidase mobility shift assay.
In conclusion, alteration in the signal peptide of DKK2 affects the different amount of intermediates in the DKK2 protein sorting pathway. We deduce a hypothisis that the SNP will cause different rate of DKK2 modification or secretion. These post-translational events may affect the function of DKK2 and, therefore, the Wnt-signaling.
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