Title : Human
Dickkopf-1 (
huDKK1 )
protein : characterization of glycosylation and determination of disulfide linkages in the two
cysteine-rich
domains
Abstract :
- Human Dickkopf-1 ( huDKK1 ), an inhibitor of the canonical Wnt-signaling pathway that has been implicated in bone metabolism and other diseases, was expressed in engineered Chinese hamster ovary cells and purified
- HuDKK1 is biologically active in a TCF/lef-luciferase reporter gene assay and is able to bind LRP6 coreceptor
- In SDS-PAGE, huDKK1 exhibits molecular weights of 27-28 K and 30 K at ∼ 1 :9 ratio
- By MALDI-MS analysis, the observed molecular weights of 27.4K and 29.5K indicate that the low molecular weight form may contain O-linked glycans while the high molecular weight form contains both N- and O-linked glycans
- LC-MS/MS peptide mapping indicates that ∼ 92% of huDKK1 is glycosylated at Asn²²⁵ with three N-linked glycans composed of two biantennary forms with 1 and 2 sialic acid (23% and 60%, respectively), and one triantennary structure with 2 sialic acids (9%)
- HuDKK1 contains two O-linked glycans, GalNAc (sialic acid)-Gal-sialic acid (65%) and GalNAc-Gal[sialic acid] (30%), attached at Ser³⁰ as confirmed by β-elimination and targeted LC-MS/MS
- The 10 intramolecular disulfide bonds at the N- and C-terminal cysteine-rich domains were elucidated by analyses including multiple proteolytic digestions, isolation and characterization of disulfide-containing peptides , and secondary digestion and characterization of selected disulfide-containing peptides
- The five disulfide bonds within the huDKK1 N-terminal domain are unique to the DKK family proteins ; there are no exact matches in disulfide position ing when compared to other known disulfide clusters
- The five disulfide bonds assigned in the C-terminal domain show the expected homology with those found in colipase and other reported disulfide clusters