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 positioning 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.
authors.affiliation: Department of Protein Sciences, Amgen, Inc., One Amgen Center Dr., Thousand Oaks, California 91320, USA.
authors.forename: Mitsuru
authors.initials: M
authors.lastname: Haniu
authors.forename: Tom
authors.initials: T
authors.lastname: Horan
authors.forename: Chris
authors.initials: C
authors.lastname: Spahr
authors.forename: John
authors.initials: J
authors.lastname: Hui
authors.forename: Wei
authors.initials: W
authors.lastname: Fan
authors.forename: Ching
authors.initials: C
authors.lastname: Chen
authors.forename: William G
authors.initials: WG
authors.lastname: Richards
authors.forename: Hsieng S
authors.initials: HS
authors.lastname: Lu
mesh_terms: D000595:Amino Acid Sequence; D000818:Animals; D016466:CHO Cells; D002236:Carbohydrate Conformation; D002240:Carbohydrate Sequence; D002460:Cell Line; D006224:Cricetinae; D003545:Cysteine / Q000737:chemistry / Q000378:metabolism; D004220:Disulfides; D004591:Electrophoresis, Polyacrylamide Gel; D006031:Glycosylation; D036341:Intercellular Signaling Peptides and Proteins / Q000737:chemistry / Q000378:metabolism*; D008969:Molecular Sequence Data; D010449:Peptide Mapping; D011134:Polysaccharides / Q000737:chemistry / Q000378:metabolism; D017433:Protein Structure, Secondary; D017434:Protein Structure, Tertiary; D020539:Sequence Analysis, Protein; D019032:Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization; D060449:Wnt Signaling Pathway
chemical_list: C501126:DKK1 protein, human; D004220:Disulfides; D036341:Intercellular Signaling Peptides and Proteins; D011134:Polysaccharides; D003545:Cysteine