PMID: 21805521

 

    Legend: Gene, Sites

Title : Human Dickkopf-1 ( huDKK1 ) protein : characterization of glycosylation and determination of disulfide linkages in the two cysteine-rich domains

Abstract :
  1. 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
  2. HuDKK1 is biologically active in a TCF/lef-luciferase reporter gene assay and is able to bind LRP6 coreceptor
  3. In SDS-PAGE, huDKK1 exhibits molecular weights of 27-28 K and 30 K at ∼ 1 :9 ratio
  4. 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
  5. 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%)
  6. 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
  7. 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
  8. 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
  9. The five disulfide bonds assigned in the C-terminal domain show the expected homology with those found in colipase and other reported disulfide clusters
Output (sent_index, trigger, protein, sugar, site):
  • 5. Asn²²⁵, , -, three N-linked glycans, Asn²²⁵
  • 5. glycosylated, , huDKK1, -, Asn²²⁵
  • 6. attached, , -, GalNAc (sialic acid)-Gal-sialic acid, Ser³⁰
  • 6. attached, , -, GalNAc-Gal[sialic acid], Ser³⁰
  • 6. attached, , -, two O-linked glycans, Ser³⁰
Output(Part-Of) (sent_index, protein, site):
  • 8. huDKK1, domain
*Output_Site_Fusion* (sent_index, protein, sugar, site):

 

 

Protein NCBI ID SENTENCE INDEX
Human Dickkopf-1 22943 1
Dickkopf-1 22943 0