Disulfide Bond Assignments of Secreted Frizzled-related Protein-1 Provide Insights about Frizzled Homology and Netrin Modules*

Abstract

Secreted Frizzled-related protein-1 (sFRP-1), a soluble protein that binds to Wnts and modulates Wnt signaling, contains an N-terminal domain homologous to the putative Wnt-binding site of Frizzled (Fz domain) and a C-terminal heparin-binding domain with weak homology to netrin. Both domains are cysteine-rich, having 10 and 6 cysteines in the Fz and heparin-binding domains, respectively. In this study, the disulfide linkages of recombinant sFRP-1 were determined. Numbering sFRP-1 cysteines sequentially from the N terminus, the five disulfide linkages in the Fz domain are 1–5, 2–4, 3–8, 6–10, and 7–9, consistent with the disulfide pattern determined for homologous domains of several other proteins. The disulfide linkages of the heparin-binding domain are 11–14, 12–15, and 13–16. This latter set of assignments provides experimental verification of one of the disulfide patterns proposed for netrin (NTR) modules and thereby supports the prediction that the C-terminal heparin-binding domain of sFRP-1 is an NTR-type domain. Interestingly, two subsets of sFRPs appear to have alternate disulfide linkage patterns compared with sFRP-1, one of which involves the loss of a disulfide due to deletion of a single cysteine from the NTR module, whereas the remaining cysteine may pair with a new cysteine introduced in the Fz domain of the protein. Analysis of glycosylation sites showed that sFRP-1 contains a relatively large carbohydrate moiety on Asn172 (∼2.8 kDa), whereas Asn262, the second potential N-linked glycosylation site, is not modified. No O-linked carbohydrate groups were detected. There was evidence of heterogeneous proteolytic processing at both the N and C termini of the recombinant protein. The predominant N terminus was Ser31, although minor amounts of the protein with Asp41 and Phe50 as the N termini were observed. The major C-terminal processing event was removal of the terminal amino acid (Lys313) with only a trace amount of unprocessed protein detected.

  • Abbreviations:
    Fz
    Frizzled
    LRP
    low density lipoprotein receptor-related protein
    sFRP
    secreted Frizzled related protein
    CRD
    cysteine-rich domain
    MALDI
    matrix-assisted laser desorption/ionization
    MS
    mass spectrometry
    TCEP
    tris-(2-carboxyethyl)-phosphine
    MDCK
    Madin-Darby canine kidney
    CNBr
    cyanogen bromide
    NTR
    netrin
    PCOLCE
    procollagen C-proteinase enhancer protein
    WFIKKN
    WAP, Fs, Ig, Ku, and NTR protein
    TIMP
    tissue inhibitors of metalloproteinases
    Szl
    Sizzled
    RP-HPLC
    reversed phase-high performance liquid chromatography
    GSK-3β
    glycogen synthesis kinase 3β
    Tricine
    N-tris(hydroxymethyl)methylglycine
    • Received September 5, 2001.
    • Revision received December 7, 2001.
    Table of Contents

    This Article

    1. The Journal of Biological Chemistry 277, 5134-5144.
    1. All Versions of this Article:
      1. M108533200v1
      2. 277/7/5134 (most recent)

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