Disulfide bond assignments of secreted frizzled-related protein-1 provide insights about frizzled homology and netrin modules

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 Asn(172) (similar to2.8 kDa), whereas Asn(262), 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 Ser(31), although minor amounts of the protein with Asp(41) and Phe(50) as the N termini were observed. The major C-terminal processing event was removal of the terminal amino acid (Lys(313)) with only a trace amount of unprocessed protein detected.