Title : 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 Asn(172) (approximately 2.8 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
Output (sent_index, trigger,
protein,
sugar,
site):
- 8. contains, , sFRP-1, a relatively large carbohydrate moiety, -
- 8. glycosylation, , -, -, Asn(262)
- 8. glycosylation, , -, -, site
- 8. glycosylation, , -, -, sites
- 8. moiety, , -, 172, Asn(172)
- 8. moiety, , -, Asn, Asn(172)
Output(Part-Of) (sent_index,
protein,
site):
- 1. Secreted Frizzled-related protein-1, domain
- 1. protein, domain
- 1. sFRP-1, domain
- 11. protein, Asp(41) and Phe(50)
- 4. Numbering sFRP-1, cysteines
- 4. proteins, domains
- 6. sFRP-1, domain
- 7. protein, domain
*Output_Site_Fusion* (sent_index,
protein,
sugar,
site):
- 8. sFRP-1, -, Asn(262)
- 8. sFRP-1, 172, Asn(172)
- 8. sFRP-1, Asn, Asn(172)