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) (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.
Chong
Jae Min
JM
Wistar Institute, Philadelphia, Pennsylvania 19104, USA.
Uren
Aykut
A
Rubin
Jeffrey S
JS
Speicher
David W
DW
D000595:Amino Acid Sequence; D000818:Animals; D001216:Asparagine / Q000737:chemistry; D001665:Binding Sites; D002851:Chromatography, High Pressure Liquid; D003545:Cysteine / Q000737:chemistry; D003553:Cystine / Q000737:chemistry; D004220:Disulfides; D004591:Electrophoresis, Polyacrylamide Gel; D051157:Frizzled Receptors; D006031:Glycosylation; D006493:Heparin / Q000737:chemistry; D006801:Humans; D008239:Lysine / Q000737:chemistry; D013058:Mass Spectrometry; D051379:Mice; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D000075386:Netrin Receptors; D010649:Phenylalanine / Q000737:chemistry; D011485:Protein Binding; D017434:Protein Structure, Tertiary; D011506:Proteins / Q000737:chemistry*; D011956:Receptors, Cell Surface / Q000737:chemistry*; D011994:Recombinant Proteins / Q000378:metabolism; D017386:Sequence Homology, Amino Acid; D012694:Serine / Q000737:chemistry; D019032:Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization; D020860:Subtilisin / Q000737:chemistry; D013997:Time Factors; D014981:Xenopus
D004220:Disulfides; D051157:Frizzled Receptors; D000075386:Netrin Receptors; D011506:Proteins; D011956:Receptors, Cell Surface; D011994:Recombinant Proteins; D012694:Serine; D010649:Phenylalanine; D003553:Cystine; D001216:Asparagine; D006493:Heparin; D020860:Subtilisin; D008239:Lysine; D003545:Cysteine