| The Wnt genes encode a large family of glycoproteins that are essential in development and tissue maintenance.1 Members of the Frizzled family of proteins serve as receptors for the Wnt signaling pathway (2). Ten mouse and human Frizzled genes have been identified to date. The predicted structure of Frizzled proteins is similar among all family members, containing a divergent N-terminal signal peptide, a highly conserved extracellular cysteine-rich domain (CRD), a variable-length linker region, a seven-pass transmembrane region, and a variable-length C-terminal cytoplasmic domain. The CRD, which comprises 642 amino acid residues and shares 95% identity with the human orthologue in the CRD. Frizzled-1 mRNA has been detected in relatively large amounts in adult heart, placenta, lung, kidney, pancreas, prostate, and ovary, and in fetal lung and kidney (3). Several Frizzled-dependent signaling pathways exist (2). Their activation depends on the Wnt ligand and the cell context. Members of the low density lipoprotein receptor-related protein (LRP) are co-receptors for the Wnt ligands. LRP5/6 serve as co-receptors in the Wnt/Frizzled canonical pathway that alters gene expression via the stabilization of beta-catenin (4,5). LRP1 may down regulate Wnt3a/Frizzled-1 signaling in the canonical pathway by sequestering Frizzled-1 (6). Frizzled-1 is one of the purported Wnt10b receptors whose signaling inhibits adipogenesis in preadipocytes (7). Frizzled-1 may be part of a feedback mechanism to modulate the effects of BMP-2 in mesenchymal cells since upregulation of its expression by BMP-2 counteracts the effects of BMP-2 and Wnt3a in inducing the expression of the osteoblast differentiatiation marker, alkaline phosphatase (8). Source: N- Human CD33 Signal Peptide (Met1-Ala16)- Mouse Frizzled-1 (Gln72-His248)- IEGRMD- Human IgG1 (Pro100-Lys330)- C A DNA sequence encoding the signal peptide from human CD33 joined with the extracellular Cysteine Rich Domain (CRD) of mouse Frizzled-1, amino acid residues 72-187 (Accession O70421) was fused to the Fc region of human IgG1 via a peptide linker. The chimeric protein was expressed in a mouse myeloma cell line, NS0. Molecular Mass: The mature recombinant mouse Frizzled-1/Fc chimera, generated by proteolytic removal of the signal peptide, is a disulfide-linked homodimeric protein. Based on amino acid sequencing, the N-terminus of the recombinant protein was mostly blocked, suggesting that the majority of the recombinant protein may start at Gln72 as predicted. A secondary sequence starting at Ala74 was also present. Full-length recombinant protein has a calculated molecular mass of ~46kD. As a result of glycosylation, the recombinant protein migrates as an approximately 55-62kD protein in SDS-PAGE under reducing conditions. Endotoxin: 1.0 EU per 1ug of the cytokine as determined by the LAL method. Activity: Measured by its ability to bind biotinylated Wnt-3a in a functional ELISA. When 100ul of the reaction mixture is captured on a streptavidin-coated plate, 200ng/mL of biotinylated rmWnt-3a will bind rmFrizzled-1/Fc with a range of 8-500ng/ml. Reconstitution: It is recommended that sterile PBS be added to the vial to prepare a working stock solution of no less than 100ug/ml. The carrier-free protein should be used immediately upon reconstitution to avoid losses in activity due to non-specific binding to the inside surface of the vial. For long term storage as a dilute solution, a carrier protein (e.g. 0.1% HSA or BSA) should be added to the vial. Storage: Lyophilized samples are stable for up to twelve months from date of receipt at -20C to -70C. Upon reconstitution, this cytokine, in the presence of a carrier protein, can be stored under sterile conditions at 2-8C for one month or at -20C to -70C in a manual defrost freezer for three months without detectable loss of activity. Avoid repeated freeze-thaw cycles. |
