MFRP (membrane-type frizzled-related protein) is a 65kD, type II transmembrane protein related to both Tolloid proteases and frizzled-domain containing Wnt pathway proteins (1-4). Human MFRP is 579 amino acids (aa) in length (3, 5, 6). It contains a 69aa cytoplasmic region, a 21aa transmembrane segment, and a 489aa extracellular domain (ECD). The ECD is characterized by the presence of two LDLR class A repeats, two CUB domains, and a C-terminal cysteine-rich/frizzled domain. The mRNA for MFRP is highly unusual in that it is dicistronic; that is, it contains two independent ORFs, one for MFRP and one for a functionally- related protein termed CTRP5/C1qTNF5 (4, 7). CTRP5 is a secreted, 25kD short-chain collagen that contains a C1q-type domain (6, 8). In prokaryotes, polycistronic transcripts exist that contain functionally-interactive molecules. This would also appear to be the case for MFRP and CTRP5. CTRP5 is suggested to bind to membrane MFRP via the C1q and CUB domains, respectively. This is posited to generate a receptor-coreceptor complex that binds select Wnts such as Wnt-1 and/or Wnt-10b (4, 6, 7). MFRP has multiple documented mutations. In human, these are associated with hyperopia (severe farsightedness). The mutations result in premature truncations (3, 9). MFRP is expressed in retinal pigment epithelium, ciliary epithelium, and keratinocytes (4, 7, 10, 11). Human MFRP ECD is 70% aa identical to mouse ECD.
Source:Human CD33 signal (Met 1-Ala 16), HHHHHHHHHHH, Human MRFP (Ser 101-Pro 579); A DNA sequence encoding the signal peptide of human CD33 was fused to the N-terminal 10X histidine-tagged extracellular domain of human MFRP (Ser 101-Pro 579; Accession # Q9BY79) (Katoh, M., 2001, Biochem. Biophys. Res. Commun. 282:116). The protein was expressed in a mouse myeloma cell line, NS0.
Molecular Mass:Based on N-terminal amino acid sequencing, the recombinant human MFRP begins with the histidine tag. It has a predicted molecular mass of approximately 52.7kDa As a result of glycosylation, the recombinant monomer migrates as an approximately 90-105kD protein in SDS-PAGE under reducing conditions.
Activity:Measured by its ability to inhibit the activity of rhBMP-1 on the cleavage of a fluorogenic peptide substrate, Mca-Y-V-A-D-A-P-K(Dnp)-OH. Cleavage of ES007 can be measured using excitation and emission wavelengths of 320nm and 405nm, respectively. 30ug/ml of rhMFRP reduces rhBMP-1 activity by 30-60% when measured with 1ug of BMP-1 and 10uM ES007 in 100ul of 10 mM HEPES, 0.01% Brij 35, pH 7.5 at RT.
Storage and Stability:Lyophilized powder may be stored at -20°C. Stable for 12 months at -20°C. Reconstitute with sterile PBS, 0.1% HSA or BSA. Aliquot to avoid repeated freezing and thawing. Store at -20°C. Reconstituted product is stable for 6 months at -20°C. For maximum recovery of product, centrifuge the original vial after thawing and prior to removing the cap. Further dilutions can be made in assay buffer.
