| MCAD (MCADherin or cadherin-15) is a 124 kD type I transmembrane glycoprotein of the cadherin superfamily of calcium-dependent homotypic adhesion molecules. 1-4 Like other classical cadherins, the 814 amino acid (aa) human MCAD contains a signal sequence (21 aa), a propeptide (29 aa), an extracellular domain with five cadherin domain repeats (ECD, 556 aa), a transmembrane segment (20 aa) and a cytoplasmic domain (188 aa). 1 A splice variant that diverges within the third cadherin repeat has been sequenced. The cadherin repeats are responsible for cell-cell adhesion by homophilic binding on opposing cells. 1-4 Intracellularly, MCAD binds beta-catenin or plakoglobin (gamma-catenin), which in turn bind alpha-catenin. 4 MCAD also binds p120 catenin. 5 Connection of cadherin/catenin complexes to the actin cytoskeleton is in question, but is possible through a linker. 6 Connection to microtubules has been shown. 7 MCAD is present during early stages of skeletal muscle development and is thought to align myoblasts for fusion. 8 It is also present in muscle satellite cells and participates in muscle regeneration. 9 MCAD is also expressed in the granule cell layer of the cerebellar glomerulus. 10 Deletion of mouse MCAD has little effect in vivo, most likely due to compensation by N-cadherin. 11 However, MCAD upregulation and adhesion between myoblasts during induction of differentiation in vitro is required for their fusion. 8, 12, 13 MCAD activity is later downregulated by sequestering to caveoli, p120 catenin/RhoA-induced ubiquitination and/or cleavage by calpain-3. This terminates fusion and allows sarcomere formation. 5, 12, 13 Human MCAD ECD shows 88% aa identity with mouse, rat or cow, and 85% aa identity with dog MCAD ECD. MCAD is an outlier among classical cadherins, with 40% aa identity or less in the ECD. 4 Source: Human MCAD (Met 1-Ala 606), IEGRMD, Human IgG1 (Pro 100-Lys 330), A DNA sequence encoding the extracellular domain of human M-cadherin (Met 1-Ala 606, Accession P55291) (Shibata, T. et al., 1997, J. Biol. Chem. 272:5236-40) was fused to the Fc region of human IgG1 via a polypeptide linker. The chimeric protein was expressed in a mouse myeloma cell line, NS0. Molecular Mass: The recombinant human MCAD/Fc is a disulfide-linked homodimeric protein. Based on N-terminal sequencing, the recombinant pro-protein starts at Val 22 and has a calculated molecular mass of 90.5 kD. As a result of glycosylation, the recombinant human MCAD/Fc monomer migrates as an approximately 122 kD protein in SDS-PAGE under reducing conditions. Endotoxin Level: <0.10 EU per 1ug of the protein as determined by the LAL method. Activity: Measured by the ability of the immobilized protein to support the adhesion of C2C12 mouse myoblast cell line stimulated for 36 hours with 10% equine serum. When 3 x 10e4 cells/well are added to rhMCAD/Fc coated plates (6ug/ml, 100uL/well), approximately 35 - 60% will adhere after 1 hour at 37 C. Storage: Lyophilized samples are stable for up to twelve months at -20C. Upon reconstitution, this protein, in the presence of a carrier protein, can be stored under sterile conditions at 2-8 C for one month or at -20C in a manual defrost freezer for three months without a significant loss of activity. Avoid repeated freeze-thaw cycles. |
