CAR, Fc Chimera, Recombinant, Mouse (Coxsackie Adenovirus Receptor)

CAR (coxsackie and adenovirus receptor), also known as CXADR, is a 46 kD type I transmembrane glycoprotein that belongs to the CTX family of the Ig superfamily (1-3). CAR has received attention as a receptor that facilitates gene transfer mediated by most adenoviruses (1, 2). It is also an adhesion molecule within junctional complexes, notably between epithelial cells lining body cavities and within myocardial intercalated discs (1, 2, 4). CAR is essential for normal cardiac development in the mouse (7). It is expressed throughout brain neuroepithelium during development, but mainly in ependymal cells in the adult (4-6). The 365 amino acid (aa) mouse CAR contains a 19 aa signal sequence, a 218 aa extracellular domain (ECD) with a V-type (D1) and a C2-type (D2) Ig-like domain, a 21 aa transmembrane segment and a 107 aa intracellular domain. D1 is thought to be responsible for homodimer formation in trans within tight junctions (2). The fiber knob of adenoviruses attaches at a similar site, and evidence suggests that disruption of tight junctions facilitates virus binding (1, 2). A PDZ binding motif at the C-terminus interacts with several cytoplasmic junctional proteins (1). The ECD of mouse CAR shares 97%, 90%, 89%, 89% and 88% aa sequence identity with the corresponding regions of rat, human, bovine, porcine and canine CAR, respectively. An alternately spliced isoform (CAR2) that diverges in the C-terminal 15 aa shows the same expression pattern, but may show different subcellular localization (4, 8). Transcription of other splice variants has been detected, but not their translation. A secreted form identified in serum and pleural fluid can block viral infection (9). Source: Human CD33 Signal Peptide (Met 1-Ala 16), Mouse CAR (Leu 20-Gly 237) IEGRMDP, Mouse IgG2a (Glu 98-Lys 330). A DNA sequence encoding the signal peptide from human CD33 joined with the extracellular domain of mouse CAR (Leu 20-Gly 237, Accession # P97792) (Bergelson, J.M. et al., 1997, Science 275(5304):1320) was fused to the Fc region of mouse IgG 2A via a peptide linker. The chimeric protein was expressed in a mouse myeloma cell line, NS0. Molecular Mass: The recombinant mouse CAR/Fc chimera, generated by proteolytic removal of the signal peptide, is a disulfide-linked homodimer. Based on N-terminal amino acid sequencing, the recombinant mouse CAR/Fc starts at Leu 20. The predicted molecular mass of the monomer is approximately 51.2kD. As a result of glycosylation, the recombinant protein migrates as a 61-65kD protein in SDS-PAGE under reducing conditions. Endotoxin Level: < 1.0 EU per 1 µg of the protein as determined by the LAL method. Activity: Measured by the ability of the immobilized protein to support the adhesion of human neutrophils. When 2 x 10e5 cells/well are added to CAR-coated plates (10ug/ml, 100µL/well), 40-60% of the cells will adhere after 20 minutes at 37° C. 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 and Stability:, Lyophilized powder may be stored at -20°C. Reconstitute to nominal volume by adding sterile PBS, 0.1% carrier protein. Aliquot and store at -20°C. Reconstituted product is stable for 3 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