Carcinoembryonic antigen-related cell adhesion molecule 6 (CEACAM-6), previously called nonspecific crossreacting antigen (NCA) or CD66c, is one of seven human CEACAM family members within the immunoglobulin superfamily (1-4). In humans, CEACAMs include type I transmembrane proteins (CEACAM-1, -3, and -4) and GPI-linked molecules (CEACAM-5 through -8) (1). There is no human CEACAM-2. Human CEACAM-6 is a 90 kD, GPI-linked membrane protein that contains a 34 amino acid (aa) signal sequence, a 286 aa extracellular domain (ECD), and a 24 aa hydrophobic C-terminal propeptide. The GPI membrane anchor is attached at the C terminus following cleavage of the propeptide. CEACAM-6 contains one N-terminal V-type Ig-like domain (N domain), followed by two C2-type Ig-like domains (2-4). It shows considerable glycosylation, including (sialyl) Lewis X , which mediates binding to E-selectin, galectins and some bacterial fimbrae (1, 2). Mature human CEACAM-6 shows 84%, 85%, 80%, 87% and 51% aa identity to the equivalent extracellular regions of human CEACAMs 1, 5 (CEA) and 8, rhesus CEACAM-2, and bovine CEACAM-6, respectively. CEACAM-6 is expressed by granulocytes and their precursors. Activation enhances surface expression by mobilizing CEACAM-6 from storage in azurophilic granules (5, 6). It often shows aberrant expression in acute lymphocytic leukemias (10). CEACAM-6 is also expressed epithelia of various organs and is upregulated in pancreatic and colon adenocarcinomas and hyperplastic polyps (7, 8). Over-expression confers resistance to adhesion-related apoptosis (anoikis) in tumor cells (8, 9). CEACAM-6 is an intercellular adhesion molecule, forming both homotypic, and heterotypic bonds with CEACAM-1, -5 and -8 through interaction of the V-type Ig-like domains (11, 12). Cross-linking of neutrophil CEACAM-6 augments α vβ3 and β2 integrin-mediated adhesion, apparently by src and caveolin-mediated inside-out integrin activation (8, 13, 14).
Source:Human CD33 Signal Peptide (Met 1-Ala 16) Human CEACAM-6 (Lys 35-Gly 320) HHHHHH. A DNA sequence encoding the mature human CEACAM-6 (Lys 35-Gly 320; Accession # P40199) (Barnett, T. et al., 1998, Genomics 3(1):59) was fused to the signal peptide from human CD33 at the amino terminus and to a polyhistidine tag at the carboxy-terminus. The chimeric protein was expressed in a mouse myeloma cell line, NS0.
Molecular Mass:Based on N-terminal sequencing, the recombinant human CEACAM-6 starts at Lys 35 and has a calculated molecular mass of approximately 32.0kD. As a result of glycosylation, the recombinant protein migrates as an approximately 57-75kD protein in SDS-PAGE under reducing conditions.
Endotoxin Level:≤ 1 EU/ug of the protein as determined by the LAL method.
Activity:Measured by the ability of the immobilized protein to support the adhesion of 1 μM calcium ionosphere treated human neutrophils. When 2x10e5 cells/well are added to CEACAM-6-coated plates (10ug/ml, 100ul/well), 35-60% 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:Lyophilized samples are stable for up to twelve months at -20°C. 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 -20°C in a manual defrost freezer for three months without detectable loss of activity. Avoid repeated freeze-thaw cycles.
