LAIR-1, Recombinant, Mouse (Leukocyte-associated Ig-like Receptor-1)

Leukocyte-associated Ig-like receptor-1 (LAIR-1) is an inhibitory receptor of the Ig superfamily that is structurally related to inhibitory members of KIR and ILT/CD85 families (1-3). It is expressed on immune cells, including NK cells, T cells, B cells, monocytes, immature neutrophils, dendritic cells and most thymocytes (2-4). The 253 amino acid (aa) type I transmembrane (TM) protein contains a 21 aa signal sequence, a 124 aa extracellular domain (ECD), a 20 aa TM domain and a 98 aa cytoplasmic domain. The ECD includes one C2-type Ig-like domain and two potential N-linked glycosylation sites. Tyrosine phosphorylation of two cytoplasmic ITIM motifs results in recruitment of phosphatases and downregulation of signaling through activating receptors (2, 3, 5). Crosslinking of LAIR-1 inhibits processes such as B cell receptor-mediated activation, NK cell and T cell cytotoxicity and basophil degranulation (1-3). Four mouse LAIR-1 splice variants have been identified, but it is not known whether they are expressed as proteins (3). LAIR1b, which is the major alternate transcript, lacks most of the ECD. Of the minor transcripts, LAIR-1c lacks a signal sequence, and LAIR-1d and 1e lack a TM sequence. All mouse splice forms are identical in the last 90 aa of the cytoplasmic domain. LAIR-1 shows high-affinity binding of collagens that results in inhibition of degranulation in a basophilic leukemia cell line (6). Human and mouse LAIR-1 ECD share only 32% aa identity but, where studied, sites of expression and activity are similar (3-6). Mouse LAIR-1 ECD also shares 62%, 31% and 28% aa identity with rat, dog, and cow orthologs, respectively. Source: Human CD33 signal (Met 1-Ala 16), Mouse LAIR-1 (Gln 22-Tyr 141), HHHHHH, A DNA sequence encoding the mature extracellular domain of mouse Leukocyte-Associated Immunoglobulin-like Receptor 1 (LAIR-1, Gln 22-Tyr 141, Accession # Q8BG84) (Lebbink, R.J., et al., 2004, J. Immunol. 172(9):5535) was fused at the N-terminus to the signal sequence of human CD33 and at the C-terminus to a polyhistidine tag. The protein was expressed in a mouse myeloma cell line, NS0. , Molecular Mass: The recombinant mouse LAIR-1, generated by proteolytic removal of the signal peptide region, is a monomeric protein. Based on N-terminal amino acid sequencing, the major sequence was blocked, suggesting that the majority of the recombinant protein in the preparation may start at Gln 22 as predicted. A small amount of the recombinant protein in the preparation starts at Ser 25. The recombinant mouse LAIR-1 has a calculated molecular mass of approximately 14.3 kD. As a result of glycosylation, the recombinant protein migrates as an approximately 20-35 kD 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 HT-29 cells. When 5 x 10e4 cells/well are added to recombinant mouse LAIR-1-coated plates (50ug/ml with 100 µL/well), approximately 30-60% will adhere after 10 minutes at 37° C. Optimal concentration depends on cell type as well as the application or research objectives. 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 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.