| Signal regulatory protein alpha (SIRPA, designated CD172a), also called SHPS-1 (SHP substrate 1) and previously, MyD-1 (Myeloid/Dendritic-1), is a monomeric ~90kD type I transmembrane glycoprotein that belongs to the SIRP/SHPS (CD172) family of the immunoglobulin superfamily.1-4 SIRPs are paired receptors, with similar extracellular domains but differing C-termini and functions.1, 2 The 503 amino acid (aa) human SIRPA contains a 342 aa extracellular domain (ECD), with one V-type, and two C1-type Ig domains, and three potential N-glycosylation sites. It has a 110 aa cytoplasmic sequence with ITIM motifs that recruit tyrosine phosphatases SHP-1 and SHP-2 when phosphorylated.4 Human SIRPA has more than 40 described polymorphisms, including the prominent BIT (Brain Ig-like molecule with Tyrosine-based activation motifs, also called SIRPA2 or PTPNS).5 One reported isoform lacks aa 1-101, which eliminates most of the V-type Ig domain. Human SIRPA ECD shares 61%, 60%, 71%, 72% and 73% aa identity with mouse, rat, porcine, bovine and equine SIRPA, respectively, it shares 84% and 76% aa identity with human SIRPB1 and SIRPy, respectively.2 SIRPA is expressed mainly on myeloid cells, including macrophages, neutrophils, dendritic and Langerhans cells.3-6 It is also found on neurons, smooth muscle and endothelial cells.7-9 SIRPA shows adhesion to the ubiquitous CD47/IAP (integrin-associated protein), while SIRPY binds more weakly and SIRPA1 does not bind at all.1, 2 Mouse and human SIRPA-CD47 binding only cross-reacts for specific polymorphisms and influences engraftment of xenotransplanted stem cells.6, 10 SIRPA engagement generally produces a negative regulatory signal.4 Low SIRPA recognition of CD47, which occurs on aged erythrocytes or platelets or xenogenic cells, promotes clearance of CD47low cells from circulation.11-13 SIRPA Arecognition of surfactants SP-A and SP-D in the lung can inhibit alveolar macrophage cytokine production.14 CD47- integrin-SIRPA interaction is reported to promote macrophage fusion during osteoclastogenesis.15 A DNA sequence encoding the extracellular domain of human SIRPa (Gly 27-Arg 370 & Glu31-Arg370, Accession NP_542970) was fused to the Fc region of human IgG1 (Pro100-Lys330) via a polypeptide linker. The chimeric protein was expressed in Chinese hamster ovary cells. Calculated molecular mass of ~ 64kD. As a result of glycosylation, the recombinant protein migrates as an ~ 70-105kD protein in SDS-PAGE under reducing conditions. Activity: Measured by the ability of the immobilized protein to support the adhesion of red blood cells (Subramanian S., et al., 2006, Blood 107:2548-2556). Red blood cells added at 10x10e6 cells/ml (100ul/well) to SIRPa coated plated will adhere after 1hour at 37C. The ED50 for this effect is 0.6-2.4ug/ml. Storage and Stability: Lyophilized and reconstituted products are stable for 6 months after receipt at -20C. Reconstitute with sterile PBS. Aliquot to avoid repeated freezing and thawing. Store at -20C. 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. |
