Phylogenetic Divergence of CD47 Interactions with Human Signal Regulatory Protein α Reveals Locus of Species Specificity

Abstract

Cell-cell interactions between ubiquitously expressed integrinassociated protein (CD47) and its counterreceptor signal regulatory protein (SIRP␣) on phagocytes regulate a wide range of adhesive signaling processes, including the inhibition of phagocytosis as documented in mice. We show that CD47-SIRP␣ binding interactions are different between mice and humans, and we exploit phylogenetic divergence to identify the species-specific binding locus on the immunoglobulin domain of human CD47. All of the studies are conducted in the physiological context of membrane protein display on Chinese hamster ovary (CHO) cells. Novel quantitative flow cytometry analyses with CD47-green fluorescent protein and soluble human SIRP␣ as a probe show that neither human CD47 nor SIRP␣ requires glycosylation for interaction. Human CD47-expressing CHO cells spread rapidly on SIRP␣-coated glass surfaces, correlating well with the spreading of primary human T cells. In contrast, CHO cells expressing mouse CD47 spread minimally and show equally weak binding to soluble human SIRP␣. Further phylogenetic analyses and multisite substitutions of the CD47 Ig domain show that human to cow mutation of a cluster of seven residues on adjacent strands near the middle of the domain decreases the association constant for human SIRP␣ to about onethird that of human CD47. Direct tests of cell-cell adhesion between human monocytes and CD47-displaying CHO cells affirm the species specificity as well as the importance of the newly identified binding locus in cell-cell interactions.Cell-cell interactions are of course critical in immune function but can have important and revealing differences between species as well as nonlinear dependences on molecular parameters, such as affinity. We explore such general issues with integrin-associated protein (IAP), 2 or CD47, which is a ubiquitous but unique immunoglobulin superfamily receptor with a single Ig domain, a pentaspan transmembrane segment, and a variably spliced cytoplasmic tail (1). The known functional roles of CD47, originally limited to integrin activation (2), have expanded considerably since the identification of SIRP␣ as a physiological ligand (3-6). SIRP␣ is another Ig superfamily member with three Ig domains and a single span transmembrane segment. SIRP␣ is also broadly expressed, present at high levels on myeloid lineages (monocytes, neutrophils) and neurons but absent from many if not most other cells (7). Both CD47 and SIRP␣ are found in many mammals, but the differences in sequence (Fig. 1A) across species have yet to be evaluated. CD47-SIRP␣ interactions regulate transmigration of monocytes and neutrophils (8 -11) with severe impairment in neutrophil recruitment to sites of infection in CD47 knock-out mice (12). CD47 on "self " red cells, platelets, lymphocytes, and other nonapoptotic cells inhibits clearance by macrophages in mice by signaling through SIRP␣ (13-16). However, in humans as opposed to mice, severe reductions of CD47 levels on red cells, as found on various Rh-deficient patient ...

Inhibition of “self” engulfment through deactivation of myosin-II at the phagocytic synapse between human cells

“…Affi nity, signaling, and cytoskeleton at the phagocytic synapse with and without CD47 Consistent with recent results showing species-specifi c CD47 -SIRP ␣ interactions ( Subramanian et al, 2007 ), soluble human-SIRP ␣ is found here to bind much less to sheep RBCs than to human RBCs ( Fig. 1 A ).…”

Signal regulatory protein alpha (SIRPα)/CD47 interaction and function

“…In general there is little cross reaction between CD47 from one species and SIRPα of another [21,22]. However the allele of SIRPα in the NOD mouse is sufficiently different from other mouse strains that it can bind human CD47 and give protection of the human cells in this mouse strain from host macrophages and xenogeneic engraftment occurs.…”

CD47 deficiency confers cell and tissue radioprotection by activation of autophagy

“…Plasmid construction and stable transfection of CD47 in CD47-deficient cells was performed as follows: Plasmid CD47-GFP containing human CD47 isoform 2 (NM_002020.4) fused to EGFP in plasmid vector pEGFPN3 52 was obtained from Dr. Dennis Discher, and used to construct an internal V5 epitope tag in CD47. PCR primer pairs M185 ATT CTC GAG ATG TGG CCC CTG GTA GCG G (XhoI underlined), M190 CGT AGA ATC TAG ACC GAG GAG AGG GTT AGG GAT AGG CTT ACC AAC ACG ATA TTT TAG CTC GAT GAT CG (XbaI underlined) and M191 GGT AAG CCT ATC CCT AAC CCT CTC CTC GGT CTA GAT AGC ACG GTT TCA TGG TTT TCT CCA AAT G (Xba1 underlined), M182 ACT AGT CTA TCA GTT ATT CCT AGG AGG (Spe1 underlined) with pCD47-GFP as template, were used to produce PCR products of the 5' proximal and distal regions of the CD47 gene, respectively.…”