Isolation and characterization of CD47 glycoprotein: a multispanning membrane protein which is the same as integrin-associated protein (IAP) and the ovarian tumour marker OA3

Mawby, W. J.; Holmes, C H; Anstee, David J.; Spring, Frances A.; Tanner, M. J. A.

doi:10.1042/bj3040525

Cited by 132 publications

(94 citation statements)

References 17 publications

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“…Glycomodulation of Human CD47-SIRP␣ Interactions-CD47 is heavily glycosylated, with all five N-linked sequons probably occupied (34), but no definitive role of glycosylation had yet been determined. Core glycosylation would seem sufficient for the export of at least the soluble form of CD47, since it could be prepared in insect cells (48).…”

Section: Discussionmentioning

confidence: 99%

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

Subramanian

Boder

Discher

2007

Journal of Biological Chemistry

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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 ...

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Section: Discussionmentioning

confidence: 99%

“…Glycosylation Is Not Required for CD47 Export or SIRP␣ Interactions-Both CD47 and SIRP␣ are highly glycosylated proteins (5,34). SIRP␣ glycosylation is already known to moderate its interactions with CD47 (25,35).…”

Section: Display Of Wild-type Cd47 and Cd47-gfp Fusion-full-mentioning

confidence: 99%

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

Subramanian

Boder

Discher

2007

Journal of Biological Chemistry

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“…5,6 CD47 interacts with signal regulatory protein-a (SIRPa), thrombospondin (TSP)-1 and -2 to mediate various cellular functions. 7,8 In particular, CD47 signaling through SIRPa inhibits the phagocytosis of CD47-expressing target cells by SIRPa-expressing macrophages.…”

Section: Introductionmentioning

confidence: 99%

Anti-CD47 antibodies promote phagocytosis and inhibit the growth of human myeloma cells

et al. 2012

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Multiple myeloma is a plasma cell neoplasm residing in bone marrow. Despite advances in myeloma therapies, novel therapies are required to improve patient outcomes. CD47 is highly expressed on myeloma cells and a potential therapeutic candidate for myeloma therapies. Flow cytometric analysis of patient bone marrow cells revealed that myeloma cells overexpress CD47 when compared with non-myeloma cells in 73% of patients (27/37). CD47 expression protects cells from phagocytosis by transmitting an inhibitory signal to macrophages. Here we show that blocking CD47 with an anti-CD47 monoclonal antibody increased phagocytosis of myeloma cells in vitro. In xenotransplantation models, anti-CD47 antibodies inhibited the growth of RPMI 8226 myeloma cells and led to tumor regression (42/57 mice), implicating the eradication of myeloma-initiating cells. Moreover, anti-CD47 antibodies retarded the growth of patient myeloma cells and alleviated bone resorption in human bone-bearing mice. Irradiation of mice before myeloma cell xenotransplantation abolished the therapeutic efficacy of anti-CD47 antibodies delivered 2 weeks after radiation, and coincided with a reduction of myelomonocytic cells in spleen, bone marrow and liver. These results are consistent with the hypothesis that anti-CD47 blocking antibodies inhibit myeloma growth, in part, by increasing phagocytosis of myeloma cells.

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“…Hypermannosylation by the yeast secretory pathway is a hurdle that must be overcome for functional expression of heterologous proteins. CD47 is a ubiquitous cell-surface protein with a wide-ranging functional repertoire (Mawby et al, 1994;Brown and Frazier, 2001;Mateo et al, 2002;Merdan et al, 2003) that should be useful in future structure-function studies, provided the roles of glycosylation and determinants of stability can first be carefully qualified.…”

Section: Discussionmentioning

confidence: 99%

Post‐translational regulation of expression and conformation of an immunoglobulin domain in yeast surface display

Parthasarathy

Subramanian

Boder

et al. 2005

Biotech & Bioengineering

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Display of heterologous proteins on the surface of Saccharomyces cerevisiae is increasingly being exploited for directed evolution because of straightforward cell screens. However, yeast post-translationally modifies proteins in ways that must be factored into library engineering and refinement. Here, we express the extracellular immunoglobulin domain of an ubiquitous mammalian membrane protein, CD47, which is implicated in cancer, immunocompatibility, and motility. CD47 has multiple sites of glycosylation and a core disulfide bond. We assess the effects of both of these post-translational modifications on expression and antibody binding. CD47's extracellular domain is fused to the yeast mating protein Aga2p on the cell wall, and the resulting fusion protein binds several key antibodies, including a conformation-sensitive antibody. Site-bysite mutagenesis of CD47's five N-linked glycosylation sites progressively decreases expression levels on yeast, but folding appears stable. Cysteine mutations disrupt the expected core disulfide, and also decrease protein expression levels, though not to the extent seen with complete deglycosylation. However, with the core disulfide mutants, antibody binding proves to be lower than expression levels might indicate and glycosylation is clearly reduced compared to wild-type. The results indicate that glycosylation regulates heterologous display on yeast more than core disulfides do and thus suggest bounds on directed evolution by post-translational processing. ABSTRACTDisplay of heterologous proteins on the surface of Saccharomyces cerevisiae is increasingly being exploited for directed evolution because of straightforward cell screens. However, yeast post-translationally modifies proteins in ways that must be factored into library engineering and refinement. Here, we express the extracellular immunoglobulin domain of an ubiquitous mammalian membrane protein, CD47, which is implicated in cancer, immunocompatibility, and motility. CD47 has multiple sites of glycosylation and a core disulfide bond. We assess the effects of both of these post-translational modifications on expression and antibody binding.

show abstract

Isolation and characterization of CD47 glycoprotein: a multispanning membrane protein which is the same as integrin-associated protein (IAP) and the ovarian tumour marker OA3

Cited by 132 publications

References 17 publications

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

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

Anti-CD47 antibodies promote phagocytosis and inhibit the growth of human myeloma cells

Post‐translational regulation of expression and conformation of an immunoglobulin domain in yeast surface display

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