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Surfactant protein D (Sp-D) binds to membrane-proximal domain (D3) of signal regulatory protein ? (SIRP?), a site distant from binding domain of CD47, while also binding to analogous region on signal regulatory protein ? (SIRP?).

ABSTRACT: Signal regulatory protein ? (SIRP?), a highly glycosylated type-1 transmembrane protein, is composed of three immunoglobulin-like extracellular loops as well as a cytoplasmic tail containing three classical tyrosine-based inhibitory motifs. Previous reports indicate that SIRP? binds to humoral pattern recognition molecules in the collectin family, namely surfactant proteins D and A (Sp-D and Sp-A, respectively), which are heavily expressed in the lung and constitute one of the first lines of innate immune defense against pathogens. However, little is known about molecular details of the structural interaction of Sp-D with SIRPs. In the present work, we examined the molecular basis of Sp-D binding to SIRP? using domain-deleted mutant proteins. We report that Sp-D binds to the membrane-proximal Ig domain (D3) of SIRP? in a calcium- and carbohydrate-dependent manner. Mutation of predicted N-glycosylation sites on SIRP? indicates that Sp-D binding is dependent on interactions with specific N-glycosylated residues on the membrane-proximal D3 domain of SIRP?. Given the remarkable sequence similarity of SIRP? to SIRP? and the lack of known ligands for the latter, we examined Sp-D binding to SIRP?. Here, we report specific binding of Sp-D to the membrane-proximal D3 domain of SIRP?. Further studies confirmed that Sp-D binds to SIRP? expressed on human neutrophils and differentiated neutrophil-like cells. Because the other known ligand of SIRP?, CD47, binds to the membrane-distal domain D1, these findings indicate that multiple, distinct, functional ligand binding sites are present on SIRP? that may afford differential regulation of receptor function.

SUBMITTER: Fournier B

PROVIDER: S-EPMC3365977 | biostudies-literature | 2012 Jun

REPOSITORIES: biostudies-literature

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Surfactant protein D (Sp-D) binds to membrane-proximal domain (D3) of signal regulatory protein α (SIRPα), a site distant from binding domain of CD47, while also binding to analogous region on signal regulatory protein β (SIRPβ).

Fournier Bénédicte B Andargachew Rakieb R Robin Alexander Z AZ Laur Oskar O Voelker Dennis R DR Lee Winston Y WY Weber Dominique D Parkos Charles A CA

The Journal of biological chemistry 20120416 23

Signal regulatory protein α (SIRPα), a highly glycosylated type-1 transmembrane protein, is composed of three immunoglobulin-like extracellular loops as well as a cytoplasmic tail containing three classical tyrosine-based inhibitory motifs. Previous reports indicate that SIRPα binds to humoral pattern recognition molecules in the collectin family, namely surfactant proteins D and A (Sp-D and Sp-A, respectively), which are heavily expressed in the lung and constitute one of the first lines of inn ...[more]

PMID: 22511785

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Project description:CD47 is a cell surface protein that transmits an anti-phagocytic signal, known as the "don't-eat-me" signal, to macrophages upon engaging its receptor signal regulatory protein α (SIRPα). Molecules that antagonize the CD47-SIRPα interaction by binding to CD47, such as anti-CD47 antibodies and the engineered SIRPα variant CV1, have been shown to facilitate macrophage-mediated anti-tumor responses. However, these strategies targeting CD47 are handicapped by large antigen sinks in vivo and indiscriminate cell binding due to ubiquitous expression of CD47. These factors reduce bioavailability and increase the risk of toxicity. Here, we present an alternative strategy to antagonize the CD47-SIRPα pathway by engineering high affinity CD47 variants that target SIRPα, which has restricted tissue expression. CD47 proved to be refractive to conventional affinity maturation techniques targeting its binding interface with SIRPα. Therefore, we developed a novel engineering approach, whereby we augmented the existing contact interface via N-terminal peptide extension, coined "Velcro" engineering. The high affinity variant (Velcro-CD47) bound to the two most prominent human SIRPα alleles with greatly increased affinity relative to wild-type CD47 and potently antagonized CD47 binding to SIRPα on human macrophages. Velcro-CD47 synergizes with tumor-specific monoclonal antibodies to enhance macrophage phagocytosis of tumor cells in vitro, with similar potency as CV1. Finally, Velcro-CD47 interacts specifically with a subset of myeloid-derived cells in human blood, whereas CV1 binds all myeloid, lymphoid, and erythroid populations interrogated. This is consistent with the restricted expression of SIRPα compared with CD47. Herein, we have demonstrated that "Velcro" engineering is a powerful protein-engineering tool with potential applications to other systems and that Velcro-CD47 could be an alternative adjuvant to CD47-targeting agents for cancer immunotherapy.

Dataset Information

Surfactant protein D (Sp-D) binds to membrane-proximal domain (D3) of signal regulatory protein ? (SIRP?), a site distant from binding domain of CD47, while also binding to analogous region on signal regulatory protein ? (SIRP?).

Publications

Surfactant protein D (Sp-D) binds to membrane-proximal domain (D3) of signal regulatory protein α (SIRPα), a site distant from binding domain of CD47, while also binding to analogous region on signal regulatory protein β (SIRPβ).

Similar Datasets

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