SIRPA Human

What is SIRPA and what is its primary function in human biology?

SIRPA (also known as CD172a, SHPS-1, or BIT) is an immunoglobulin superfamily transmembrane protein with intracellular docking sites for Src homology domain-containing tyrosine phosphatases. It is most abundantly expressed in neurons and myeloid cells, particularly macrophages . SIRPA functions as a critical immune inhibitory receptor by interacting with CD47, creating a "don't eat me" signal that prevents phagocytosis of cells expressing CD47 . This interaction serves as a cellular self-recognition mechanism that protects healthy cells from inappropriate removal by the immune system.

In the immune system, SIRPA regulates myeloid cell activities including phagocytosis, migration, and cytokine production. Beyond immune regulation, SIRPA also demonstrates functions in the nervous system, where it has been shown to have roles in presynaptic organization and neuronal activities .

What are the key structural domains of SIRPA that enable its function?

SIRPA contains several critical structural domains that facilitate its signaling function:

• Extracellular region: Contains three immunoglobulin-like domains, with the N-terminal IgV domain being responsible for CD47 binding .

• Transmembrane region: A single-pass transmembrane domain anchoring the protein to the cell membrane.

• Cytoplasmic region: Contains immunoreceptor tyrosine-based inhibition motifs (ITIMs) that become phosphorylated upon CD47 binding, serving as docking sites for SHP-1 and SHP-2 tyrosine phosphatases .

When SIRPA on macrophages engages CD47 on target cells, the cytoplasmic ITIMs become phosphorylated, recruiting phosphatases that inhibit signaling pathways that would otherwise promote phagocytosis, thus creating the inhibitory effect that prevents cell engulfment.

How does human SIRPA polymorphism affect macrophage function?

Human SIRPA is highly polymorphic in the IgV domain, with research identifying two major variants (V1 and V2) that differ by 13 amino acid residues . These polymorphisms can significantly modulate macrophage-mediated functions, particularly in the context of hematopoiesis.

Studies have demonstrated that human macrophages exert suppressive effects on hematopoiesis, and SIRPA polymorphism modulates this macrophage-mediated suppression . Different SIRPA variants can result in varying levels of suppressive activity when assessed in long-term culture-initiating cells (LTC-IC) assays. This suggests that SIRPA polymorphism affects how macrophages interact with hematopoietic stem and progenitor cells, potentially influencing their proliferation, differentiation, and maintenance .

The functional differences between SIRPA variants highlight the importance of considering genetic variation when studying macrophage biology and designing experiments involving macrophage-mediated processes.

What is known about SIRPA polymorphism across species and its implications?

SIRPA polymorphism extends beyond humans and has profound implications for cross-species interactions, particularly in xenotransplantation research. Most notably, the SIRPA from Non-Obese Diabetic (NOD) mice has significantly greater reactivity with human CD47 than SIRPA from other mouse strains .

This unique property of NOD-derived SIRPA has been identified as a critical factor enabling successful xenogeneic engraftment of human hematopoietic cells in NOD mice. Specifically, NOD mouse SIRPA binds human CD47 with approximately 10-fold higher affinity (KD ≈ 0.08 μM) than human SIRPA binds to human CD47 (KD ≈ 0.6 μM) , as shown in the following affinity measurements:

This table demonstrates the significantly higher affinity of NOD mouse SIRPA for human CD47 compared to other mouse strains or even the human-human interaction . These affinity differences explain why NOD-derived immunodeficient mouse strains show superior engraftment of human hematopoietic cells.

What are the most effective methods for studying SIRPA-CD47 interactions?

Several robust methods have been developed to study SIRPA-CD47 interactions at both molecular and cellular levels:

• Surface Plasmon Resonance (SPR): The gold standard for determining binding affinities and kinetics between SIRPA and CD47. Using the BIAcore system, researchers can immobilize SIRPA proteins via biotin tags and measure real-time interactions with soluble CD47 at physiological temperatures (37°C) .

• Recombinant Protein Production: Expression of the extracellular domains of SIRPA and CD47 in mammalian cell lines, followed by affinity purification and gel filtration, provides high-quality proteins for binding studies .

• Long-term Culture-Initiating Cells (LTC-IC) Assays: Used to examine how SIRPA variants affect hematopoiesis through co-culture of hematopoietic progenitors with stromal cells and macrophages expressing different SIRPA variants .

• Phagocytosis Assays: Direct measurement of macrophage phagocytic activity against target cells, which can assess the functional consequences of SIRPA-CD47 interactions and the effects of blocking this pathway.

These complementary approaches enable comprehensive analysis from molecular binding kinetics to functional cellular outcomes in relevant biological contexts.

What approaches have been most successful for generating human SIRPA transgenic models?

Several genetic engineering approaches have been evaluated for creating human SIRPA transgenic animal models:

• PiggyBac Transposase/Transposon System: This approach has demonstrated superior efficiency compared to classical BAC transgenesis, with better success rates for complete BAC integration and predictable end sequences that facilitate insertion site identification through simple PCR approaches .

• BAC Transgenesis: Involving embryonic injection of bacterial artificial chromosomes containing the entire human SIRPA gene with its regulatory elements, though this classical approach shows lower efficiency than piggyBac-mediated methods .

• CRISPR/Cas9 and TALEN Approaches: Both have been evaluated but were not found to be effective for the large targeted insertions required for BAC-based transgenics of human SIRPA .

• Knock-in Strategies: Creating precise replacements of endogenous SIRPA with human SIRPA has been successful in some mouse models, ensuring physiological expression patterns .

The piggyBac approach currently represents the most effective method for generating human SIRPA BAC transgenic rat models, faithfully expressing human SIRPA and allowing for improved human cell engraftment and enhanced functionality of the human adaptive immune system in vivo .

How do human SIRPA transgenic animal models enhance xenotransplantation research?

Human SIRPA transgenic animal models have revolutionized xenotransplantation research by significantly improving human cell engraftment capabilities. These models demonstrate:

• Enhanced Engraftment Efficiency: Human SIRPA transgenic models show substantially improved human cell engraftment compared to wild-type counterparts. For example, BRGShuman mice (human SIRPA knock-in models) demonstrated significantly greater engraftment and maintenance of human hematopoiesis .

• Improved Immune System Functionality: These models show enhanced development of multiple human hematopoietic lineages and better maintenance of long-term hematopoietic stem cells .

• Reduced Rejection: The human SIRPA expressed in these models binds to human CD47 on engrafted cells, providing inhibitory signals to host macrophages that prevent phagocytosis of human cells, resulting in longer survival and better proliferation of transplanted human cells.

These advantages make human SIRPA transgenic models invaluable tools for studying human immune diseases, evaluating therapeutic strategies, and examining human stem cell biology in vivo .

What are the implications of SIRPA-CD47 interactions for xenotransplantation?

The SIRPA-CD47 interaction has profound implications for xenotransplantation, particularly for overcoming species barriers:

• Species Compatibility Barriers: Pig CD47 does not interact with human SIRPA, creating a major barrier for pig-to-human xenotransplantation . This incompatibility leads to rapid recognition and clearance of pig cells by human macrophages.

• Engineering Solutions: Manipulating porcine cells to express human CD47 or adding soluble human CD47-Fc fusion proteins can significantly reduce the susceptibility of porcine cells to phagocytosis by human macrophages .

• Transgenic Approaches: The development of transgenic pigs expressing human CD47 represents a promising strategy for improving the compatibility of pig organs for human transplantation .

• Binding Affinity Considerations: The ideal binding affinity for xenotransplantation may not necessarily be the highest possible. The observation that NOD mouse SIRPA binds human CD47 with 10-fold higher affinity than the human-human interaction raises questions about the optimal affinity for xenograft success .

Understanding these molecular interactions provides a foundation for rationally designing improved xenotransplantation strategies and developing better animal models for preclinical testing of human therapies.

How do alternative ligands affect SIRPA-CD47 interactions?

Beyond CD47, SIRPA can interact with alternative ligands that introduce another layer of regulatory complexity:

• Surfactant Proteins: Lung surfactant proteins A and D (SP-A and SP-D) can interact with SIRPA and block its binding to CD47 . SIRPA engagement through SP-A or SP-D leads to decreased uptake of apoptotic cells by alveolar macrophages under normal conditions.

• Context-Dependent Regulation: During inflammation, newly recruited macrophages can escape this blocking effect, allowing efficient clearance of apoptotic cells in inflammatory responses .

• Competitive Binding Effects: The presence of these alternative ligands means that SIRPA may not always be available for CD47 binding, particularly in tissues where these surfactant proteins are present.

• Experimental Implications: When studying SIRPA-CD47 interactions in systems where alternative ligands are present, their competing effects should be considered to accurately interpret results.

These interactions highlight the importance of considering tissue context and the presence of alternative ligands when designing experiments to study SIRPA function or developing therapeutic approaches targeting this pathway.

How is SIRPA expression regulated in different physiological contexts?

SIRPA expression undergoes dynamic regulation that significantly impacts its availability and function:

• Tissue-Specific Expression: SIRPA is most abundantly expressed in myeloid cells (particularly macrophages) and neurons, indicating tissue-specific transcriptional regulation .

• Inflammatory Regulation: In macrophages, SIRPA expression can be downregulated by inflammatory stimuli such as lipopolysaccharide (LPS) . This reduction releases the inhibitory regulation of macrophage activity, allowing increased phagocytosis and inflammatory responses during infection.

• Post-Translational Regulation: In neural cells, SIRPA/CD47 can be removed from the cell surface by endocytosis, providing a mechanism for switching off this interaction after it has occurred . Whether this occurs in myeloid cells remains to be fully elucidated.

• Receptor Density Effects: The level of SIRPA expression directly impacts the strength of inhibitory signaling, with higher expression generally correlating with stronger inhibition of phagocytosis .

Understanding these regulatory mechanisms is essential for researchers working with SIRPA, as they influence experimental design and interpretation of results across different cell types and experimental conditions.