HLA-F Human
Description
Molecular Structure and Genetic Features
HLA-F is encoded on chromosome 6p21.3 and shares structural homology with classical HLA class I molecules but features distinct modifications :
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Gene Structure: Comprises eight exons, but exon 7 remains untranslated, resulting in a truncated cytoplasmic tail (~2 kDa) .
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Protein Architecture:
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α1, α2, and α3 domains form a peptide-binding groove with a volume of ~1,250 ų, larger than classical MHC-I molecules .
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A glycine residue at position 97 creates space in the groove, enabling accommodation of longer peptides (14–22 residues) .
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Unique histidine residues (His114-His116) and conserved tyrosine residues in the groove floor influence peptide binding .
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Biological Functions
HLA-F regulates immunity through dual mechanisms:
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Peptide Presentation: Binds unconventional-length endogenous peptides, enabling interactions with T cells and NK cells .
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Immune Modulation:
Table 2: HLA-F Associations in Pathologies
Clinical and Therapeutic Implications
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Diagnostic Challenges: Monoclonal antibodies (mAbs) against HLA-F often cross-react with HLA-E or HLA-G, complicating immunodiagnostics .
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Therapeutic Targets:
Evolutionary Insights
The R62W mutation in HLA-F, absent in non-human primates, enabled its capacity to bind longer peptides—a trait linked to placental evolution and immune adaptation in humans .
Unresolved Questions
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How do HLA-F OCs and peptide-bound forms dynamically regulate immune responses?
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What drives HLA-F’s tissue-specific expression in activated lymphocytes and tumors?
Product Specs
Q&A
What forms can HLA-F adopt on cell surfaces and how do they differ functionally?
HLA-F can exist in multiple forms on cell surfaces, primarily:
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Peptide-loaded β2M-HLA-F complexes: These are conventional trimeric structures containing HLA-F heavy chain, β2M, and bound peptide.
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HLA-F open conformers (OC): These lack β2M and peptide.
These different forms appear to have distinct functional properties and receptor recognition profiles. Empty HLA-F open conformers have been shown to heterodimerize with other MHC-I molecules . Peptide-loaded β2M-HLA-F, but not HLA-F OC, binds with high affinity to the inhibitory LIR1 receptor . Additionally, tetramers composed of peptide-loaded HLA-F or HLA-F OC differentially stain leukocytes, suggesting peptide-dependent engagement with receptors .
How can researchers detect and visualize HLA-F expression on cells?
Several methodological approaches can be employed to detect HLA-F expression:
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Antibody-based detection: Monoclonal antibodies specific to HLA-F, such as the 3D11 antibody, can be used for immunostaining and flow cytometry .
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Chimeric protein detection: Recombinant KIR3DS1-Fc chimeric proteins can bind to HLA-F, with binding detected using anti-human IgG Fc secondary antibodies .
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Tetramer staining: Tetramers made from HLA-F produced in 293T cells (loaded with endogenous peptides) or Hi5 cells (bound with insect peptides) or empty HLA-F OC can be used to stain potential receptor-expressing cells .
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Acid pulse verification: To verify that acid pulsing produces HLA open conformers, antibodies that recognize conformational epitopes dependent on β2M association (such as W6/32, 3D12, and 2M2) can be used as negative controls, as they don't recognize open conformers .
What methodologies can be used to study the interaction between HLA-F and NK cell receptors?
Investigating HLA-F interactions with NK cell receptors requires sophisticated methodological approaches:
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Biolayer interferometry (BLI): This technique can be used to measure binding kinetics and affinities between HLA-F and receptors such as LIR1. The preparation of different forms of HLA-F (OC through refolding and peptide-loaded β2M-HLA-F in expression systems like 293T or Hi5 cells) allows for comparative binding studies .
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Crystallographic analysis: X-ray crystallography of complexes between HLA-F and receptors provides detailed structural information about binding interfaces. For example, the complex structure of LIR1 bound to HLA-F revealed that LIR1 adopts a conserved docking orientation on the side of the β2M-HLA-F complex, making contacts with both HLA-F and β2M .
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NK cell functional assays: To assess functional outcomes of receptor-ligand interactions, researchers can evaluate NK cell activation markers, cytokine production, or cytotoxicity after co-culture with cells expressing different forms of HLA-F .
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Mutagenesis studies: Site-directed mutagenesis of key residues in HLA-F or NK receptors, followed by binding and functional assays, can help identify critical contact points and structure-function relationships.
How should researchers approach the purification and refolding of HLA-F for structural and functional studies?
The purification and refolding of HLA-F presents unique challenges that require specific methodological considerations:
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Expression systems selection: Different forms of HLA-F require different expression approaches:
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Fusion construct design: For crystallization, a β2M-HLA-F fusion construct has been successfully used in the baculovirus insect cell expression system .
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Quality control: Circular dichroism and thermal shift assays can verify proper folding. Properly folded, peptide-loaded HLA-F should display melting curves consistent with other MHC-I molecules bound with endogenous peptides .
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Peptide elution and analysis: For peptide identification, mass spectrometry analysis following acid elution from purified HLA-F can reveal the nature of bound peptides. This has shown that HLA-F can present peptides with a length distribution reminiscent of MHC class II molecules rather than the typical 8-10 amino acid peptides of classical MHC-I .
What experimental approaches can delineate the role of HLA-F in pregnancy and fetal-maternal interface regulation?
Investigating HLA-F's role in pregnancy requires multidisciplinary approaches:
How can researchers investigate the evolutionary significance of the R62W mutation in HLA-F?
The R62W mutation in HLA-F represents a fascinating evolutionary adaptation that has profoundly affected its function. Researchers can investigate this using several approaches:
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Comparative genomics: Sequence analysis across primate species can determine when the R62W mutation arose and whether it emerged independently in different lineages. Current evidence suggests it arose separately in human and orangutan lineages .
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Ancestral sequence reconstruction: Computational methods can infer ancestral HLA-F sequences, which can then be synthesized and functionally compared to modern variants.
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Site-directed mutagenesis: Creating W62R reversion mutants in human HLA-F can directly test the functional impact of this substitution on peptide binding, receptor interactions, and cellular localization.
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Structural analysis: Molecular dynamics simulations comparing wild-type HLA-F with the R62W variant can elucidate how this single amino acid change altered the peptide-binding groove structure and dynamics.
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Selection pressure analysis: Statistical methods (dN/dS ratios) can determine if the R62W mutation was under positive selection during primate evolution, suggesting functional advantage.
What are the optimal experimental designs to study HLA-F's role in viral infection and autoimmunity?
To investigate HLA-F's roles in viral infection and autoimmunity, researchers should consider:
For viral infection studies:
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In vitro infection models: Infect relevant cell types with viruses of interest (e.g., HIV-1) and monitor HLA-F expression, localization, and conformational changes.
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NK cell co-culture systems: Assess how HLA-F on virus-infected cells modulates NK cell functions, particularly focusing on KIR3DS1+ NK cells which have been shown to elicit anti-viral responses that inhibit HIV-1 replication through HLA-F recognition .
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Viral evasion mechanism identification: Investigate whether specific viruses encode proteins that interfere with HLA-F expression or function, similar to known viral evasion strategies targeting classical MHC molecules.
For autoimmunity studies:
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Patient cohort analysis: Compare HLA-F expression patterns and polymorphisms in tissues and peripheral blood from patients with autoimmune conditions versus healthy controls.
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Motor neuron models for ALS: Since HLA-F recognition by inhibitory KIR3DL2 has been shown to prevent motor neuron death in ALS development , motor neuron cell cultures with manipulated HLA-F expression can help elucidate protective mechanisms.
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Transgenic animal models: Develop mice expressing human HLA-F and relevant NK receptors to study in vivo relevance in autoimmune disease models.
Methods for HLA-F Detection and Characterization
Experimental Systems for Studying HLA-F Function
Product Science Overview
The Major Histocompatibility Complex (MHC) is a set of genes that play a crucial role in the immune system by presenting peptide fragments to T cells. These genes are highly polymorphic and are essential for the adaptive immune response. MHC Class I molecules are one of the two primary classes of MHC molecules, the other being MHC Class II .
MHC Class I molecules are heterodimers consisting of two polypeptide chains: an alpha chain and a beta-2 microglobulin (B2M) chain . These molecules are expressed on the surface of almost all nucleated cells and are responsible for presenting endogenous peptides, typically derived from cytosolic proteins, to cytotoxic T lymphocytes (CTLs) . This presentation is crucial for the immune system to recognize and eliminate infected or cancerous cells .
MHC Class I F (HLA-F in humans) is a non-classical MHC Class I molecule. Unlike classical MHC Class I molecules (HLA-A, HLA-B, and HLA-C), which are highly polymorphic and ubiquitously expressed, HLA-F has limited polymorphism and a more restricted expression pattern . HLA-F is primarily expressed in the placenta and certain immune cells, and it plays a role in immune regulation and tolerance .
Recombinant HLA-F refers to the laboratory-produced version of the HLA-F protein. This recombinant protein is used in various research applications to study its structure, function, and interactions with other molecules. The production of recombinant HLA-F involves cloning the HLA-F gene into an expression vector, which is then introduced into a host cell line to produce the protein .
Recombinant HLA-F is used in several research areas, including:
- Immunology: To study the role of HLA-F in immune regulation and its interactions with other immune cells.
- Transplantation: To understand the role of HLA-F in transplant tolerance and rejection.
- Cancer Research: To investigate the potential role of HLA-F in tumor immune evasion and its potential as a therapeutic target .
