ULBP1 Human, Sf9

What is ULBP1 and what is its primary function in human biology?

ULBP1 (UL16 Binding Protein 1) is a glycoprotein that functions as a ligand for the NKG2D receptor, which is primarily expressed on natural killer (NK) cells and certain T cell subsets . Structurally, ULBP1 belongs to the MHC class I family . Its primary function is to bind and activate the KLRK1/NKG2D receptor, thereby mediating natural killer cell cytotoxicity against target cells . ULBP1 is expressed on the surface of stressed or infected cells and plays a crucial role in the immune system's ability to recognize and eliminate abnormal cells . When ULBP1 binds to NKG2D receptors on NK cells, it triggers a cascade of signaling events that ultimately result in the activation of NK cells and the destruction of the target cell expressing ULBP1 .

What are the alternative names and classifications for ULBP1?

ULBP1 is known by several alternative names in scientific literature and databases:

• N2DL1 (NKG2D Ligand 1)

• RAET1I (Retinoic Acid Early Transcript 1I)

• ALCAN-beta

• N2DL-1

• NKG2DL1

• UL16-binding protein 1

The protein is classified as part of the MHC class I family and functions within the immune recognition system . Its gene identifier information includes:

• HGNC: 14893

• OMIM: 605697

• KEGG: hsa:80329

• STRING: 9606.ENSP00000229708

• UniGene: Hs.653255

What experimental applications are most suitable for recombinant ULBP1 Human, Sf9?

Recombinant ULBP1 Human produced in Sf9 cells is suitable for multiple research applications, including:

• Receptor-ligand interaction studies: Particularly for examining NKG2D-ULBP1 binding dynamics and functional consequences .

• Structural biology investigations: The purified protein can be used for crystallography studies to understand structure-function relationships .

• Immunological assays: Using ULBP1 as a standard in assays measuring ULBP1 levels in patient samples, such as ELISA for serum ULBP1 quantification .

• Cell-based functional assays: Testing NK cell activation and cytotoxicity in response to ULBP1 stimulation .

• Antibody validation: As a positive control for validating the specificity and sensitivity of anti-ULBP1 antibodies .

The protein's high purity (≥90%) makes it particularly valuable for applications requiring minimal contaminants, and its baculovirus-Sf9 expression system ensures proper folding and post-translational modifications compared to bacterial expression systems .

What are the recommended storage and handling conditions for ULBP1 Human, Sf9?

To maintain optimal activity of recombinant ULBP1 Human produced in Sf9 cells, the following storage and handling conditions are recommended:

• Short-term storage (2-4 weeks): Store at 4°C if the entire vial will be used within this timeframe .

• Long-term storage: Store frozen at -20°C for periods exceeding 4 weeks. For optimal long-term stability (up to 1 year from receipt), storage at -70°C is recommended .

• Formulation considerations: The protein is typically formulated in a buffer containing Phosphate Buffered Saline (pH 7.4) with 10% glycerol .

• Stability enhancement: For long-term storage, adding a carrier protein (0.1% HSA or BSA) is recommended to prevent protein adhesion to vial surfaces and maintain stability .

• Avoid freeze-thaw cycles: Repeated freeze-thaw cycles should be avoided as they can lead to protein denaturation and activity loss .

• Working dilutions: Prepare fresh dilutions before use in experiments, as diluted solutions may have reduced stability compared to the stock.

What methodological approaches are most effective for detecting ULBP1 expression in research settings?

Several methodological approaches have proven effective for detecting ULBP1 expression in various research contexts:

• Enzyme-Linked Immunosorbent Assay (ELISA):

  • Sandwich ELISA has been successfully used to measure ULBP1 in serum samples of patients with hepatocellular carcinoma and other conditions .

  • This method can detect circulating ULBP1, with studies establishing clinical thresholds (e.g., >2000 pg/ml) associated with specific outcomes .

• Western Blotting (WB):

  • Recommended antibody dilutions for WB range from 1:500 to 1:2000 when using recombinant monoclonal antibodies against ULBP1 .

  • This approach allows for protein size verification and semi-quantitative analysis.

• Flow Cytometry:

  • Particularly useful for detecting cell-surface expression of ULBP1 on live cells.

  • Can distinguish between intracellular and surface expression.

• Immunohistochemistry (IHC):

  • Valuable for examining ULBP1 expression in tissue samples, particularly tumor specimens.

• RT-PCR and qPCR:

  • For detecting ULBP1 mRNA expression and alternative splicing variants .

  • Particularly important when investigating transcriptional regulation.

• Exosome isolation:

  • Size exclusion techniques have been used to compare ULBP1 concentration in exosomes versus free protein .

  • Studies show ULBP1 is predominantly present as free protein rather than bound to exosomes .

When selecting a detection method, researchers should consider the specific research question, sample type, and whether protein or mRNA detection is more appropriate for their experimental design.

How is ULBP1 expression altered in cancer, and what are the implications for cancer immunosurveillance?

ULBP1 expression undergoes significant alterations in cancer with important implications for immunosurveillance:

• Upregulation on tumor cells: ULBP1 is frequently upregulated on tumor cells, which can lead to tumor clearance by activating NK cells through the NKG2D receptor . This represents a natural defense mechanism against cancer development.

• Elevation in hepatocellular carcinoma (HCC): Serum ULBP1 levels are significantly elevated in patients with HCC regardless of the underlying liver disease etiology (HBV, HCV, or other causes) . This elevation appears to be independent of markers of cirrhosis such as platelet count or serum albumin .

• Prognostic significance: High serum ULBP1 levels (>2000 pg/ml) have been associated with significantly reduced survival in HCC patients. In one cohort, median survival was 26 vs. 74 days for patients with high vs. low ULBP1 levels (HR 2.37) . This effect remained significant after adjustment for BCLC staging .

• Mechanism of immune evasion: Soluble ULBP1 released from cancer cells may act as a decoy that binds to NKG2D receptors on NK cells without triggering cellular cytotoxicity, potentially impairing anti-tumor immune responses .

• Distinct from AFP: There is no association between serum ULBP1 and alpha-fetoprotein (AFP) levels in HCC patients, suggesting ULBP1 secretion involves mechanisms distinct from AFP production . This indicates ULBP1 could serve as a complementary biomarker.

These findings suggest ULBP1 could be both a potential prognostic marker and therapeutic target in various cancers, particularly HCC .

What is the relationship between ULBP1 and viral hepatitis, and how does antiviral therapy affect ULBP1 levels?

ULBP1 shows a notable relationship with viral hepatitis and responds to antiviral therapy:

• Elevation in chronic hepatitis B (CHB): Patients with active or cirrhotic CHB exhibit higher ULBP1 levels compared to inactive carriers or healthy controls, though these levels remain significantly lower than in HBV-related HCC .

• Independence from viral replication markers: In the context of HBV-related HCC, serum ULBP1 levels were not associated with HBV viral load, suggesting ULBP1 production in established HCC is independent of HBV replication .

• Response to antiviral therapy: In patients with HBV without HCC treated with tenofovir according to EASL guidelines, ULBP1 levels significantly decreased following 12 months of treatment compared to levels at treatment initiation . This indicates that successful viral suppression may reduce the stress signals that trigger ULBP1 expression.

• Independence from liver fibrosis markers: Serum ULBP1 was not associated with markers of liver disease including platelet count, alanine transaminase, or serum albumin in HCC patients, suggesting that in the context of clinical HCC, ULBP1 production was independent of liver fibrosis and hepatocyte dysfunction .

These findings suggest that ULBP1 may serve as a marker of immune activation in viral hepatitis and could potentially be used to monitor response to antiviral therapy. The reduction in ULBP1 levels following successful antiviral treatment indicates that controlling viral infection may help normalize the stress response that triggers ULBP1 expression.

What molecular pathways and factors regulate ULBP1 expression in cancer cells?

Research has identified several key molecular pathways and factors that regulate ULBP1 expression in cancer cells:

• Transcription factor ATF4: Forward genetic screening has revealed that the transcription factor ATF4 drives ULBP1 gene expression in cancer cell lines . ATF4 is a stress-responsive transcription factor often activated in tumor cells.

• RNA-binding protein RBM4: This protein supports ULBP1 expression by suppressing a novel alternatively spliced isoform of ULBP1 mRNA . This post-transcriptional regulation represents an important control mechanism.

• Stepwise contribution of independent pathways: Research indicates that multiple independent pathways work at various stages of ULBP1 biogenesis, suggesting a complex regulatory network .

• Stress response pathways: ULBP1 expression is tied to cellular stress pathways that alert the immune system to danger, particularly in the context of malignant transformation .

• Polymorphic variants: The existence of polymorphic variants of ULBPs with different binding affinities to NKG2D can significantly affect immune recognition. For example, the R>L polymorphism in ULBP0602 creates an energetic hotspot within the NKG2D-ULBP6 interaction, resulting in an affinity of 15.5 nM, which is 10-1000 fold greater than other ULBP-NKG2D interactions .

• Paradoxical effects of high-affinity binding: Interestingly, very strong interaction between NKG2D and certain ULBP variants (like ULBP0602) can actually impair NKG2D-mediated effector functions, likely due to impaired serial engagement or through release of high-affinity soluble protein isoforms that block NKG2D binding .

Understanding these regulatory mechanisms provides insight into how cancer cells modulate their immunogenicity and suggests potential targets for therapeutic intervention aimed at enhancing immune recognition of tumor cells.

What is known about alternative splicing of ULBP1 and its functional consequences?

Alternative splicing of ULBP1 represents an important regulatory mechanism with significant functional consequences:

• Novel alternatively spliced isoform: Research has identified a previously uncharacterized alternatively spliced isoform of ULBP1 mRNA that affects protein expression . This discovery came from deeper investigation of selected hits from a forward genetic screen.

• Regulation by RBM4: The RNA-binding protein RBM4 has been shown to support ULBP1 expression by suppressing this alternative splicing event . When RBM4 is absent or reduced, the alternative splicing may predominate, potentially reducing functional ULBP1 expression.

• Potential impact on immune recognition: Alternative splicing could affect the structure, stability, or surface expression of ULBP1, potentially altering its ability to engage with NKG2D receptors on immune cells and affecting immune surveillance.

• Cancer relevance: Since alternative splicing is often dysregulated in cancer, this mechanism may represent one way that tumor cells evade immune recognition by modifying the expression of functional ULBP1.

• Therapeutic implications: Understanding these alternative splicing events could potentially lead to therapeutic strategies aimed at modulating ULBP1 splicing to enhance immune recognition of cancer cells.

This area represents a cutting-edge aspect of ULBP1 biology that merits further investigation, particularly regarding the structural differences between splice variants and their differential abilities to activate or inhibit immune responses.

How do the structural characteristics of ULBP1 influence its binding affinity to NKG2D?

The structural characteristics of ULBP1 significantly influence its binding affinity to the NKG2D receptor:

Understanding these structure-function relationships is critical for developing strategies to modulate ULBP1-NKG2D interactions for therapeutic purposes, particularly in the context of enhancing anti-tumor immunity.

What is the potential of ULBP1 as a prognostic biomarker in cancer?

ULBP1 shows considerable promise as a prognostic biomarker in cancer, particularly hepatocellular carcinoma (HCC):

• Survival prediction in HCC: Elevated serum ULBP1 (>2000 pg/ml) has been associated with significantly shorter survival in HCC patients. In a Gambian cohort, patients with high ULBP1 had a median survival of 26 days versus 74 days for those with lower levels (HR 2.37, p=0.0029) . This finding was validated in a larger UK-based cohort (HR 2.1) .

• Independence from other markers: The prognostic value of ULBP1 remained significant after adjustment for Barcelona Clinic Liver Cancer (BCLC) staging (p=0.03), indicating it provides independent prognostic information .

• Not simply a reflection of tumor size: ULBP1 levels did not correlate with HCC volume estimated by ultrasound scan, suggesting its prognostic value isn't merely a reflection of tumor burden .

• Complementary to existing markers: No association was found between serum ULBP1 and alpha-fetoprotein (AFP), suggesting ULBP1 secretion is mechanistically distinct from AFP production . This indicates ULBP1 could provide additional prognostic information to current markers.

• Applicability across diverse etiologies: ULBP1 was similarly elevated in HCC resulting from various underlying causes (HBV, HCV, or other etiologies), suggesting broad applicability as a biomarker regardless of disease etiology .

• Potential for monitoring treatment response: The observation that ULBP1 levels decrease following antiviral therapy in chronic hepatitis B patients suggests it might also serve as a marker of treatment response .

These findings suggest ULBP1 merits further investigation as a prognostic marker in HCC across diverse settings and potentially in other cancers where the NKG2D-ULBP1 axis plays a role in disease progression.

How might the ULBP1-NKG2D axis be therapeutically targeted in cancer immunotherapy?

The ULBP1-NKG2D axis presents several promising avenues for therapeutic targeting in cancer immunotherapy:

• Enhancing ULBP1 expression on tumor cells: Given that upregulation of ULBP1 on tumor cells can lead to tumor clearance , strategies to increase ULBP1 expression could enhance immune recognition. This might involve:

  • Targeting transcriptional regulators like ATF4 that drive ULBP1 expression

  • Modulating RNA-binding proteins like RBM4 that affect ULBP1 splicing

  • Using epigenetic modifiers to reverse potential silencing of ULBP1 genes

• Neutralizing soluble ULBP1: Since elevated soluble ULBP1 is associated with poorer outcomes and may act as a decoy reducing NKG2D function, antibodies that bind soluble ULBP1 without interfering with membrane-bound ULBP1 recognition could enhance immune surveillance.

• Bispecific antibodies: Designing antibodies that simultaneously bind to ULBP1 on tumor cells and to immune effector cells could enhance targeting of ULBP1-expressing tumors.

• CAR-NK cells: Chimeric antigen receptor NK cells designed to recognize ULBP1 could provide targeted killing of ULBP1-expressing tumor cells, bypassing potential NKG2D downregulation.

• Combination approaches: ULBP1-targeting approaches might be particularly effective when combined with checkpoint inhibitors or other immunotherapies that address different aspects of tumor immune evasion.

• Monitoring therapy response: Given that ULBP1 levels decrease following successful treatment of underlying conditions (e.g., antiviral therapy in HBV) , ULBP1 could serve as a biomarker for monitoring response to cancer therapies.

The complexity of ULBP1 regulation and the paradoxical effects observed with high-affinity binding variants highlight the need for careful targeting strategies that enhance immune recognition without triggering compensatory immune evasion mechanisms.

What methodological challenges exist in studying the alternative splice variants of ULBP1?

Investigating alternative splice variants of ULBP1 presents several methodological challenges that researchers must address:

• Detection specificity: Designing primers or antibodies that specifically recognize particular splice variants can be challenging, especially if the variants share substantial sequence homology. This requires careful design of splice junction-spanning primers for PCR or epitope-specific antibodies.

• Low abundance transcripts: Some alternative splice variants may be expressed at very low levels, making their detection difficult without highly sensitive techniques like digital PCR or deep RNA sequencing.

• Tissue-specific expression patterns: Alternative splicing can vary between different tissues or cell types, requiring comprehensive analysis across multiple samples to fully characterize splicing patterns.

• Functional validation: Determining the functional consequences of alternative splicing requires expression of individual splice variants in appropriate cellular contexts, which can be technically demanding.

• Regulation complexity: Understanding the regulatory mechanisms controlling alternative splicing of ULBP1, such as the role of RNA-binding protein RBM4 , requires sophisticated approaches like RNA immunoprecipitation and splicing reporter assays.

• Translational relevance: Correlating the expression of specific splice variants with clinical outcomes requires large, well-characterized patient cohorts with appropriate sample collection and storage protocols.

• Model systems limitations: Studying human ULBP1 splicing in animal models is complicated by species differences in NKG2D ligand expression and regulation.

• Single-cell heterogeneity: Alternative splicing can vary at the single-cell level, necessitating single-cell RNA sequencing approaches to fully capture this heterogeneity.

Addressing these challenges requires an integrated approach combining molecular biology, biochemistry, immunology, and clinical research to fully understand the role of ULBP1 alternative splicing in health and disease.