SPP1 Human, HEK

What purification methods are most effective for SPP1 expressed in HEK cells?

Purification of recombinant SPP1 from HEK cells typically employs a multi-step chromatographic approach. The initial capture step usually leverages the polyhistidine tag engineered at the C-terminus through immobilized metal affinity chromatography (IMAC) using nickel or cobalt resins. Secondary purification often includes ion exchange chromatography to separate based on charge properties, followed by size exclusion chromatography to achieve high purity. When removing the His-tag is required, enzymatic cleavage with specific proteases followed by a second IMAC step is recommended. Throughout the purification process, conditions must be optimized to preserve SPP1's glycosylation state, as these post-translational modifications are critical for maintaining biological activity .

How should researchers account for SPP1 glycosylation in experimental design?

When designing experiments with SPP1, researchers must consider several glycosylation-related factors:

• Molecular weight variations: Expect a significant difference between the theoretical weight (34.5kDa) and observed size (60-65kDa) in SDS-PAGE and western blotting .

• Functional implications: Glycosylation affects SPP1's binding properties, stability, and biological activities. Using deglycosylated SPP1 as a control can help determine glycosylation-dependent functions.

• Expression system selection: HEK293 cells provide human-like glycosylation patterns preferable for studies requiring physiologically relevant modifications.

• Batch consistency: Implement robust quality control measures to ensure consistent glycosylation patterns between protein preparations using techniques like lectin blotting or mass spectrometry.

• Deglycosylation protocols: For studies requiring deglycosylated SPP1, enzymatic treatment with PNGase F (for N-linked glycans) or O-glycosidase (for O-linked glycans) under non-denaturing conditions can preserve protein structure while removing carbohydrate moieties.

How does SPP1 expression vary across different cancer types?

SPP1 expression shows significant variation across cancer types when compared to normal tissues. According to TCGA RNA expression data analyzed through TIMER2 and GEPIA2 platforms, SPP1 is notably overexpressed in multiple cancers including breast adenocarcinoma, colon adenocarcinoma, glioblastoma multiforme, head and neck squamous cell cancer, liver hepatocellular cancer, cholangiocarcinoma, lung adenocarcinoma, lung squamous cell cancer, and stomach adenocarcinoma .

Interestingly, SPP1 shows downregulation in kidney renal clear cell carcinoma compared to normal tissues, suggesting tissue-specific regulatory mechanisms . SPP1 expression levels also correlate with tumor pathological stages in several cancers including bladder urothelial carcinoma, cervical squamous cell carcinoma, esophageal carcinoma, and liver hepatocellular cancer, making it a potential biomarker for disease progression .

What is the prognostic significance of SPP1 expression in human cancers?

• Adrenocortical carcinoma (HR = 2.3, P = 0.042)

• Cervical squamous cell carcinoma (HR = 1.8, P = 0.014)

• Head and neck squamous cell cancer (HR = 1.3, P = 0.045)

• Brain lower grade glioma (HR = 2.2, P = 2.3e-05)

• Liver hepatocellular cancer (HR = 2.0, P = 0.00011)

• Lung adenocarcinoma (HR = 1.4, P = 0.015)

These findings indicate that SPP1 could serve as a prognostic biomarker in multiple cancers, though its predictive value appears to be context-dependent.

How does phosphorylation affect SPP1 function in tumor tissues?

Phosphorylation at S234 of SPP1 shows significantly higher levels in tumor tissues compared to normal tissues across multiple cancer types, including breast cancer, colon adenocarcinoma, lung adenocarcinoma, and uterine corpus endometrial carcinoma, as revealed by Clinical Proteomic Tumor Analysis Consortium (CPTAC) dataset analysis .

This post-translational modification represents a critical regulatory mechanism that likely alters SPP1's functional properties in the tumor microenvironment. While the specific functional consequences of S234 phosphorylation aren't extensively characterized, phosphorylation generally affects:

• Protein-protein interactions

• Subcellular localization

• Signaling pathway activation

• Stability and turnover rates

For researchers investigating SPP1 phosphorylation in cancer, methodological approaches should include:

• Using phospho-specific antibodies for detection in tissue samples

• Employing phosphomimetic (S234D/E) or phospho-deficient (S234A) mutations in functional studies

• Analyzing kinase activity in tumor samples to identify upstream regulators

• Correlating phosphorylation status with patient outcomes and treatment responses

What role does SPP1 play in immune infiltration in human cancers?

SPP1 significantly influences immune cell infiltration in the tumor microenvironment. Analysis using TIMER2 algorithms with purity-adjusted Spearman's rank correlation tests demonstrates that SPP1 expression correlates with the infiltration of multiple immune cell populations within tumors .

Furthermore, TISIDB analysis reveals relationships between SPP1 expression and various immunomodulators, including immune inhibitors, immune stimulators, MHC molecules, and chemokines. This suggests SPP1 functions as a key regulator of immune responses within tumors .

The correlation patterns vary across different cancer types, indicating context-dependent immune regulatory functions. This has important implications for immunotherapy, as SPP1 may influence response to immune checkpoint inhibitors and other immune-based treatments.

What methodologies are recommended for studying SPP1's role in the tumor microenvironment?

To comprehensively investigate SPP1's role in the tumor microenvironment, researchers should consider these methodological approaches:

• Spatial transcriptomics and proteomics: Technologies like Visium or imaging mass cytometry can map SPP1 expression with spatial resolution, revealing relationships with different cell types in the tumor microenvironment.

• Single-cell multi-omics: Integrating single-cell RNA-seq with epigenomic profiling identifies cell populations expressing SPP1 and the regulatory mechanisms controlling its expression.

• Immune correlation analysis: As demonstrated in the research using TIMER2 and TISIDB databases, computational methods can examine SPP1's associations with specific immune cell subsets .

• In situ protein interaction detection: Proximity ligation assays can identify SPP1 interaction partners directly in tissue sections.

• Functional genomics: CRISPR-based screens in cancer models can systematically identify genes that modify SPP1-dependent phenotypes.

• Patient-derived models: Organoids with SPP1 manipulation can evaluate its functional impact on tumor growth, immune infiltration, and therapy response.

• Integrated multi-platform analysis: Similar to the pan-cancer analysis that integrated "DNA methylation data, RNA expression, immune infiltration, immunohistochemistry, protein phosphorylation, patients' survival status, and biological pathway data" .

What protein complexes does SPP1 form and how can researchers distinguish between them?

SPP1 forms distinct protein complexes depending on cellular context, most notably:

• The Set1 complex: In vegetative cells, SPP1 associates with the Set1 histone methyltransferase complex, where it contributes to H3K4 methylation .

• The Mer2-SPP1 complex: In meiotic cells, SPP1 interacts with Mer2, where it plays a role in meiotic recombination initiation .

To distinguish between these complexes experimentally, researchers can employ:

• Co-immunoprecipitation with complex-specific markers: The research demonstrates that "Swd1 immunoprecipitated Spp1 from meiotic cells, but not Mer2, and reciprocally, Mer2 pulled down Spp1, but not Swd1" , confirming physically distinct complexes.

• ChIP-seq/ChIP-qPCR analysis: The genomic distribution patterns differ significantly. In wild-type cells, SPP1 localization shows limited correlation with RNA polymerase II. In mer2Δ cells, SPP1 binding becomes positively correlated with RNA pol II (Pcorr = 0.57) .

• Genetic approaches: Mutants that disrupt specific interactions can help delineate complex-specific functions.

• Functional assays: Monitoring histone H3K4 methylation versus meiotic DSB formation provides functional readouts for different complexes.

What experimental challenges exist when studying SPP1's protein-protein interactions?

Studying SPP1's protein-protein interactions presents several methodological challenges:

• Context-dependent interactions: SPP1 forms distinct complexes depending on cellular context. Experimental conditions must preserve the physiological context relevant to the interaction being studied .

• Post-translational modifications: Extensive glycosylation and phosphorylation significantly affect protein-protein interactions. Researchers must consider whether to use bacterially-expressed (non-modified) or eukaryotic-expressed (modified) proteins .

• Transient interactions: Some SPP1 interactions may be transient or condition-specific. Techniques like crosslinking prior to immunoprecipitation or proximity labeling approaches may be necessary.

• Complex stability: When isolating SPP1-containing complexes, buffer conditions significantly impact complex stability. Optimization of salt concentration, detergent type/concentration, and pH is critical.

• Specificity verification: Confirming interaction specificity requires appropriate controls, including SPP1-null cells, competition with recombinant proteins, and reciprocal co-immunoprecipitation .

• Structural constraints: For detailed interface mapping, structural biology approaches face challenges due to SPP1's flexible regions and heterogeneous post-translational modifications.

How can researchers study SPP1's dual role in histone methylation and meiotic recombination?

To effectively study SPP1's dual role in histone methylation and meiotic recombination, implement this multi-faceted experimental strategy:

• Separation of function mutants: Develop mutations that specifically disrupt either the SPP1-Set1 interaction or the SPP1-Mer2 interaction. Research shows that "Disrupting the Spp1-Set1 interaction mildly decreases H3K4me3 levels and does not affect meiotic recombination initiation. Conversely, the Spp1-Mer2 interaction is required for normal meiotic recombination initiation, but dispensable for Set1 complex-mediated histone H3K4 methylation" .

• Chromatin immunoprecipitation: Perform ChIP-seq to map SPP1 binding sites in relation to histone H3K4me3 marks, RNA polymerase II occupancy, and chromosome axis proteins. Compare patterns between wild-type and mutant backgrounds (set1Δ, mer2Δ) .

• Protein interaction studies: Use co-immunoprecipitation followed by mass spectrometry to identify SPP1-associated proteins under different conditions.

• Functional readouts: Monitor both H3K4me3 levels and meiotic DSB formation to assess the impact of experimental manipulations.

• Time course analysis: Perform temporal analyses to capture dynamic changes in SPP1's interactions and functions throughout meiosis.

• Domain mapping: Create truncation or point mutants to identify specific domains responsible for interactions with Set1 complex components versus Mer2.

What methods are most reliable for detecting different forms of SPP1 in tissue samples?

For reliable detection of different SPP1 forms in tissue samples, researchers should consider these methodological approaches:

• Immunohistochemistry with validated antibodies: The Human Protein Atlas used antibody CAB002212 for IHC analysis across multiple tumor and normal tissue types, showing variable staining patterns. This antibody demonstrated high expression in liver hepatocellular cancer and normal liver tissue, medium staining in head and neck squamous cell cancer compared to low staining in normal oral tissue, and medium staining in lung adenocarcinoma with no detection in normal lung tissue .

• Form-specific antibodies: For post-translationally modified SPP1, use:

  • Phospho-specific antibodies targeting S234 phosphorylation

  • Glycoform-specific antibodies or lectin-based detection methods

  • Antibodies specific to cleaved versus full-length SPP1

• Multi-antibody approach: Use antibodies targeting different SPP1 epitopes to confirm staining patterns, particularly for quantitative comparisons.

• Validation controls:

  • Positive and negative tissue controls

  • Absorption controls with recombinant SPP1

  • SPP1 knockdown tissues or cell lines

  • Western blotting correlation

• Mass spectrometry-based proteomics: For unambiguous identification of SPP1 forms, including post-translational modifications, targeted MS approaches can quantify specific peptides and modifications.