TPST2 Human, sf9

What is TPST2 and what is its biological function?

TPST2 is one of two isoforms of tyrosylprotein sulfotransferase enzymes (TPST1 and TPST2) that catalyze the post-translational modification of proteins through tyrosine sulfation. This modification plays a crucial role in extracellular protein-protein interactions implicated in inflammation, hemostasis, and viral infection . TPST2 functions as a Golgi-resident enzyme that transfers a sulfonate group from the co-factor 3'-phosphoadenosine 5'-phosphosulfate (PAPS) to specific tyrosine residues within target proteins . The enzyme recognizes accessible tyrosine residues that are typically surrounded by acidic amino acids within -5 to +5 positions, although no strict consensus sequence has been defined . While TPST1 and TPST2 are co-expressed in all human tissues examined, their expression levels vary significantly across different tissues, suggesting distinct physiological functions .

What are the structural characteristics of TPST2?

The crystal structure of human TPST2 has been solved at 1.9Å resolution, revealing important structural insights. The TPST2 catalytic domain comprises a single α/β motif with a five-stranded parallel β-sheet flanked by α helices . Central to this structural motif is the 5'-phosphosulfate-binding (5'PSB) motif (75-GVPRSGTTL-83) within a strand-loop-helix consisted of β3 and α1, which forms extensive interactions with the 5'-phosphosulfate of PAPS . Additionally, β6 and α7 are key elements that include the 3'-phosphate binding (3'PB) motif for PAPS recognition. These 5'PSB and 3'PB motifs are conserved among all members of the sulfotransferase family . Interestingly, the substrate peptide recognition mode observed in TPST2 resembles that of receptor-type tyrosine kinases, despite their different cellular locations and substrate proteins .

How does recombinant TPST2 Human, sf9 differ from native human TPST2?

Recombinant TPST2 Human produced in Sf9 Baculovirus cells is engineered as a single, glycosylated polypeptide chain containing 361 amino acids (residues 26-377) with a molecular mass of 40.4kDa, although it migrates at 40-57kDa on SDS-PAGE under reducing conditions due to glycosylation . For research applications, it's typically expressed with a 6-amino acid His tag at the C-terminus to facilitate purification by chromatographic techniques . In contrast, the native membrane-bound TPST2 contains a transmembrane domain that anchors it to the Golgi membrane. For structural studies, researchers have used a proteolytically-defined 38kDa core domain of human TPST2 (designated TPST2ΔC18; encompassing Gly43–Leu359), which lacks the transmembrane domain but retains catalytic activity . When working with recombinant TPST2, it's important to consider that the recombinant form may have different post-translational modifications compared to the native enzyme, which could potentially affect its activity or substrate specificity.

What differences exist between TPST1 and TPST2?

TPST1 and TPST2 exhibit several key differences that influence their functional characteristics:

• Substrate specificity: TPST1 and TPST2 have slightly different substrate preferences. TPST1 displayed considerably lower Km and Vmax for the majority of tested peptide substrates compared to TPST2, suggesting differential substrate specificity .

• Response to divalent cations: While both enzymes display distinct acidic pH optima and are stimulated by Mn²⁺, TPST2 activity is uniquely stimulated in the presence of Mg²⁺, whereas TPST1 is not .

• Substrate preference patterns: Experimental evidence shows that TPST1 efficiently sulfates peptides derived from human FGF7 and CCR8, while TPST2 shows little to no activity toward these substrates . In contrast, both enzymes can sulfate a peptide derived from complement C4, though TPST1 demonstrates higher efficiency .

• Structural differences: While the majority of residues constituting the substrate binding region are conserved between TPST1 and TPST2, several variable residues likely dictate their differences in substrate specificity .

What assays are available for studying TPST2 activity?

Several assay methods have been developed to study TPST2 activity:

• Mobility-based enzymatic assay: A non-radioactive method that uses synthetic fluorescent peptides as substrates. This assay exploits the charge difference between sulfated and non-sulfated peptides, which results in different electrophoretic mobilities when analyzed on a microfluidic platform . This approach allows for rapid quantification of tyrosine sulfation and is suitable for high-throughput screening.

• Thermal stability assay (TSA): This assay examines the thermal stability of TPST1 or TPST2 in the presence or absence of biochemical ligands using Differential Scanning Fluorimetry (DSF) . It can be used to identify compounds that bind to and potentially inhibit TPST2.

• Radioactive assays: Traditional methods often use [³⁵S]-PAPS as a sulfate donor to measure the incorporation of radioactive sulfate into peptide or protein substrates.

• HPLC-based assays: These methods separate sulfated and non-sulfated peptides based on their hydrophobicity differences and can be coupled with mass spectrometry for precise identification.

When selecting an assay, researchers should consider factors such as the specific research question, available equipment, safety considerations (especially for radioactive methods), and the need for quantitative versus qualitative data .

What is known about TPST2 substrate recognition and specificity?

TPST2 substrate recognition involves four key features:

• Specific recognition of acceptor tyrosine: This recognition is mediated by hydrogen-bond interactions with glutamic acid residues and hydrophobic interactions with proline residues in TPST2 .

• Recognition of peptide backbone: TPST2 recognizes backbone atoms at -2 and -1 positions relative to the acceptor tyrosine through interactions with arginine and threonine residues. The backbone atoms of the acceptor tyrosine are recognized via a short parallel β-sheet type interaction .

• Binding mode: The substrate peptide binds in a deep cleft of TPST2 and forms an L-shaped structure when bound .

• Acidic context preference: TPST2 preferentially recognizes tyrosines surrounded by acidic amino acids, typically within -5 to +5 positions, although no strict consensus sequence has been defined .

Interestingly, this substrate recognition mode resembles that observed for receptor-type tyrosine kinases, suggesting possible convergent evolution since both enzyme types modify tyrosine residues post-translationally . When designing experiments to study TPST2 specificity, researchers should consider using multiple peptide substrates with varying amino acid compositions around the acceptor tyrosine to gain a comprehensive understanding of specificity determinants.

What inhibitors are known to affect TPST2 activity and how can they be used in research?

Several classes of small molecules have been identified as TPST2 inhibitors:

• Suramin: This anti-angiogenic compound has been shown to inhibit both TPST1 and TPST2 .

• Rottlerin: Originally identified as a protein kinase C inhibitor, rottlerin also inhibits TPST activity .

• RAF kinase inhibitors: Oxindole-based inhibitors of the Ser/Thr kinase RAF function as low micromolar inhibitors of TPST1/2. Additionally, unrelated RAF inhibitors such as the dual BRAF/VEGFR2 inhibitor RAF265 also inhibit TPSTs in vitro .

These inhibitors offer valuable research tools for several applications:

• As chemical probes to study TPST function in cellular systems

• As starting points for developing more specific TPST inhibitors through structure-activity relationship studies

• To investigate the role of tyrosine sulfation in specific biological processes

When using these inhibitors, researchers should be aware of their potential off-target effects, especially since many were originally developed for other targets. Control experiments should be performed to distinguish between effects due to TPST inhibition versus effects on other cellular targets .

How can experimental conditions be optimized for TPST2-mediated sulfation reactions?

Optimizing TPST2-mediated sulfation reactions requires careful consideration of several parameters:

• Buffer composition: TPST2 activity is influenced by buffer composition, with distinct acidic pH optima being optimal .

• Divalent cations: TPST2 activity is stimulated by both Mn²⁺ and Mg²⁺ ions . Including these divalent cations in reaction buffers can enhance enzymatic activity.

• PAPS concentration: As the sulfate donor, optimal PAPS concentration is critical for efficient sulfation. Researchers should perform concentration-dependent studies to determine the optimal PAPS level for their specific experimental system.

• Substrate design: Design peptide substrates with acidic residues surrounding the target tyrosine, as TPST2 preferentially recognizes tyrosines in an acidic context .

• Enzyme-to-substrate ratio: Optimize the ratio of TPST2 to substrate to ensure efficient sulfation without wasting enzyme.

• Reaction time and temperature: These parameters should be optimized based on the specific substrate and experimental goals.

• Prevention of product inhibition: In some cases, the sulfated product may inhibit further enzyme activity. This can be addressed by using continuous-flow systems or by removing the product during the reaction.

When optimizing conditions, a systematic approach testing one variable at a time while keeping others constant is recommended to identify the optimal conditions for specific research purposes .

What are the challenges in expressing and purifying active TPST2?

Expression and purification of active TPST2 present several challenges that researchers should be aware of:

• Membrane protein nature: Native TPST2 is a type I transmembrane protein located in the Golgi apparatus, making full-length expression challenging .

• Post-translational modifications: TPST2 is glycosylated, which may affect its stability and activity. Expression systems that support appropriate glycosylation (such as insect cells or mammalian cells) are often preferred .

• Solubility issues: The catalytic domain of TPST2 can form inclusion bodies when expressed in bacterial systems, requiring refolding from guanidine hydrochloride .

• Maintaining proper folding: The correct folding of TPST2 is critical for its activity. Careful attention to buffer composition, pH, and reducing agents during purification is essential.

• Co-factor binding: TPST2 binds PAPS, and proper folding of the binding pocket is necessary for activity.

• Expression system selection: Various expression systems have been used for TPST2, including Sf9 baculovirus cells and bacterial systems with subsequent refolding . The choice depends on the specific research requirements for yield, activity, and post-translational modifications.

To address these challenges, researchers have developed several strategies, including:

• Using a truncated form of TPST2 lacking the transmembrane domain

• Adding affinity tags (such as His-tags) to facilitate purification

• Employing specialized expression systems like Sf9 insect cells that support proper folding and glycosylation

• Developing refolding protocols for protein expressed in inclusion bodies

How should peptide substrates for TPST2 be designed?

Designing effective peptide substrates for TPST2 requires consideration of several key factors:

• Acidic context: Include acidic residues (Asp, Glu) surrounding the target tyrosine, particularly within positions -5 to +5. The presence of these acidic residues enhances recognition by TPST2 .

• Peptide length: Optimal peptide substrates typically range from 9-14 amino acids, as demonstrated by effectively sulfated peptides derived from complement C4, FGF7, and CCR8 . This length provides sufficient context for enzyme recognition while remaining manageable for synthesis.

• Target tyrosine position: Place the target tyrosine centrally within the peptide sequence to ensure adequate flanking residues on both N- and C-terminal sides .

• Fluorescent labeling: For non-radioactive assays, incorporate a fluorophore (such as 5-FAM) at the N-terminus of the peptide. This enables detection of both sulfated and non-sulfated forms using microfluidic platforms .

• Natural substrate-based design: Base peptide sequences on known TPST2 substrates, such as those derived from complement C4, which has been shown to be sulfated by both TPST1 and TPST2 .

• Control peptides: Design control peptides where the target tyrosine is replaced with phenylalanine to confirm specificity of sulfation .

When synthesizing these peptides, high purity is essential for accurate activity measurements. Additionally, researchers should verify peptide identity and purity using mass spectrometry and HPLC before use in enzymatic assays .

What expression systems are recommended for producing recombinant TPST2?

Several expression systems have been used successfully for producing recombinant TPST2, each with advantages and limitations:

• Sf9 Baculovirus system: This is a widely used system for producing glycosylated TPST2. It yields a single glycosylated polypeptide chain that retains enzymatic activity . The Sf9 system supports proper folding and post-translational modifications similar to those in mammalian cells.

• Bacterial expression systems: E. coli has been used to express the catalytic domain of TPST2, although the protein typically forms inclusion bodies requiring refolding from guanidine hydrochloride into a Tris-based buffer . While this approach may offer higher yields, the refolding process can be challenging and may result in lower specific activity.

• Mammalian cell expression: HEK293 or CHO cells can be used for expressing TPST2 with mammalian-specific post-translational modifications, although yields are typically lower than insect or bacterial systems.

Key considerations when selecting an expression system include:

• Required yield and scale of production

• Need for specific post-translational modifications

• Downstream applications and purity requirements

• Available resources and expertise

• Whether full-length or truncated protein is needed

For most research applications requiring active enzyme, the Sf9 baculovirus system offers a good balance of yield, activity, and appropriate post-translational modifications . For structural studies, bacterial expression with subsequent refolding has been successfully employed .

How can TPST2 activity be validated in experimental settings?

Validating TPST2 activity in experimental settings is crucial for ensuring reliable research outcomes. Several complementary approaches can be used:

• Mobility-based enzymatic assays: These assays use fluorescently labeled peptide substrates and track the appearance of a distinctly migrating product band representing the sulfated peptide . This approach provides direct evidence of enzymatic activity.

• Mass spectrometry: LC-MS/MS analysis can confirm the addition of sulfate groups (+80 Da) to specific tyrosine residues in peptide or protein substrates, providing both qualitative and site-specific information .

• Control experiments:

  • Omit PAPS co-factor to confirm its requirement

  • Use heat-inactivated enzyme as a negative control

  • Compare wild-type TPST2 with catalytically inactive mutants

  • Use known TPST inhibitors to confirm specificity of the observed activity

• Substrate specificity profiling: Test multiple peptide substrates with varying sequences to confirm expected specificity patterns. TPST2 should preferentially sulfate tyrosines in an acidic context .

• Thermal stability assays: These can indirectly confirm the functionality of TPST2 by demonstrating proper folding and ligand binding capabilities .

• Comparative analysis with TPST1: TPST2 shows distinct substrate preferences compared to TPST1. For example, while both enzymes sulfate the C4 peptide, TPST1 more efficiently sulfates FGF7 and CCR8 peptides compared to TPST2 . These differential activities can serve as a fingerprint to validate TPST2 identity and function.

What are emerging applications of TPST2 in research?

Emerging applications of TPST2 in research span multiple fields:

• Structural biology: The crystal structure of TPST2 complexed with a substrate peptide and PAP has provided unprecedented insights into the mechanism of tyrosine sulfation . Future research may leverage this structural information to design highly specific substrates or inhibitors.

• Drug discovery: The finding that certain protein kinase inhibitors, particularly RAF inhibitors, can inhibit TPST2 opens new avenues for drug repurposing or redesign . This cross-reactivity could be exploited to develop dual-action therapeutics or more specific TPST inhibitors.

• Chemoenzymatic synthesis: TPST2 can be used for the site-specific sulfation of proteins and peptides, enabling the production of sulfated biomolecules for studying protein-protein interactions in processes like inflammation, hemostasis, and viral infection .

• Proteomics: Tools for evaluating protein tyrosine sulfation can help map the "sulfotyrosyl proteome," enhancing our understanding of this important post-translational modification . This could reveal new roles for tyrosine sulfation in cellular physiology and pathology.

• Cell signaling research: Given the similarities in substrate recognition between TPST2 and receptor tyrosine kinases, research on TPST2 may provide insights into evolutionary relationships between different post-translational modification systems .

As analytical technologies continue to advance, we can expect more comprehensive characterization of tyrosine-sulfated proteins and a deeper understanding of the physiological roles of TPST2 in various tissues .

How can small molecule inhibitors of TPST2 be developed?

Development of small molecule inhibitors of TPST2 represents an exciting frontier in chemical biology. Several approaches can be pursued:

• Structure-based design: The crystal structure of TPST2 provides a foundation for rational design of inhibitors targeting the active site or substrate binding cleft . In silico docking and molecular dynamics simulations can guide the design of compounds that complement the binding pocket.

• Repurposing protein kinase inhibitors: Given that certain RAF kinase inhibitors also inhibit TPST2, these compounds could serve as starting points for developing more specific TPST inhibitors . Structure-activity relationship studies can identify modifications that enhance selectivity for TPST2 over kinases.

• High-throughput screening: The development of non-radioactive mobility-based enzymatic assays enables screening of large compound libraries for TPST2 inhibitors . This approach can identify novel chemical scaffolds with inhibitory activity.

• Fragment-based drug discovery: This approach involves screening small molecular fragments and then growing or linking them to create more potent inhibitors. Thermal stability assays can identify fragments that bind to TPST2 .

• Peptidomimetic approach: Designing inhibitors that mimic the structure of TPST2 substrate peptides but resist sulfation could yield competitive inhibitors of the enzyme.

Challenges in inhibitor development include:

• Achieving selectivity for TPST2 over TPST1 and other sulfotransferases

• Balancing potency with physicochemical properties needed for cellular penetration

• Developing assays to validate target engagement in cellular contexts

Successful development of specific TPST2 inhibitors would provide valuable tools for investigating the biological roles of tyrosine sulfation and potentially lead to new therapeutic approaches for conditions where aberrant sulfation contributes to disease .