Recombinant Xenopus tropicalis Synaptotagmin-17 (syt17)

What is Synaptotagmin-17 and what is its genomic classification in Xenopus tropicalis?

Synaptotagmin-17 (syt17) is a protein-coding gene in Xenopus tropicalis identified by Entrez Gene ID 100125097. It belongs to the synaptotagmin family, a group of membrane-trafficking proteins that generally function as calcium sensors in regulated exocytosis and neurotransmitter release . The full gene name is "synaptotagmin 17" and it is classified as a protein-coding gene in the tropical clawed frog (Xenopus tropicalis) genome .

How does syt17 expression change during X. tropicalis development?

While specific syt17 expression patterns during X. tropicalis development aren't detailed in available research, related synaptotagmin family members show distinct developmental regulation. For example, synaptotagmin II exhibits peak expression at Nieuwkoop and Faber (NF) stage 63 during metamorphosis climax when tail regression is prominent . This expression pattern aligns with other synapse-related proteins including myelin-associated glycoprotein, myelin basic protein, and other neural components, suggesting a coordinated upregulation during critical developmental transitions .

To characterize syt17 expression specifically:

• Perform RT-qPCR analysis across developmental stages from embryo to adult

• Conduct in situ hybridization to identify tissue-specific expression patterns

• Compare expression before, during, and after metamorphosis climax

• Analyze potential thyroid hormone responsiveness, as many neuronal genes in X. tropicalis show thyroid hormone-dependent regulation during metamorphosis

How can researchers distinguish between syt17 and other synaptotagmin family members?

Methodological approach:

• Sequence alignment analysis:

  • Compare conserved domains and variable regions across synaptotagmin family members

  • Focus on C2 domains which may have distinct calcium-binding properties in syt17

• Expression pattern analysis:

  • Unlike classical synaptotagmins primarily expressed at synaptic terminals, syt17 may show different subcellular localization

  • Use co-localization studies with markers for different cellular compartments

• Functional differential analysis:

  • Develop isoform-specific antibodies targeting unique regions

  • Design PCR primers targeting unique sequences for isoform-specific detection

  • Perform systematic phosphorylation analysis using techniques like PhosTag SDS-PAGE to identify distinct modification patterns

What are the optimal expression systems for producing functional recombinant X. tropicalis syt17?

Methodological recommendations:

For bacterial expression:

• Use solubility-enhancing fusion tags (MBP, SUMO, GST)

• Express individual domains separately for higher solubility

• Optimize induction temperature (16-18°C often improves folding)

For mammalian expression:

• Consider the N1E-115 or HEK293T cell systems as used for similar membrane protein studies

• Use strong promoters (CMV) for high expression

• Include appropriate trafficking signals for proper subcellular localization

How can researchers optimize purification of recombinant X. tropicalis syt17?

A multi-step purification strategy yields the highest purity:

• Initial capture:

  • Immobilized metal affinity chromatography (IMAC) for His-tagged constructs

  • Anti-FLAG immunoaffinity for FLAG-tagged proteins, similar to the PLPPR3-N-Flag purification described in comparable studies

• Intermediate purification:

  • Ion exchange chromatography based on theoretical pI

  • Apply salt gradient elution for separation of differentially modified forms

• Final polishing:

  • Size exclusion chromatography to separate monomeric protein from aggregates

  • Also enables buffer exchange for downstream applications

Critical considerations:

• Include phosphatase inhibitors if studying phosphorylated forms

• Consider membrane protein-specific detergents if working with full-length syt17

• Validate purification with SDS-PAGE and western blotting

• Confirm identity with mass spectrometry

What methods can verify proper folding and activity of purified recombinant syt17?

Structural integrity assessment:

• Circular dichroism (CD) spectroscopy to analyze secondary structure content

• Thermal shift assays to evaluate stability and proper folding

• Limited proteolysis to assess compact domain structure

Functional verification:

• Membrane binding assays using liposomes of defined composition

• Calcium binding studies if calcium sensitivity is expected

• PhosTag SDS-PAGE analysis to evaluate phosphorylation state, which can be critical for function

Activity comparison:

• Compare with native protein isolated from X. tropicalis brain tissue

• Assess binding to known interacting partners

• Compare wild-type and mutant forms to identify critical residues

How can researchers effectively study phosphorylation states of X. tropicalis syt17?

Based on phosphorylation studies of similar membrane proteins, researchers should employ a multi-faceted approach:

• PhosTag SDS-PAGE analysis:

  • Incorporate PhosTag molecules into acrylamide gels to separate proteins based on phosphorylation states

  • Compare migration patterns before and after phosphatase treatment

  • Identify phosphorylated forms by band shifts, as demonstrated in phosphorylation studies of related proteins

• Site identification strategy:

  • Use phosphorylation prediction tools like NetPhos3.1 to identify potential sites

  • Generate phospho-dead (S/T→A) and phospho-mimetic (S/T→D/E) mutants

  • Verify with mass spectrometry to identify specific modified residues

• Kinase regulation analysis:

  • Test the effects of PKA activators like Forskolin or 8-Br-cAMP, which have been shown to induce phosphorylation of related membrane proteins

  • Use kinase inhibitors like H89 to block specific phosphorylation events

  • Compare phosphorylation patterns in membrane versus cytosolic fractions

• Functional correlation:

  • Analyze how phosphorylation affects protein-protein interactions

  • Determine if phosphorylation alters subcellular localization

  • Assess effects on calcium sensitivity and membrane binding

How can recombinant syt17 be utilized in neuronal culture experiments?

For meaningful neuronal culture experiments:

• Expression system optimization:

  • Transfect primary hippocampal neurons using electroporation or viral transduction

  • Use neuron-specific promoters (synapsin) for appropriate expression levels

  • Co-express with fluorescent markers for visualization

• Subcellular localization analysis:

  • Perform immunofluorescence co-localization with synaptic markers like synaptophysin-1 and VGLUT1

  • Compare distribution patterns in axons versus dendrites

  • Quantify synaptic versus extrasynaptic localization similar to methods used for other synaptic proteins

• Functional assays:

  • Analyze effects on synaptic vesicle cycling using pHluorin-based sensors

  • Measure impact on axonal branching and synapse formation

  • Assess calcium-dependent activities in different subcellular compartments

• Protein-protein interactions:

  • Perform co-immunoprecipitation experiments to identify binding partners

  • Use proximity labeling approaches to identify the syt17 interactome

  • Verify interactions with co-localization studies in neuronal contexts

What approaches can identify protein interaction partners of X. tropicalis syt17?

Based on successful protein interaction studies with related proteins, researchers should:

• Co-immunoprecipitation strategy:

  • Express tagged versions of syt17 (FLAG, His, or GFP tags)

  • Immunoprecipitate protein complexes using tag-specific antibodies

  • Identify interacting partners by mass spectrometry

  • This approach has proven effective for identifying interactions between membrane proteins and binding partners like BASP1

• Affinity column approach:

  • Generate peptide or protein domains representing specific regions of syt17

  • Create affinity columns with immobilized syt17 fragments

  • Incubate with brain lysate and analyze eluates by mass spectrometry

  • Similar approaches successfully identified interaction partners of phosphorylated peptides from related proteins

• Subcellular co-localization analysis:

  • Perform immunofluorescence staining in neurons or heterologous cells

  • Quantify co-localization with potential partners

  • Validate with super-resolution microscopy techniques

  • This approach has been used to characterize synaptic protein complexes in primary hippocampal neurons

How might syt17 function during X. tropicalis metamorphosis-related neuronal remodeling?

Xenopus tropicalis undergoes dramatic TH-dependent remodeling during metamorphosis, including significant brain restructuring. To investigate syt17's role:

• Expression analysis during metamorphosis:

  • Assess syt17 mRNA and protein levels across metamorphic stages

  • Compare with expression patterns of other synaptic proteins that show developmental regulation

  • Many synaptic proteins like synaptotagmin II show peak expression at NF stage 63 during metamorphosis climax

• Thyroid hormone regulation assessment:

  • Examine if syt17 expression is TH-responsive like other neuronal genes

  • Some brain genes show significantly reduced expression in TRα-knockout tadpoles but not in TRβ-knockout animals, suggesting TRα-dependent regulation

  • Experimentally manipulate TH levels and observe effects on syt17 expression

• Neuronal remodeling correlation:

  • Determine if syt17 expression aligns with specific remodeling events

  • Compare aquatic versus terrestrial neuronal adaptations

  • Assess regional differences in expression across brain areas undergoing different degrees of remodeling

What is the conservation profile of syt17 across species, and how can this inform functional studies?

Understanding evolutionary conservation can provide insights into functional domains:

• Multiple sequence alignment analysis:

  • Compare syt17 sequences across species including mouse, human, zebrafish, X. tropicalis, rhesus monkey, and chicken

  • Identify conserved phosphorylation sites, which may be functionally significant

  • Similar conservation analysis for phosphorylation sites in related proteins revealed evolutionary preservation of key regulatory sites

• Structure-function relationship:

  • Identify highly conserved domains that likely perform essential functions

  • Map variable regions that may confer species-specific properties

  • Generate chimeric constructs to test domain-specific functions

• Conservation-based experimental design:

  • Focus mutational studies on highly conserved residues

  • Design antibodies targeting conserved epitopes for cross-species studies

  • Develop assays that can be standardized across model organisms

How can researchers determine if X. tropicalis syt17 is subject to alternative splicing or post-translational modifications?

For comprehensive characterization of syt17 variants and modifications:

• Alternative splicing analysis:

  • Perform RT-PCR across different tissues and developmental stages

  • Use RNA-seq data to identify potential splice variants

  • Verify variants with isoform-specific primers

• Post-translational modification mapping:

  • Use PhosTag SDS-PAGE to detect phosphorylated forms, as this technique effectively separates proteins based on phosphorylation state

  • Compare migration patterns before and after phosphatase treatment

  • Analyze different subcellular fractions, as modification patterns may differ between membrane-associated and cytosolic pools

• Mass spectrometry characterization:

  • Perform proteomic analysis of purified native and recombinant syt17

  • Enrich for specific modifications using affinity techniques

  • Compare modification patterns across developmental stages and tissues

• Functional significance assessment:

  • Generate modification-specific antibodies for specific detection

  • Create non-modifiable mutants to assess functional consequences

  • Compare modification patterns in different physiological and pathological conditions

What are the technical challenges in producing full-length versus truncated forms of recombinant X. tropicalis syt17?

Technical optimizations:

• For full-length protein:

  • Use mild detergents during extraction and purification

  • Consider nanodiscs or liposomes for membrane protein stabilization

  • Apply membrane-targeting sequences as used in studies of similar proteins

• For domain-specific constructs:

  • Design careful domain boundaries based on structural predictions

  • Use solubility-enhancing fusion tags

  • Consider the approach used for PLPPR3 where separate membrane-tagged (ICDm) and cytosolic (ICDc) variants revealed different phosphorylation patterns

• Expression verification:

  • Confirm proper folding with functional assays

  • Verify subcellular localization in cell models

  • Compare properties with native protein from X. tropicalis brain tissue

How does X. tropicalis syt17 research contribute to understanding comparative neurodevelopment?

Xenopus tropicalis provides a valuable model for evolutionary and comparative neurodevelopmental studies:

• Evolutionary insights:

  • X. tropicalis represents an important evolutionary position between fish and mammals

  • Comparing syt17 function across species can reveal conserved neuronal mechanisms

  • Research suggests the rodent brain undergoes TH-dependent remodeling during the first three postnatal weeks similar to X. tropicalis during metamorphosis

• Developmental model advantages:

  • External development allows easy manipulation and observation

  • Metamorphosis provides a natural model of extensive neuronal remodeling

  • The expression of brain genes during metamorphosis climax in X. tropicalis parallels expression patterns observed during postnatal development in rodents

• Research integration strategy:

  • Compare syt17 function across model organisms

  • Leverage X. tropicalis to study aspects of neurodevelopment difficult to access in mammals

  • Apply insights from X. tropicalis to understand human neurodevelopmental processes