Recombinant Bombyx mori Trehalase, partial

What is the molecular function of Bombyx mori trehalase and how does it differ from mammalian disaccharidases?

Bombyx mori trehalase (EC 3.2.1.28) is an enzyme that catalyzes the hydrolysis of trehalose (α-D-glucopyranosyl-(1,1)-α-D-glucopyranoside) into two glucose molecules. Unlike mammalian disaccharidases that primarily process dietary sugars, trehalase in B. mori serves as a critical regulator of hemolymph trehalose levels, which is the main circulating sugar in insects . The enzyme exists in two distinct forms: soluble trehalase (BmTreh-1) and membrane-bound trehalase (BmTreh-2), with the latter containing a transmembrane domain that enables it to penetrate the cell membrane . This functional specialization allows precise regulation of trehalose metabolism across different tissues and developmental stages, with soluble trehalase predominantly found in goblet cell cavities and membrane-bound trehalase primarily located on visceral muscle surrounding the midgut .

How are trehalase isoforms regulated differently during the developmental stages of Bombyx mori?

The regulation of trehalase isoforms in B. mori shows distinct stage-specific patterns throughout development. Research has revealed that trehalase activity is dynamically controlled through both transcriptional regulation and post-translational modifications . During the feeding period of the fifth larval instar, soluble trehalase activity is modulated by two types of inhibitors in the hemolymph: a proteinaceous inhibitor (inhibitor-P) and an inhibitor extractable with methanol and ethanol (inhibitor-M) . These inhibitors show differential temporal expression patterns, with inhibitor-M exhibiting high activity during early to middle feeding periods followed by a sudden decrease, while inhibitor-P activity increases throughout the feeding period and markedly decreases two days after gut purge . This coordinated inhibitory activity ensures precise control of trehalose levels during critical developmental transitions.

What experimental approaches have been successful for cloning and characterizing the Bombyx mori trehalase gene?

Several successful approaches for cloning and characterizing B. mori trehalase genes have been documented:

• Promoter Analysis: Researchers have cloned the promoter of B. mori trehalase (BmTreh) and identified that estrogen-related receptor (ERR) binds directly to core response elements within the promoter .

• cDNA Cloning: Full-length cDNA cloning has been used to identify novel trehalases, including the membrane-penetrating type-2 trehalase that lacks the omega site typically found in GPI-anchored proteins .

• Bioinformatics Analysis: Computational approaches have been employed to identify conserved domains, transmembrane regions, and signal peptides in trehalase sequences .

• RT-PCR and qRT-PCR: These techniques have been utilized to analyze tissue-specific expression patterns and developmental regulation of trehalase genes .

Which expression systems are most effective for producing active recombinant Bombyx mori trehalase?

The baculovirus-silkworm expression system has proven most effective for producing active recombinant B. mori trehalase . This system offers several advantages:

• Preservation of enzymatic activity: The baculovirus-silkworm system maintains the native post-translational modifications essential for trehalase activity .

• Tag orientation effects: Studies have demonstrated that only N-terminally tagged trehalase shows high activity, while C-terminally tagged versions exhibit significantly reduced activity . This suggests that the C-terminus may be critical for proper folding or substrate binding.

• Fusion protein design: Successful expression has been achieved using hexahistidine tags for purification, with the bombyxin signal peptide (MKILLAIALMLSTVMWVST) serving as an effective secretion sequence for both conventional and unconventional secretory proteins .

When expressing recombinant trehalase, researchers should consider:

• Optimizing codon usage for the expression host

• Ensuring proper signal peptide cleavage

• Maintaining appropriate glycosylation patterns

• Preserving disulfide bond formation

How can researchers troubleshoot issues with recombinant trehalase activity after purification?

When encountering reduced activity of purified recombinant trehalase, researchers should systematically address several potential issues:

• Tag interference: As demonstrated in previous studies, the position of affinity tags can significantly impact enzyme activity . If activity is low, consider:

  • Repositioning the tag from C-terminus to N-terminus

  • Using a cleavable tag system

  • Testing different tag types (His, Strep, GST)

• Protein folding: Ensure proper folding through:

  • Optimizing expression temperature (typically lower temperatures promote proper folding)

  • Adding folding chaperones

  • Including appropriate cofactors during purification

• Post-translational modifications: Verify that essential modifications are preserved by:

  • Using eukaryotic expression systems

  • Analyzing glycosylation patterns

  • Confirming signal peptide cleavage sites

• Storage conditions: Prevent activity loss during storage by:

  • Testing various buffer compositions

  • Adding stabilizers (glycerol, trehalose)

  • Determining optimal pH and temperature for stability

• Inhibitor contamination: Be aware that B. mori tissues contain endogenous trehalase inhibitors that might copurify with the recombinant enzyme .

What analytical methods are essential for characterizing the structural integrity of recombinant Bombyx mori trehalase?

Complete characterization of recombinant B. mori trehalase requires multiple analytical approaches:

• Mass spectrometry:

  • For confirming protein identity

  • Identifying post-translational modifications

  • Verifying signal peptide cleavage sites and potential microheterogeneity

• Circular dichroism spectroscopy:

  • Assessing secondary structure content

  • Monitoring thermal stability

  • Evaluating conformational changes upon substrate binding

• Enzymatic activity assays:

  • Determination of kinetic parameters (Km, Vmax)

  • pH optimum and temperature stability profiles

  • Inhibitor sensitivity testing

• Immunoblotting:

  • Detection of specific trehalase isoforms

  • Assessment of expression levels

  • Verification of tag accessibility

• Immunohistochemistry:

  • Localization of expressed protein

  • Comparison with native trehalase distribution patterns

How does the estrogen-related receptor influence trehalase expression and hemolymph glucose levels in Bombyx mori?

The estrogen-related receptor (ERR) in B. mori serves as a direct transcriptional regulator of trehalase gene expression, with significant metabolic consequences. Research has revealed several key mechanisms:

• Direct promoter binding: ERR binds directly to core response elements (CREs) in the trehalase promoter, specifically ERRE-like CRE 1, 2, 4, and 6, as demonstrated through electrophoretic mobility shift assays (EMSA) and luciferase reporter assays .

• Transgenic overexpression effects: Studies with transgenic silkworms overexpressing BmERR showed:

  • Significantly increased expression of BmTreh in the midgut

  • Elevated glucose content in the hemolymph

  • Reduced trehalose content in the midgut

  • Changes in development and weight of last instar larvae

Experimental data from transgenic studies showed the following effects:

This relationship between ERR and trehalase expression represents a novel regulatory mechanism controlling carbohydrate metabolism in silkworms, with potential implications for understanding similar processes in other insects .

What is the significance of membrane-bound versus soluble trehalase in Bombyx mori tissue-specific energy metabolism?

The differential distribution and regulation of membrane-bound (BmTreh-2) and soluble (BmTreh-1) trehalase in B. mori tissues reveals specialized roles in energy metabolism:

• Spatial distribution:

  • Soluble trehalase-1 is predominantly located in goblet cell cavities of the midgut

  • Membrane-bound trehalase-2 is primarily found on visceral muscle surrounding the midgut

• Structural differences:

  • Membrane-bound trehalase contains a transmembrane domain absent in soluble trehalase

  • Unlike mammalian intestinal trehalases, which use GPI-anchors with omega sites, B. mori membrane-bound trehalase directly penetrates the cell membrane

• Metabolic implications:

  • The distinct localization suggests complementary functions in trehalose metabolism

  • Membrane-bound trehalase likely facilitates direct uptake of hemolymph trehalose into muscle tissue

  • Soluble trehalase may be involved in digestive processes within the midgut lumen

• Regulatory differences:

  • The two forms show differential sensitivity to inhibitors

  • Proteinaceous inhibitor-P inhibits soluble trehalase more effectively

  • Inhibitor-M (extractable with methanol/ethanol) inhibits membrane-bound trehalase slightly more effectively

This compartmentalization of trehalase activity enables precise tissue-specific regulation of carbohydrate metabolism, allowing different tissues to respond appropriately to varying energetic demands during development and feeding states .

How do peptide hormones like bombyxin and sulfakinin coordinate with trehalase to regulate energy homeostasis?

Peptide hormones play crucial roles in coordinating trehalase activity with broader energy homeostasis in B. mori through complex signaling networks:

• Bombyxin (Bombyx insulin-like peptide):

  • Reduces trehalose concentration in hemolymph

  • Decreases glycogen content in tissues

  • Increases oxygen consumption rate

  • Enhances fructose 2,6-bisphosphate content (a glycolysis activator) in growing tissues

  • Functions analogously to vertebrate insulin in promoting cellular energy production for tissue growth

• Sulfakinin signaling pathway:

  • Acts through the BNGR-A9 receptor

  • Significantly elevates hemolymph trehalose levels

  • Decreases food consumption and body weight

  • Triggers intracellular signaling involving IP3, Ca²⁺, and ERK1/2 phosphorylation

  • Represents a critical regulator of both feeding behavior and energy balance

• Coordination mechanism:

  • These hormonal systems create a balanced regulatory network

  • Bombyxin promotes tissue glucose utilization

  • Sulfakinin increases circulating trehalose levels

  • Together they maintain appropriate energy distribution between storage and utilization

Experimental evidence shows that sulfakinin administration significantly increases hemolymph trehalose levels, but this effect is markedly reduced by BNGR-A9 receptor knockdown using dsRNA, demonstrating the receptor-mediated nature of this regulation .

What controls are essential when measuring trehalase activity in transgenic Bombyx mori models?

When designing experiments to measure trehalase activity in transgenic B. mori models, researchers should implement comprehensive controls to ensure reliable results:

• Genetic controls:

  • Non-transgenic siblings from the same genetic background

  • Empty vector transgenics to control for insertion effects

  • Multiple independent transgenic lines to account for position effects

• Developmental controls:

  • Precise staging of larvae (e.g., specific day of last instar)

  • Standardized feeding conditions

  • Consistent sampling times to control for circadian effects

• Tissue-specific considerations:

  • Separate analysis of different tissues (midgut, fat body, muscle)

  • Microdissection techniques to isolate specific cell types

  • Recognition of distinct trehalase isoform distribution patterns

• Enzymatic assay controls:

  • Substrate blanks (no enzyme)

  • Enzyme blanks (no substrate)

  • Standard curves with pure glucose

  • Positive controls using commercial trehalase

  • Inclusion of known inhibitors as reference points

• Validation approaches:

  • Corroboration of enzymatic activity with gene expression levels

  • Complementary measurements of trehalose and glucose levels

  • Physiological outcomes (weight, development rate)

In the study of ERR overexpression, researchers screened transgenic lines and designated them as OE-BmERR for detection experiments, using fluorescent markers to identify positive transformants . They then measured multiple parameters including trehalase expression, trehalose/glucose content, and developmental outcomes to comprehensively assess the phenotypic effects.

How can researchers effectively distinguish between the functions of soluble and membrane-bound trehalase in vivo?

Distinguishing between the functions of soluble and membrane-bound trehalase in vivo requires sophisticated experimental approaches:

• Isoform-specific RNA interference:

  • Design dsRNA targeting unique regions of each trehalase isoform

  • Verify knockdown specificity using qRT-PCR

  • Assess physiological impacts of selective knockdown

• Immunolocalization with isoform-specific antibodies:

  • Generate antibodies against unique epitopes of each isoform

  • Perform immunohistochemistry to confirm differential tissue distribution

  • Use confocal microscopy for precise subcellular localization

• Biochemical fractionation:

  • Separate membrane-bound from soluble proteins using ultracentrifugation

  • Extract membrane proteins with detergents

  • Measure trehalase activity in different fractions

• Selective inhibition:

  • Utilize the differential sensitivity to inhibitor-P and inhibitor-M

  • Treat with specific inhibitor concentrations that preferentially affect one isoform

  • Monitor resulting changes in trehalose metabolism

• Transgenic rescue experiments:

  • Create double knockdown models depleted of both trehalase isoforms

  • Separately reintroduce each isoform using tissue-specific promoters

  • Evaluate which physiological functions are restored by each isoform

Previous research has successfully employed immunoblotting and immunohistochemical analyses to demonstrate that in midgut tissue, soluble trehalase-1 is present mainly in goblet cell cavities, while membrane-bound trehalase-2 is predominantly found on visceral muscle surrounding the midgut .

What methodological approaches can resolve contradictory findings about trehalase regulation across different tissues?

When faced with contradictory findings regarding trehalase regulation across different tissues, researchers should employ these methodological approaches:

• Tissue-specific transcriptomics and proteomics:

  • Perform RNA-seq on isolated tissues to identify transcriptional differences

  • Use proteomics to detect post-translational modifications

  • Analyze tissue-specific regulatory elements in trehalase genes

• Single-cell analysis techniques:

  • Employ single-cell RNA-seq to identify cell-specific expression patterns

  • Use laser capture microdissection to isolate specific cell populations

  • Perform in situ hybridization to visualize mRNA distribution patterns

• Time-course studies:

  • Conduct fine-grained temporal analysis across development

  • Sample multiple timepoints within the same developmental stage

  • Track dynamic changes in trehalase activity, inhibitor levels, and regulatory hormone concentrations

• Conditional genetic manipulations:

  • Develop tissue-specific and temporally regulated transgenic systems

  • Use GAL4/UAS or similar systems for targeted gene expression

  • Create inducible knockdown models to determine tissue-specific functions

• Systems biology approach:

  • Integrate transcriptomic, proteomic, and metabolomic data

  • Develop mathematical models of trehalase regulation

  • Identify feedback loops and compensatory mechanisms

Research has shown that trehalase inhibitors exhibit tissue-specific and stage-specific activities. Inhibitor-P inhibited soluble trehalase more effectively than membrane-bound trehalase, while inhibitor-M inhibited membrane-bound trehalase slightly more than soluble trehalase . This differential regulation helps explain seemingly contradictory observations in different tissues and developmental stages.

How might CRISPR/Cas9 genome editing advance our understanding of trehalase function in Bombyx mori?

CRISPR/Cas9 genome editing offers transformative potential for elucidating trehalase function in B. mori through several innovative applications:

• Precise modification of trehalase genes:

  • Creation of isoform-specific knockout models

  • Introduction of point mutations in catalytic domains

  • Modification of regulatory elements in promoter regions

  • Insertion of reporter tags for live imaging of expression patterns

• Regulatory network dissection:

  • Targeted modification of ERR binding sites in the trehalase promoter

  • Editing of hormone receptor genes (e.g., BNGR-A9) to disrupt signaling pathways

  • Creation of transcription factor binding site variants to alter expression patterns

• Structure-function analysis:

  • Generation of domain-specific mutations to identify critical functional regions

  • Introduction of amino acid substitutions to alter substrate specificity

  • Creating chimeric proteins between soluble and membrane-bound isoforms

• Physiological studies:

  • Development of conditional knockout models to study tissue-specific functions

  • Creation of reporter lines to visualize trehalose utilization in real-time

  • Engineering of trehalase variants with altered inhibitor sensitivity

• Evolutionary insights:

  • Introduction of trehalase variants from other insect species to test functional conservation

  • Recreation of ancestral trehalase sequences to study evolutionary trajectories

The genetic tractability of B. mori combined with CRISPR/Cas9 technology would allow researchers to move beyond correlative studies toward definitive causal relationships between trehalase function and physiological outcomes.

What are the implications of trehalase research for understanding metabolic disorders in non-insect systems?

Trehalase research in B. mori has broader implications for understanding metabolic disorders in non-insect systems:

• Evolutionary conservation of carbohydrate metabolism:

  • Trehalose metabolism shares regulatory mechanisms with glucose homeostasis in mammals

  • Insulin-like peptides (bombyxin) have functional parallels with mammalian insulin signaling

  • The ERR pathway represents a conserved nuclear receptor signaling system

• Novel regulatory mechanisms:

  • The sulfakinin/BNGR-A9 pathway provides insights into peptide hormone regulation of metabolism

  • Dual inhibitor systems (proteinaceous and non-proteinaceous) suggest sophisticated metabolic control mechanisms

  • Membrane-penetrating trehalase offers a model for transmembrane enzyme function

• Therapeutic applications:

  • Understanding trehalase inhibition could inform approaches to modulating carbohydrate metabolism

  • The bombyxin signal peptide has proven effective for recombinant protein production with potential biopharmaceutical applications

  • Discovery of novel regulatory mechanisms may identify new therapeutic targets for metabolic disorders

• Methodological advances:

  • The silkworm expression system represents an efficient platform for producing recombinant proteins, including potential therapeutic agents

  • Techniques developed for studying trehalase may be applicable to other metabolic enzymes

Research has demonstrated that the silkworm expression system can effectively produce both conventionally and unconventionally secreted human growth factors with full biological functionality, highlighting its potential for biopharmaceutical applications .

How can integrative multi-omics approaches advance our understanding of trehalase's role in hormonal regulation of metabolism?

Integrative multi-omics approaches offer powerful strategies to comprehensively understand trehalase's role in the hormonal regulation of metabolism:

• Multi-level systems analysis:

  • Combine genomics, transcriptomics, proteomics, and metabolomics data

  • Integrate tissue-specific and temporal dimensions

  • Develop computational models of trehalase regulation within broader metabolic networks

• Hormone-response profiling:

  • Perform transcriptomic and proteomic analysis after bombyxin or sulfakinin administration

  • Identify direct and indirect targets of hormonal signaling

  • Map signaling cascade from receptor activation to metabolic enzyme regulation

• Developmental metabolomics:

  • Track comprehensive metabolite profiles throughout development

  • Correlate trehalose/glucose dynamics with broader metabolic changes

  • Identify metabolic signatures associated with different developmental transitions

• Interactome mapping:

  • Use proteomics to identify trehalase-interacting proteins

  • Characterize protein complexes involved in trehalase regulation

  • Map physical interactions between signaling components and metabolic enzymes

• Cross-species comparative analysis:

  • Compare trehalase regulation across insect orders

  • Identify conserved and divergent regulatory mechanisms

  • Trace evolutionary history of trehalase regulation