Recombinant Mouse Diacylglycerol O-acyltransferase 2-like protein 6 (Dgat2l6)

What is Dgat2l6 and what is its functional role in lipid metabolism?

Dgat2l6 (Diacylglycerol O-acyltransferase 2-like protein 6) is a member of the diacylglycerol acyltransferase 2 family. It functions as a putative acyltransferase, likely involved in the synthesis of di- or triacylglycerol, though its specific substrate preferences remain under investigation . The protein exhibits diacylglycerol O-acyltransferase activity and is primarily localized to the endoplasmic reticulum membrane . Dgat2l6 plays a role in lipid metabolism pathways similar to other DGAT2 family members, which catalyze the formation of triglycerides by creating covalent bonds between activated fatty acids (FA-CoA) and diacylglycerol .

Unlike DGAT1, which has multiple functions, DGAT2 family members (including Dgat2l6) appear more specific to triacylglycerol synthesis, making them interesting targets for metabolic research . Current evidence suggests Dgat2l6 contributes to cellular lipid homeostasis, though its exact physiological significance compared to other family members requires further characterization.

How does Dgat2l6 relate structurally and functionally to DGAT2?

Dgat2l6 belongs to the DGAT2 gene family but displays distinct characteristics from the canonical DGAT2. While DGAT2 was identified by its homology to a DGAT in the fungus Mortierella rammaniana , Dgat2l6 represents one of several mammalian homologues that evolved subsequently.

The human DGAT2L6 protein consists of 337 amino acids and shares the following functional domains with DGAT2 :

While both proteins share core enzymatic functions, Dgat2l6's specific activity, regulation, and physiological importance remain less characterized than DGAT2, which has demonstrated crucial importance for cell membrane stability and lipid metabolism .

What is the genetic location and evolutionary conservation of Dgat2l6?

In mice, the Dgat2l6 gene is located on chromosome X (Chr X:99568444-99589714) . The human ortholog (DGAT2L6, Gene ID: 347516) is also X-linked. This X-chromosome localization may have implications for sex-specific expression patterns that warrant investigation in research studies.

Evolutionarily, Dgat2l6 appears to be part of a family that expanded from an ancestral DGAT2-like gene. The protein contains several functionally important domains that show varying degrees of conservation across species, including the catalytic domain responsible for acyltransferase activity. When designing cross-species studies, researchers should account for species-specific variations in protein sequence and function, particularly when translating findings between mouse models and human applications.

What are the optimal conditions for reconstituting and storing recombinant Dgat2l6?

Proper reconstitution and storage of recombinant Dgat2l6 is critical for maintaining protein activity. Based on established protocols, researchers should follow these guidelines:

Reconstitution Protocol:

• Briefly centrifuge the vial containing lyophilized protein before opening to bring contents to the bottom

• Reconstitute in deionized sterile water to a concentration of 0.1-1.0 mg/mL

• Add glycerol to a final concentration of 5-50% (the standard recommendation is 50%) for long-term storage

• Aliquot to minimize freeze-thaw cycles

Storage Recommendations:

• Store working aliquots at 4°C for up to one week

• For long-term storage, maintain at -20°C/-80°C

• Avoid repeated freeze-thaw cycles as they significantly reduce protein activity

• Shelf life is approximately 6 months for liquid formulations at -20°C/-80°C

• Lyophilized form has an extended shelf life of approximately 12 months at -20°C/-80°C

These conditions help preserve the native conformation and enzymatic activity of the protein for experimental applications.

What expression systems are most effective for producing functional recombinant Dgat2l6?

Multiple expression systems have been utilized to produce recombinant Dgat2l6, each with advantages for different experimental applications:

For producing full-length mouse Dgat2l6, both E. coli and yeast systems have been successfully employed. The choice depends on the specific research application:

• For structural studies or applications where high purity is prioritized over native folding, E. coli systems may be preferred

• For functional studies where enzymatic activity is critical, mammalian or yeast expression systems typically produce more biologically relevant protein

When designing expression constructs, commonly used tags include His-tag, Fc-tag, and Avi-tag, which facilitate purification while minimally impacting protein function .

How can researchers assess the enzymatic activity of Dgat2l6 in vitro?

Assessing Dgat2l6 enzymatic activity requires methods that measure its ability to catalyze the transfer of an acyl group to diacylglycerol. Based on established protocols for related DGAT enzymes, the following methods can be adapted for Dgat2l6:

In vitro DGAT Activity Assay:

• Prepare cellular membranes (typically microsomal fractions) containing recombinant Dgat2l6

• Incubate with diacylglycerol substrate and fatty acyl-CoA (labeled with radioisotope or fluorescent tag)

• Measure the formation of triacylglycerol products using:

  • Thin-layer chromatography (TLC) for separation

  • Liquid scintillation counting (for radioisotope-labeled substrates)

  • HPLC or mass spectrometry for detailed product analysis

Key Assay Parameters:

• Optimal buffer: Tris/PBS-based buffer, pH 8.0

• Temperature: 37°C (mammalian) or 30°C (if stability is an issue)

• Presence of appropriate cofactors

• Controls should include reactions without enzyme or without one substrate

• MgCl₂ concentration affects activity (high concentrations ~100mM may inhibit activity, based on DGAT2 data)

Researchers should note that Dgat2l6's substrate specificity is not fully characterized, so assays may need to test multiple potential acyl donors and diacylglycerol species to comprehensively assess enzymatic function.

How can CRISPR-Cas9 technology be optimized for Dgat2l6 gene editing?

CRISPR-Cas9 gene editing of Dgat2l6 requires careful design and validation strategies:

sgRNA Design Considerations:

• Target sequence selection should avoid regions with high homology to other DGAT family members

• For complete knockout, target early exons or essential catalytic domains

• For each target, design multiple sgRNAs to increase success probability

• Verify target sequence uniqueness through BLAST analysis

Validation Methods:

• Screen edited clones using detection systems like abm's Screen It™ CRISPR Cas9 Cleavage Detection Kit

• Confirm indels and mutations through Sanger sequencing

• Verify knockout at protein level using Western blot with validated antibodies

• Assess functional consequences through enzymatic activity assays

Commercial DGAT2L6 knockout cell lines (e.g., 293T) are available and validated by multiple methods, providing useful controls or starting points for research . For mouse studies, researchers should consider both constitutive and conditional knockout approaches, particularly given potential developmental effects.

What are the implications of Dgat2l6 in disease models and potential therapeutic applications?

While DGAT2L6-specific disease associations are still being characterized, research on the related DGAT2 provides valuable insights:

Disease Associations:

• Lipid metabolism disorders: DGAT2 family members play crucial roles in triglyceride synthesis and storage

• Neurological disorders: DGAT2 mutations have been linked to Axonal Charcot-Marie-Tooth disease

• Cancer metabolism: Alterations in lipid metabolism genes, including DGAT family members, are observed in cancer cells with increased energy demands

Mutation Analyses:
DGAT2 mutations have been extensively studied in the COSMIC database, identifying:

• D222V as a mutation hotspot that may affect enzyme activity in cancer cells

• Multiple mutations within the catalytic site with potential pathogenic effects

• A distinctive pattern between cancer-associated mutations and the Y223H mutation linked to Charcot-Marie-Tooth disease

For Dgat2l6 research, similar comprehensive mutation analyses could reveal specific disease associations and functional consequences. Given its role in lipid metabolism, Dgat2l6 represents a potential target for therapeutic development in metabolic disorders, though target validation would require further characterization of its specific physiological roles distinct from other DGAT family members.

How do species differences in Dgat2l6 affect experimental design and data interpretation?

Species differences in Dgat2l6 sequence, expression patterns, and function require careful consideration in experimental design:

Key Species Differences:

• Sequence homology: While core catalytic domains are conserved, species variations in regulatory regions may affect function

• Expression patterns: Mouse and human Dgat2l6 may have different tissue distribution patterns

• Protein-protein interactions: Species-specific interaction partners could alter functional outcomes

• Regulatory mechanisms: Transcriptional and post-translational regulation may differ between species

Experimental Design Implications:

• When using mouse models to study human disease, acknowledge limitations in translating findings

• Consider using humanized mouse models for studies focused on human-specific functions

• Always validate key findings across species when possible

• In cell culture systems, use species-matched components when studying protein-protein interactions

The BioGPS expression databases for human and mouse provide tissue-specific expression data that can guide researchers in selecting appropriate experimental systems . Species differences should be explicitly addressed in research papers to avoid overgeneralizing findings.

What approaches can be used to study Dgat2l6 protein-protein interactions and regulatory networks?

Understanding Dgat2l6's interactome and regulatory networks requires multiple complementary approaches:

Protein-Protein Interaction Methods:

• Co-immunoprecipitation (Co-IP) with tagged recombinant Dgat2l6

• Proximity labeling approaches (BioID, APEX) for membrane-associated proteins

• Yeast two-hybrid screening, with appropriate modifications for membrane proteins

• Split-luciferase complementation assays for in vivo interaction validation

• Protein-fragment complementation assays

Regulatory Network Analysis:

• ChIP-seq to identify transcriptional regulators of Dgat2l6

• RNA-seq following perturbations (knockout, overexpression) to identify downstream pathways

• Phosphoproteomics to identify post-translational modification sites

• Metabolomics to characterize impact on lipid profiles and metabolic pathways

Data Integration Approaches:
For comprehensive understanding, integrate data from:

• Transcriptomic profiling across tissues (available in resources like BioGPS and Allen Brain Atlas)

• Proteomics datasets for co-expression patterns

• Knowledge-based databases like Reactome, which already contain information on DGAT2L6 activities

The Harmonizome database indicates DGAT2L6 has 2,457 functional associations with biological entities spanning 8 categories, providing a starting point for interactome exploration .

How can researchers design experiments to distinguish Dgat2l6 functions from other DGAT family members?

Distinguishing the specific functions of Dgat2l6 from other DGAT family members requires carefully designed experiments:

Selective Inhibition Approaches:

• Gene-specific knockdown/knockout: Use siRNA, shRNA, or CRISPR targeting unique regions of Dgat2l6

• Domain-specific mutations: Introduce mutations in regions unique to Dgat2l6 while preserving common catalytic domains

• Selective inhibitors: While pan-DGAT inhibitors exist, developing Dgat2l6-specific inhibitors would be valuable

Substrate Specificity Determination:

• In vitro enzyme assays with diverse substrates to identify unique preferences

• Competitive substrate assays to determine relative affinities

• Structure-guided mutagenesis to identify residues controlling substrate specificity

Rescue Experiments:

• Knockout multiple DGAT family members and perform selective rescue with Dgat2l6

• Create chimeric proteins with domains from different DGAT family members to map function

Expression Pattern Analysis:
Compare tissue-specific and subcellular expression patterns of Dgat2l6 with other family members to identify unique physiological contexts .

What are the current limitations and contradictions in Dgat2l6 research that require resolution?

Several key limitations and contradictions exist in the current understanding of Dgat2l6:

Knowledge Gaps:

• Substrate specificity: Unlike DGAT2, which is specific to triacylglycerol synthesis, Dgat2l6's precise substrate preferences remain undefined

• Physiological role: The specific contribution of Dgat2l6 to lipid metabolism relative to other DGAT family members is unclear

• Regulation: Mechanisms controlling Dgat2l6 expression and activity in different physiological states are poorly characterized

Research Contradictions:

• Function prediction vs. experimental validation: While bioinformatic analyses predict similar functions to DGAT2, experimental verification is incomplete

• Expression data inconsistencies: Different databases and studies may show conflicting tissue expression patterns

• Disease relevance: The significance of Dgat2l6 in metabolic disorders and cancer remains to be established definitively

Technical Challenges:

• Protein solubility: As a membrane protein, Dgat2l6 presents challenges for structural studies

• Antibody specificity: Ensuring antibodies distinguish between closely related DGAT family members

• Functional redundancy: Compensatory mechanisms may mask phenotypes in single-gene perturbation studies

Addressing these limitations requires multidisciplinary approaches combining structural biology, enzymology, cell biology, and in vivo models.

How can metabolomics approaches be integrated into Dgat2l6 functional studies?

Metabolomics offers powerful approaches to elucidate Dgat2l6 function in lipid metabolism:

Targeted Lipidomics:

• LC-MS/MS analysis focusing on triglycerides, diglycerides, and related intermediates

• Stable isotope labeling to track specific lipid fluxes and turnover rates

• Comparative analysis between wild-type and Dgat2l6-modified systems

Experimental Design Considerations:

• Time-course studies to capture dynamic changes in lipid profiles

• Challenge experiments (high-fat conditions, fasting/feeding cycles) to reveal conditional functions

• Tissue-specific analyses reflecting physiological context

Integration with Other Data Types:

• Correlate metabolomic changes with transcriptomic alterations

• Link metabolite profiles to phenotypic observations

• Use computational modeling to predict pathway fluxes

Analysis Framework:

• Utilize both univariate and multivariate statistical approaches

• Consider pathway-level analysis rather than individual metabolites

• Validate key findings with targeted analyses and isotope tracing

This integrated approach can reveal not only direct substrates and products but also broader metabolic consequences of Dgat2l6 activity or deficiency.

What emerging technologies could advance our understanding of Dgat2l6 function?

Several cutting-edge technologies offer promising approaches for Dgat2l6 research:

Structural Biology Advances:

• Cryo-EM for membrane protein structural determination

• Hydrogen-deuterium exchange mass spectrometry for conformational dynamics

• Computational prediction methods incorporating AlphaFold2 for protein structure modeling

Single-Cell Technologies:

• Single-cell RNA-seq to identify cell populations with high Dgat2l6 expression

• Spatial transcriptomics to map expression in tissue context

• Mass cytometry with metabolic probes to correlate protein levels with functional outcomes

Genome Engineering:

• Base editing for precise introduction of specific mutations

• Prime editing for more complex genetic modifications

• Inducible degron systems for temporal control of Dgat2l6 function

In Situ Imaging:

• Advanced fluorescent lipid probes to track metabolism in living cells

• Super-resolution microscopy to visualize subcellular localization

• Live-cell imaging with activity-based probes to monitor enzyme function

These technologies, particularly when used in combination, could resolve current limitations in understanding Dgat2l6's precise biochemical function, physiological roles, and potential as a therapeutic target.

What interdisciplinary approaches might yield breakthrough insights into Dgat2l6 biology?

Advancing Dgat2l6 research will likely require interdisciplinary collaboration:

Systems Biology Approaches:

• Integrative multi-omics analysis combining genomics, transcriptomics, proteomics, and metabolomics data

• Network modeling to position Dgat2l6 within broader metabolic pathways

• Machine learning applications to predict functional consequences of genetic variants

Translational Research Collaborations:

• Clinical-basic science partnerships examining Dgat2l6 variants in patient cohorts

• Biobanking initiatives to correlate expression levels with metabolic parameters

• Pharmaceutical partnerships to develop specific modulators

Cross-Species Comparative Studies:

• Evolutionary analyses to understand functional conservation and divergence

• Model organism studies beyond mice (e.g., zebrafish, C. elegans) for complementary insights

• Comparative genomics to identify regulatory elements controlling expression

The fact that Dgat2l6 functions at the intersection of multiple fields—lipid metabolism, membrane biology, cellular energetics, and potentially disease pathogenesis—makes it an ideal candidate for interdisciplinary investigation that could yield insights extending beyond its specific function to broader principles of metabolic regulation.