GLYATL2 Human

What is the genomic location and structure of the human GLYATL2 gene?

The human GLYATL2 gene is located on chromosome 11q12.1 (complement strand). It spans positions 58834065 to 58909828 on NC_000011.10 and comprises 8 exons. The gene is also known by alternative identifiers GATF-B and BXMAS2-10. Understanding its genomic context is essential for designing targeted genetic studies and interpreting variants detected in research or clinical settings .

What are the key structural and functional properties of human GLYATL2?

Human GLYATL2 is a member of a family of four putative glycine conjugating enzymes. This enzyme is localized to the endoplasmic reticulum and demonstrates glycine N-acyltransferase activity. Functionally, it catalyzes the conjugation of various medium- and long-chain acyl-CoAs to glycine, with a preference for glycine as the acceptor molecule. GLYATL2 most efficiently produces N-oleoyl glycine and is involved in the catabolic processes of long-chain fatty acids, medium-chain fatty acids, and monounsaturated fatty acids .

What is the tissue distribution pattern of human GLYATL2?

Human GLYATL2 mRNA shows highest expression in salivary gland and trachea. Significant expression has also been detected in spinal cord and skin fibroblasts. This distribution pattern suggests potential roles in barrier function and immune response, particularly given the presence of N-acyl glycines in skin and lung tissues. When designing experiments to study GLYATL2 function, selecting appropriate cellular models that reflect this tissue distribution is critical for physiologically relevant results .

What are effective methods for recombinant expression and purification of GLYATL2?

For recombinant expression of GLYATL2, research suggests that bacterial expression systems using E. coli can be effective when optimized. Based on successful approaches with similar enzymes like mouse GLYAT, researchers should consider:

• Using a bacterial expression vector containing an N-terminal His-tag for affinity purification

• Conducting expression at lower temperatures (16-20°C) to enhance proper folding

• Employing nickel affinity chromatography for purification to homogeneity

This approach typically yields 2-3 mg/L of pure protein from bacterial culture. When expressing human GLYATL2, researchers should pay particular attention to codon optimization for bacterial expression and the potential need for chaperone co-expression to improve folding efficiency .

How can enzymatic activity of GLYATL2 be measured in laboratory settings?

GLYATL2 activity can be measured spectrophotometrically by following the release of free CoA-SH using Ellman's reagent (DTNB, ε412 = 13,600 M^-1cm^-1). A typical reaction buffer consists of:

• 300 mM Tris-HCl pH 8.0

• 150 μM DTNB

• Variable concentrations of acyl-CoA substrates (as amino acceptors)

• Variable concentrations of glycine (as amino donor)

For kinetic characterization, steady-state parameters can be determined by:

• Holding the concentration of acyl-CoA constant (e.g., 300 μM benzoyl-CoA) while varying glycine concentration to determine kinetic constants for the amino donor

• Holding glycine concentration constant (e.g., 100 mM) while varying acyl-CoA concentration to determine kinetic constants for the amino acceptor

Data can be fitted to the Michaelis-Menten equation to derive kinetic parameters .

What approaches can be used to study post-translational modifications of GLYATL2?

Research indicates that human GLYATL2 is regulated by acetylation/deacetylation of lysine 19, suggesting an important regulatory mechanism for this enzyme . To study these post-translational modifications, researchers can employ:

• Mass spectrometry-based approaches: Including both bottom-up and top-down proteomics to identify and quantify acetylation sites

• Site-directed mutagenesis: Creating K19R (non-acetylatable) or K19Q (acetylation-mimicking) mutants to assess functional consequences

• Biochemical assays with recombinant enzyme: Comparing activity of native versus acetylated forms

• Deacetylase inhibitor studies: Using HDAC inhibitors in cellular systems to modulate acetylation status and observe effects on enzyme activity

Understanding these regulatory mechanisms is crucial for comprehensive characterization of GLYATL2 function in physiological contexts.

How can researchers investigate the physiological roles of GLYATL2-derived N-acyl glycines?

N-acyl glycines produced by GLYATL2 demonstrate diverse biological activities including antinociceptive, anti-inflammatory, and antiproliferative effects, and can activate G-protein-coupled receptors . To investigate these physiological roles:

• Target identification: Use affinity-based proteomics or GPCR activation screening to identify molecular targets of specific N-acyl glycines

• Tissue-specific knockdown/knockout: Generate conditional GLYATL2 knockout models in tissues with high expression to observe phenotypic consequences

• Metabolomics profiling: Employ LC-MS/MS to quantify changes in N-acyl glycine profiles in different physiological and pathological states

• Barrier function assessment: Given expression in skin and trachea, evaluate epidermal/epithelial barrier integrity using transepithelial electrical resistance (TEER) measurements in GLYATL2-modulated systems

• Immune response characterization: Assess effects on inflammatory cytokine production and immune cell function using ex vivo and in vivo approaches

These multidisciplinary approaches can help elucidate the complex biological functions of this enzymatic system .

What experimental designs are most suitable for studying substrate specificity of human GLYATL2?

To characterize GLYATL2 substrate specificity comprehensively:

Previous characterization suggests GLYATL2 efficiently conjugates oleoyl-CoA, arachidonoyl-CoA, and other medium/long-chain acyl-CoAs specifically to glycine, with preference for producing N-oleoyl glycine . Researchers should design experiments that explore beyond these known substrates, potentially identifying novel biological activities.

How can researchers resolve contradictory findings regarding GLYATL2 function?

When faced with contradictory experimental results regarding GLYATL2 function:

• Harmonize experimental conditions: Standardize buffer compositions, substrate concentrations, and assay methods across laboratories

• Control for post-translational modifications: Given the known regulation by lysine acetylation, ensure consistent preparation methods that preserve or control for PTM status

• Account for species differences: Compare human GLYATL2 with orthologs from model organisms to identify divergent properties

• Validate with multiple methodologies: Employ both in vitro biochemical approaches and cellular systems to corroborate findings

• Consider tissue context: Evaluate whether contradictions might arise from tissue-specific cofactors or regulatory mechanisms

• Examine isozyme contributions: Investigate potential functional overlap with other GLYAT family members (GLYAT, GLYATL1, GLYATL3)

Rigorous application of these approaches can help resolve apparent contradictions and develop a more unified understanding of GLYATL2 biology .

What strategies can be employed to study GLYATL2 in the context of lipid signaling networks?

GLYATL2-produced N-acyl glycines represent an emerging class of lipid signaling molecules. To study GLYATL2 within broader lipid signaling networks:

• Lipidomics integration: Implement comprehensive LC-MS/MS workflows to simultaneously profile multiple lipid classes in response to GLYATL2 modulation

• Receptor de-orphanization: Screen N-acyl glycines against GPCR libraries to identify specific receptor interactions

• Signaling pathway analysis: Use phosphoproteomics to map downstream effects of N-acyl glycine application in cellular systems

• In vivo microdialysis: Collect and analyze N-acyl glycines from extracellular fluid in specific tissues under various physiological states

• Biosensor development: Create fluorescent or bioluminescent sensors for real-time monitoring of GLYATL2 activity or N-acyl glycine production

These approaches enable researchers to position GLYATL2 within the complex landscape of lipid-based intercellular communication systems .

How can structural biology approaches enhance understanding of GLYATL2 function?

Structural biology offers powerful tools to understand GLYATL2 mechanism and regulation:

• X-ray crystallography/Cryo-EM: Determine three-dimensional structure of GLYATL2, particularly in complex with substrates or products

• Homology modeling: Leverage structures of related GNAT superfamily members to predict GLYATL2 structure

• Molecular dynamics simulations: Model substrate binding and catalytic mechanism in silico

• HDX-MS (Hydrogen-deuterium exchange mass spectrometry): Map conformational changes upon substrate binding or post-translational modifications

• Site-directed mutagenesis guided by structural data: Systematically alter putative catalytic residues to validate mechanistic hypotheses

Structural insights would significantly advance understanding of GLYATL2 substrate specificity and catalytic mechanism, potentially enabling structure-based drug design targeting this enzyme or its products .

What considerations are important when developing genetic models to study GLYATL2 function?

When designing genetic models to study GLYATL2:

• Model selection justification: Consider tissue expression patterns (salivary gland, trachea, spinal cord, skin) when selecting model organisms or cell types

• Compensation mechanisms: Assess potential compensatory upregulation of other GLYAT family members (GLYAT, GLYATL1, GLYATL3) in knockout models

• Temporal control: Implement inducible systems to distinguish between developmental and acute effects of GLYATL2 loss

• Rescue experiments: Include complementation with wild-type and mutant forms to confirm specificity of observed phenotypes

• Physiological relevance: Focus on phenotypes related to known N-acyl glycine functions (antinociception, inflammation, barrier function)

These considerations ensure that genetic models provide meaningful insights into GLYATL2 biology rather than artifacts of experimental design .

How might GLYATL2 research contribute to understanding pathological conditions?

Given the biological activities of N-acyl glycines, GLYATL2 research may provide insights into several pathological conditions:

• Inflammatory disorders: The anti-inflammatory properties of N-acyl glycines suggest GLYATL2 dysregulation might contribute to inflammatory conditions, particularly in tissues with high expression like trachea and skin

• Pain processing abnormalities: The antinociceptive effects of N-acyl glycines indicate potential roles in pain perception and modulation, relevant to spinal cord expression

• Barrier dysfunction diseases: Expression in tissues with important barrier functions (skin, trachea) suggests potential involvement in conditions characterized by compromised epithelial integrity

• Cancer biology: The antiproliferative effects of N-acyl glycines warrant investigation of GLYATL2 in the context of cellular proliferation control

Research methodologies should include clinical sample analysis, correlation of gene expression or enzyme activity with disease parameters, and functional studies in disease-relevant cell types .

What methodological approaches can link GLYATL2 genetic variants to functional consequences?

To investigate functional consequences of GLYATL2 genetic variants:

• Variant identification and prioritization: Use databases like ClinVar and Variation Viewer to identify reported variants, prioritizing those in conserved regions or predicted functional domains

• Recombinant expression of variant proteins: Express and purify variant forms of GLYATL2 to assess alterations in enzymatic activity, substrate specificity, or protein stability

• Cellular models: Generate isogenic cell lines expressing variant forms using CRISPR-Cas9 to observe effects on N-acyl glycine production and cellular phenotypes

• Structural modeling: Predict impacts of amino acid substitutions on protein structure and function using computational approaches

• Population-based metabolomics: Correlate variant genotypes with N-acyl glycine profiles in biological samples from population cohorts

This systematic approach connects genetic variation to molecular and cellular phenotypes, potentially revealing both pathological mechanisms and normal regulatory processes .