Recombinant Human Glycerol-3-phosphate acyltransferase 2, mitochondrial (GPAT2)

What is GPAT2 and what distinguishes it from other GPAT isoforms?

GPAT2 is a mitochondrial isoform of glycerol-3-phosphate acyltransferase that catalyzes the initial and rate-limiting step in glycerolipid synthesis. Unlike GPAT1 (which is NEM-resistant), GPAT2 demonstrates N-ethylmaleimide (NEM) sensitivity. The protein has a calculated mass of 88.8 kDa and shares approximately 27% amino acid identity with GPAT1 .

GPAT2 differs significantly from other GPAT isoforms in its tissue distribution and physiological role. While GPAT1 is predominantly expressed in lipogenic tissues and upregulated during adipocyte differentiation, GPAT2 is highly expressed in testis and is not associated with typical lipogenic functions. Instead, GPAT2 has been unexpectedly found to be required for piRNA biogenesis, functioning in a manner that appears independent of its acyltransferase activity .

Another distinguishing feature is that GPAT2 belongs to the "cancer-testis genes" (CTs) group - genes that are important during specific stages of spermatogenesis but show low or no expression in somatic tissues. These genes are often ectopically overexpressed in various cancers and contribute to the tumor phenotype .

What is the tissue distribution pattern of GPAT2?

GPAT2 exhibits a highly specialized tissue distribution pattern with dramatically higher expression in testis compared to other tissues. Quantitative RT-PCR analysis has revealed that GPAT2 mRNA expression in testis is approximately 50-fold higher than in liver or brown adipose tissue, and even lower in other tissues .

This contrasts sharply with GPAT1, which shows highest expression in metabolically active tissues including brown and white adipose tissue, liver, soleus muscle, and heart . Interestingly, despite the high mRNA expression in testis, the protein content of GPAT2 and the specific activity in mitochondria appear similar in testis and liver, indicating a potential discrepancy between mRNA and protein expression levels .

The restricted expression pattern of GPAT2 suggests a specialized function in testicular tissue, particularly in spermatogenesis, rather than a general role in lipid metabolism as seen with other GPAT isoforms .

What are the structural characteristics of GPAT2?

GPAT2 consists of 798 amino acids with a calculated molecular mass of 88.8 kDa. Structural prediction analyses have identified at least two transmembrane domains (TMDs) in the protein. One TMD is consistently predicted to be located between amino acids 446 and 474, which aligns with the first TMD of mouse GPAT1 .

The second probable TMD is predicted to encompass residues 156-177, positioned between the N-terminus and the active site domain. With these transmembrane domains, GPAT2 likely adopts a membrane topography where both the N- and C-termini are located in the intermembrane space, allowing the active site to face the cytosol where it can access its glycerol-3-phosphate and acyl-CoA substrates .

Like GPAT1, GPAT2 does not contain known mitochondrial targeting sequences, despite its mitochondrial localization. The protein contains conserved motifs (1-4) that form part of the active site, similar to other acyltransferases .

Interestingly, when expressed in cell culture systems, GPAT2 appears to undergo post-translational modification, as evidenced by the appearance of an 80 kDa protein band in addition to the expected 88 kDa band corresponding to the full-length protein. This modification appears to be tissue-specific, as brown adipose tissue expresses both the 88 kDa and 80 kDa forms, while liver and testis only express the 88 kDa form .

How is GPAT2 expression regulated during spermatogenesis?

GPAT2 expression during spermatogenesis follows a precise temporal pattern that correlates with specific developmental stages. Studies of the first wave of mouse spermatogenesis have shown that GPAT2 mRNA content and protein expression reach maximal levels at 15 days post-partum (dpp) and are restricted to pachytene spermatocytes .

This transient expression is achieved through a combination of epigenetic mechanisms and trans-acting factors. DNA methylation plays a crucial role in regulating GPAT2 expression during spermatogenesis. Bisulfite sequencing of the GPAT2 promoter in germ cells has confirmed that DNA methylation decreases dramatically at around 11 dpp, consistent with the initiation of meiosis. This demethylation appears to be a prerequisite for GPAT2 expression .

In addition to DNA methylation, histone acetylation also contributes to GPAT2 regulation. In vitro studies have demonstrated that GPAT2 expression increases following treatment with the DNA methyltransferase inhibitor 5-aza-2-deoxycitidine (DAC) and the histone deacetylase inhibitor trichostatin A (TSA), confirming the involvement of both DNA methylation and histone modifications in controlling GPAT2 expression .

Furthermore, retinoic acid (RA) has been identified as an upregulator of GPAT2 expression. This finding is particularly significant as RA is known to play a critical role in the initiation of meiosis during spermatogenesis .

What is the role of GPAT2 in piRNA biogenesis?

GPAT2 has been unexpectedly identified as a critical factor in piRNA (piwi-interacting RNA) biogenesis. piRNAs are small non-coding RNAs that protect the germ cell genome from retrotransposable elements by interacting with the Argonaute family proteins, specifically the PIWI clade proteins like MIWI, MILI, and MIWI2 .

Research has shown that knockdown of GPAT2 in germline stem cells impairs primary piRNA production. Moreover, GPAT2 protein physically interacts with MILI in both germline stem cells and mouse testis, suggesting a direct role in the piRNA pathway .

The timing of GPAT2 expression during spermatogenesis (maximal at 15 dpp in pachytene spermatocytes) aligns with the period of active piRNA synthesis, further supporting its functional importance in this process. The specific stage-limited expression pattern of GPAT2 suggests a precise temporal requirement for its activity during meiosis I prophase .

What is the relationship between GPAT2 and cancer?

GPAT2 belongs to a category of genes known as "cancer-testis genes" (CTs), which are primarily expressed during specific stages of spermatogenesis but show minimal or no expression in normal somatic tissues. These genes are frequently overexpressed ectopically in various types of cancer, where they contribute to the tumor phenotype .

Research has demonstrated that human GPAT2 is overexpressed in several cancer types and in cancer-derived human cell lines. This aberrant expression appears to contribute significantly to the tumorigenic phenotype. Experimental studies have shown that tumor cells with diminished GPAT2 expression exhibit lower proliferation and migration rates. Furthermore, in mouse xenograft models, cells with reduced GPAT2 expression demonstrate decreased tumorigenicity .

The dual nature of GPAT2 as both a critical factor in normal germ cell development and a potential oncogene when inappropriately expressed makes it an intriguing target for further cancer research.

What experimental approaches can be used to study GPAT2 activity in vitro?

Several methodological approaches have been established for investigating GPAT2 activity in vitro:

Recombinant Expression Systems:
GPAT2 can be studied using transient transfection in mammalian cell lines such as Cos-7 or HEK293 cells. The full-length mouse GPAT2 cDNA can be subcloned into a mammalian expression vector (e.g., pcDNA3.1(+)) and transfected using reagents like Fugene 6. For enhanced detection, epitope tags such as the Flag tag (DYKDDDDK) can be added to the C-terminus of the protein .

GPAT Activity Assays:
After expression, GPAT2 activity can be measured in total cell membranes. The assay typically involves incubating membrane preparations with glycerol-3-phosphate and radiolabeled acyl-CoA substrates. GPAT2 activity is distinguished from GPAT1 by its sensitivity to N-ethylmaleimide (NEM). Therefore, conducting parallel assays with and without NEM allows for the specific measurement of GPAT2 activity .

The reaction products can be analyzed by thin-layer chromatography (TLC), which typically shows a distribution of approximately 27% lysophosphatidic acid, 67% phosphatidic acid, and 6% diacylglycerol for GPAT2 activity .

Metabolic Labeling Studies:
To assess the impact of GPAT2 on lipid metabolism, transfected cells can be incubated with trace amounts of radiolabeled fatty acids (e.g., [1-14C]oleate). Subsequent extraction and analysis of cellular lipids reveal the incorporation of labeled fatty acids into various lipid classes, providing insights into the in vivo activity of GPAT2 .

How can GPAT2 promoter methylation be analyzed?

Analysis of GPAT2 promoter methylation is critical for understanding its epigenetic regulation. Several approaches can be employed:

Bisulfite Sequencing:
This represents the gold standard for DNA methylation analysis. The method involves treating DNA with bisulfite, which converts unmethylated cytosines to uracils while leaving methylated cytosines unchanged. Subsequent PCR amplification and sequencing reveal the methylation status of individual CpG sites within the GPAT2 promoter. This approach has been successfully used to confirm DNA methylation changes in germ cells during spermatogenesis .

Methylation-Specific PCR (MSP):
This technique uses primer sets designed to distinguish between methylated and unmethylated sequences after bisulfite conversion. It provides a rapid assessment of methylation status but with less resolution than bisulfite sequencing.

Pharmacological Demethylation Studies:
Treatment of cells with DNA methyltransferase inhibitors such as 5-aza-2-deoxycitidine (DAC) can be used to assess the impact of DNA methylation on GPAT2 expression. Typically, cells are treated with DAC for several days (e.g., 2 μM DAC for 3 days, replacing media and DAC every 24 hours), followed by RNA isolation and qPCR to measure changes in GPAT2 mRNA levels .

Combined Epigenetic Modification Analysis:
To study the interplay between DNA methylation and histone acetylation, cells can be treated with both DAC and histone deacetylase inhibitors such as trichostatin A (TSA). For instance, cells might be treated with DAC for 3 days followed by TSA (500 nM) on the fourth day, with cell harvesting on day 5 .

What are effective methods for detecting and measuring GPAT2 expression?

Multiple complementary approaches can be used to detect and quantify GPAT2 expression at both mRNA and protein levels:

Quantitative Real-Time PCR (qPCR):
This technique allows precise quantification of GPAT2 mRNA levels. RNA is isolated from tissues or cells using reagents like TRIzol, followed by cDNA synthesis and qPCR with GPAT2-specific primers. For accurate results, appropriate reference genes should be used for normalization .

In Situ Hybridization:
This method enables visualization of GPAT2 mRNA expression in tissue sections, providing spatial information about expression patterns. It has been successfully used to localize GPAT2 expression to specific cell types during spermatogenesis, notably pachytene spermatocytes .

Immunohistochemistry:
This technique allows visualization of GPAT2 protein expression in tissue sections. It requires a specific antibody against GPAT2. Studies have used this approach to confirm the cellular localization of GPAT2 in testicular tissues .

Western Blotting:
This method quantifies GPAT2 protein levels in tissue or cell lysates. It can reveal important information about post-translational modifications, as evidenced by the detection of both 88 kDa and 80 kDa forms of GPAT2 in certain tissues. Polyclonal antibodies raised against full-length GPAT1 have been shown to cross-react with GPAT2, allowing for its detection .

Confocal Microscopy:
This imaging technique provides high-resolution visualization of protein localization within cells. Using epitope-tagged GPAT2 constructs or specific antibodies, confocal microscopy has confirmed the mitochondrial localization of GPAT2 .

How should experiments be designed to study GPAT2's role during spermatogenesis?

Investigating GPAT2's function during spermatogenesis requires a multi-faceted experimental approach:

Developmental Time-Course Studies:
To capture the dynamic expression pattern of GPAT2 during the first wave of spermatogenesis, testis samples should be collected at different time points post-partum (e.g., 5, 10, 15, 20, 30 dpp). This allows for tracking changes in GPAT2 expression relative to key spermatogenic events .

Cell-Type Isolation:
Specific germ cell populations (spermatogonia, spermatocytes, spermatids) can be isolated using techniques such as FACS (Fluorescence-Activated Cell Sorting) or MACS (Magnetic-Activated Cell Sorting) to analyze GPAT2 expression in defined cell types.

Correlation with Meiotic Markers:
Co-expression analysis of GPAT2 with established markers of meiotic progression helps position GPAT2 function within the spermatogenic program. This can be achieved through co-immunostaining or parallel analysis of marker gene expression .

In Vivo Manipulation:
Testis-specific knockdown or overexpression of GPAT2 using viral vectors or transgenic approaches can provide insights into its functional importance. Analysis should include assessment of germ cell development, meiotic progression, and fertility outcomes.

piRNA Analysis:
Given GPAT2's role in piRNA biogenesis, experiments should include analysis of piRNA profiles in normal versus GPAT2-depleted testes. This can be achieved through small RNA sequencing and northern blot analysis for specific piRNA species .

Epigenetic Regulation Studies:
Analysis of GPAT2 promoter methylation at different developmental stages provides insights into regulatory mechanisms. This should be coupled with ChIP (Chromatin Immunoprecipitation) studies to identify transcription factors and chromatin modifications associated with GPAT2 activation .

What approaches can be used to investigate the post-translational modification of GPAT2?

The observation that GPAT2 appears as both 88 kDa and 80 kDa forms suggests important post-translational modifications that may regulate its function:

N-Terminal Sequencing:
To determine the exact nature of the 80 kDa form, N-terminal sequencing of the purified protein can identify the cleavage site. This requires immunoprecipitation of sufficient quantities of the 80 kDa GPAT2 form from cells or tissues expressing this variant .

Mass Spectrometry Analysis:
LC-MS/MS analysis of purified GPAT2 can identify various post-translational modifications including phosphorylation, acetylation, and glycosylation that might affect protein function.

Mutagenesis Studies:
Site-directed mutagenesis of potential modification sites followed by expression and functional analysis can reveal the importance of specific residues in GPAT2 processing and activity.

Pulse-Chase Experiments:
These studies can determine whether the 80 kDa form arises from proteolytic processing of the full-length protein or represents an alternative translation product. Cells expressing GPAT2 are pulse-labeled with radioactive amino acids and then "chased" with non-radioactive media, allowing tracking of protein processing over time.

In Vitro Translation:
As demonstrated in the research, in vitro transcription and translation systems can be used to compare the size of the primary translation product with the forms observed in vivo. The TNT-T7 Quick coupled transcription and non-radioactive translation system has been successfully used for this purpose .

How should discrepancies between GPAT2 mRNA and protein levels be interpreted?

Research has revealed interesting discrepancies between GPAT2 mRNA and protein expression levels that warrant careful interpretation:

Tissue-Specific Post-Transcriptional Regulation:
Despite GPAT2 mRNA being approximately 50-fold higher in testis compared to liver, the protein content and specific activity appear similar in both tissues . This suggests substantial post-transcriptional regulation, which might include:

• Differential mRNA stability or translation efficiency across tissues

• Tissue-specific microRNA regulation of GPAT2 mRNA

• Different protein turnover rates in various tissues

Experimental Approaches for Resolution:
To address these discrepancies, researchers should consider:

• Polysome profiling to assess translational efficiency of GPAT2 mRNA in different tissues

• mRNA half-life studies using actinomycin D chase experiments

• Analysis of potential regulatory microRNAs using prediction algorithms and validation studies

• Protein stability assays using cycloheximide to block new protein synthesis

Functional Implications:
The discrepancy between mRNA and protein levels may reflect a regulatory mechanism that ensures tight control over GPAT2 activity. High mRNA levels in testis might allow for rapid protein synthesis at specific developmental stages without requiring new transcription. This could be particularly important for the precise temporal control needed during spermatogenesis .

How can contradictory data about GPAT2 function be reconciled?

GPAT2 presents some seemingly contradictory aspects that require careful interpretation:

To reconcile these observations, researchers should consider:

• GPAT2 may have evolved dual functionality, with separate domains mediating acyltransferase activity and piRNA biogenesis

• The enzymatic activity might be required for creating a specific lipid environment conducive to piRNA processing

• Structure-function studies with domain deletion or mutation constructs can help delineate the regions responsible for each function

Expression Pattern vs. Activity Distribution:
The dramatically higher mRNA expression in testis compared to other tissues contrasts with the relatively similar protein levels and enzymatic activity observed in testis and liver mitochondria .

This contradiction might be resolved by:

• Considering that high mRNA levels may serve as a reservoir for rapid translation during specific developmental windows

• Investigating whether testicular GPAT2 might be subject to inhibitory post-translational modifications that reduce its specific activity

• Examining whether the protein has different subcellular distributions or binding partners across tissues that affect its apparent activity

What are the critical unanswered questions regarding GPAT2 function?

Several fundamental questions about GPAT2 remain unanswered and represent important directions for future research:

Mechanism of piRNA Biogenesis Regulation:
The precise molecular mechanism by which GPAT2 contributes to piRNA biogenesis remains unclear. Future studies should investigate:

• The specific protein domains that interact with MILI and other piRNA pathway components

• Whether GPAT2 enzymatic activity generates specific lipid species that facilitate piRNA processing complex assembly

• The composition and subcellular localization of GPAT2-containing complexes during piRNA biogenesis

Physiological Substrates and Products:
While GPAT2 has demonstrated acyltransferase activity in vitro, its physiological substrates and products in vivo, particularly in testis, remain to be fully characterized. Research should address:

• Substrate specificity analysis using various acyl-CoA species and glycerol-3-phosphate analogs

• Lipidomic analysis of GPAT2-deficient tissues to identify altered lipid species

• The potential role of specific GPAT2-generated lipids in membrane dynamics during spermatogenesis

Regulatory Mechanisms Beyond Epigenetics:
While DNA methylation and histone acetylation have been implicated in GPAT2 regulation, other potential regulatory mechanisms warrant investigation:

• Identification of specific transcription factors that drive GPAT2 expression

• The role of non-coding RNAs in post-transcriptional regulation

• Potential hormonal regulation, particularly by reproductive hormones

Cancer-Promoting Mechanisms:
The mechanisms by which GPAT2 contributes to tumorigenesis remain incompletely understood. Future studies should examine:

• Changes in global piRNA profiles in cancer cells with altered GPAT2 expression

• Potential impacts on retrotransposon silencing and genomic stability

• Alterations in lipid metabolism that might support cancer cell proliferation

What methodological advances would enhance GPAT2 research?

Advancement in GPAT2 research could be accelerated by several methodological innovations:

Improved Antibodies and Detection Systems:
Development of highly specific monoclonal antibodies against GPAT2 would greatly facilitate:

• More sensitive and specific detection of endogenous GPAT2 by Western blotting and immunohistochemistry

• Effective immunoprecipitation for interaction studies and N-terminal sequencing

• Super-resolution microscopy to precisely localize GPAT2 within mitochondrial membranes

CRISPR/Cas9-Mediated Genome Editing:
Application of CRISPR/Cas9 technology would enable:

• Generation of tissue-specific or inducible GPAT2 knockout or knockin mouse models

• Introduction of specific mutations to dissect structure-function relationships

• Endogenous tagging of GPAT2 for visualization and purification

Advanced Structural Biology Approaches:
Determining the three-dimensional structure of GPAT2 would provide crucial insights into:

• The arrangement of transmembrane domains and membrane topology

• The architecture of the active site and substrate binding pockets

• Potential interaction interfaces with piRNA pathway components

Single-Cell Analysis Technologies:
Application of single-cell approaches would allow:

• Precise mapping of GPAT2 expression dynamics during spermatogenesis at single-cell resolution

• Correlation of GPAT2 expression with global transcriptome and epigenome changes

• Identification of heterogeneous responses to GPAT2 manipulation within tumor cell populations