METTL21A Human

What is METTL21A and what is its primary molecular function?

METTL21A is a human protein methyltransferase that belongs to the seven-beta strand (7BS) methyltransferase family. It specifically catalyzes the trimethylation of lysine residues in several heat shock protein 70 (HSP70) family members, earning it the alternative designation HSPA-KMT (HSPA lysine methyltransferase). Located on chromosome 2, this protein-coding gene is also known as FAM119A and HCA557b .

The enzyme functions by transferring methyl groups from S-adenosylmethionine (SAM) to specific lysine residues in HSP70 proteins. This post-translational modification has functional consequences, as it alters the ability of HSP70 proteins to interact with their client proteins . METTL21A adopts a characteristic Rossmann fold-like twisted β-sheet structure containing conserved sequence motifs involved in methyl donor binding .

What are the verified targets of METTL21A methyltransferase activity?

METTL21A exhibits remarkable specificity in its methylation activity. Current research has identified the following specific methylation sites in human HSP70 proteins:

These methylation sites are all located within the lid domain of HSP70 proteins, a region critical for client protein interactions . Notably, experimental evidence demonstrates that METTL21A exclusively methylates 70-kDa proteins in mammalian protein extracts, highlighting its high substrate specificity .

How is METTL21A expressed across human tissues and what is its subcellular localization?

Regarding subcellular localization, immunofluorescence co-staining with TRA98 (a nuclear marker of pan-germ cells) revealed that METTL21A is mainly localized in the cytoplasm of germ cells . This cytoplasmic localization is consistent with its role in methylating cytoplasmic HSP70 proteins.

For researchers investigating METTL21A expression patterns, both transcript-level analyses (RT-qPCR) and protein-level detection methods (Western blot, immunofluorescence) should be employed to generate comprehensive expression profiles.

What essential experimental methods are used to study METTL21A activity?

Researchers investigating METTL21A employ several complementary experimental approaches:

In vitro enzymatic assays:

• Recombinant protein expression and purification of METTL21A

• Methyltransferase activity assays using purified HSP70 proteins as substrates

• Detection of methylation using S-adenosylmethionine (SAM) as a methyl donor

• ATP-dependent activity measurements to determine cofactor requirements

Cellular models:

• Generation of METTL21A knockout cell lines using CRISPR/Cas9 technology

• siRNA-mediated knockdown of METTL21A expression

• Overexpression of wild-type and mutant METTL21A constructs

Animal models:

• Generation of Mettl21a knockout mice using CRISPR/Cas9 with dual sgRNAs targeting introns flanking exon 2

• Phenotypic analysis of reproductive function and cellular development

Analytical detection methods:

• Mass spectrometry to identify methylation sites and quantify methylation states

• Extraction of MS/MS spectra using Qual Browser

• Generation of chromatograms representing various methylation states

• Relative quantification by integrating area under curves

What is the current evidence in the METTL21A vs. SETD1A controversy regarding HSPA1-K561 methylation?

A significant controversy in the field concerns which enzyme is responsible for methylating HSPA1 at lysine 561:

Cho et al. reported that SETD1A catalyzes dimethylation of HSPA1 at lysine 561 (HSPA1-K561me2), suggesting this modification localizes to the cell nucleus where it activates Aurora kinase B . Conversely, other researchers identified METTL21A as the enzyme responsible for HSPA1-K561 methylation in vitro and in vivo .

To resolve this controversy, researchers analyzed a METTL21A knockout cell line using mass spectrometry:

• HSPA1-K561 was primarily trimethylated (HSPA1-K561me3) in wild-type KBM-7 cells

• In METTL21A knockout cells (which still expressed SETD1A), HSPA1-K561 was exclusively unmethylated

These findings provide strong evidence that METTL21A, not SETD1A, is required for HSPA1-K561 methylation. This controversy highlights the importance of using genetic knockout models combined with precise analytical techniques to definitively assign enzyme-substrate relationships in protein methylation research .

How does ATP influence METTL21A methyltransferase activity and what are the structural implications?

A significant finding in METTL21A research is that its methyltransferase activity toward HSP70 proteins is stimulated by ATP . This ATP dependence has important structural and functional implications:

• HSP70 proteins are ATP-dependent molecular chaperones that undergo substantial conformational changes upon ATP binding and hydrolysis

• The ATP-dependence of METTL21A activity suggests the enzyme may preferentially methylate HSP70 proteins in specific conformational states

• This relationship provides a potential regulatory mechanism linking METTL21A enzymatic activity to the functional cycle of HSP70 chaperones

Methodologically, researchers investigating METTL21A kinetics should:

• Include ATP in reaction conditions

• Compare methylation efficiency across different nucleotide-bound states of HSP70

• Consider how conformational changes in the substrate affect enzyme accessibility to the target lysine residue

What phenotypes are associated with METTL21A knockout models and what do they reveal about functional redundancy?

Studies using METTL21A knockout models have yielded surprising findings about its physiological roles:

• Despite high expression in testes, Mettl21a knockout male mice show complete fertility with no apparent defects in germ cell development, including spermatogonial differentiation, meiosis, and sperm maturation

• The absence of METTL21A does not affect the expression levels or subcellular localization of its known target proteins (HSP70, HSC70, GRP75, and GRP78) in mouse testes

• RT-qPCR and Western blot confirmed the complete absence of both mRNA and protein of Mettl21a in knockout mice

Several hypotheses may explain this lack of phenotype:

• Functional redundancy: Other methyltransferases might compensate for METTL21A absence, although researchers did not observe significant increases in mRNA levels of other METTL21 homologs

• Context-specific function: METTL21A may play more critical roles under specific stress conditions not examined in the knockout studies

• Species-specific functions: The importance of METTL21A may differ between mice and humans

For researchers studying METTL21A function, these findings highlight the importance of considering compensatory mechanisms and examining phenotypes under various physiological and stress conditions .

How does METTL21A-mediated methylation affect HSP70 protein function at the molecular level?

METTL21A-mediated trimethylation has several functional consequences for HSP70 proteins:

• Altered client protein interactions: Trimethylation of HSPA8 (Hsc70) changes its affinity for both monomeric and fibrillar forms of α-synuclein, a protein associated with Parkinson's disease

• Regulatory impact: The methylation occurs in the lid domain of HSP70 proteins, which governs substrate binding and release cycles

• Potential localization effects: Some researchers have suggested methylation might affect subcellular distribution of HSP70 proteins, although conflicting data exist regarding nuclear localization of methylated HSP70

To investigate these functional impacts, researchers should:

• Compare client binding profiles between methylated and unmethylated HSP70 proteins

• Examine chaperone activity using ATPase assays with various client proteins

• Utilize point mutations (K-to-R) at methylation sites to create non-methylatable variants

• Employ METTL21A knockout cells as controls in functional studies

What methodological approaches provide the most accurate detection of METTL21A-mediated methylation?

Detecting and quantifying METTL21A-mediated methylation requires specialized analytical approaches:

Mass spectrometry-based methods:

• Extraction of MS/MS spectra of relevant peptides using Qual Browser

• Generation of chromatograms representing various methylation states (unmethylated, mono-, di-, and trimethylated)

• Relative quantification by integrating area under curves

• High mass accuracy (10 ppm sensitivity) to distinguish between different methylation states

Sample preparation considerations:

• Enrichment strategies for methylated proteins/peptides

• Digestion protocols optimized for recovering methylated peptides

• Consideration of peptide charge states and fragmentation patterns

Methodological limitations:

• Lack of specific antibodies against methylated targets limits immunological detection methods

• Potential for methyl mark loss during sample processing

• Challenges in distinguishing between various methylation states (mono-, di-, trimethylation)

Researchers should validate findings using multiple complementary approaches, including genetic models (METTL21A knockout) and rescue experiments with wild-type or catalytically inactive METTL21A.

What is known about the potential role of METTL21A in disease processes and pathological conditions?

The potential involvement of METTL21A in disease processes remains an emerging area of research:

• Neurodegenerative diseases: The interaction between methylated HSPA8 and α-synuclein suggests potential implications for Parkinson's disease and other protein aggregation disorders

• Cancer: Some reports suggest altered HSPA1-K561 methylation in various cancers, though direct evidence for METTL21A's role in cancer development remains limited

• Stress response modulation: As METTL21A modifies HSP70 proteins critical for cellular stress responses, its dysregulation could potentially impact how cells respond to various stressors

• Analysis of METTL21A expression and activity across various disease states

• Investigation of genetic variations in METTL21A and their potential association with disease risk

• Examination of how METTL21A-mediated methylation affects HSP70 function under pathological conditions