GYG1 Human

Abstract

This comprehensive FAQ collection addresses key research questions about the human glycogenin-1 gene (GYG1) and its encoded protein, which plays a critical role in glycogen biosynthesis. The document synthesizes current scientific understanding of GYG1 function, pathology, and research methodologies based on peer-reviewed literature. These FAQs are specifically designed for academic researchers, ranging from fundamental concepts to advanced experimental considerations, with an emphasis on methodological approaches for investigating GYG1-related disorders.

What is the function of the GYG1 gene in humans?

The human GYG1 gene encodes glycogenin-1, a critical protein that initiates glycogen synthesis through autoglucosylation. This self-glucosylating activity creates a short glucose polymer that serves as the primer for subsequent elongation by glycogen synthase (GYS1) . Glycogenin-1 is essential for normal glycogen formation, particularly in skeletal muscle where glycogen serves as an energy reserve for contraction and relaxation .

Interestingly, research has shown that glycogenin-1 is not absolutely mandatory for glycogen formation in muscle tissue. Studies of patients with GYG1 mutations demonstrate that various amounts of normal glycogen can still be found despite depletion or absence of glycogenin-1 . This observation suggests potential alternative mechanisms for glycogen priming, which may explain the relatively late onset and slow progression of GYG1-related myopathies .

How does glycogenin-1 protein interact with glycogen synthase?

The interaction between glycogenin-1 and glycogen synthase is crucial for proper glycogen synthesis. Structural studies have revealed that:

• The C-terminal domain of glycogenin-1 is essential for binding to glycogen synthase .

• In cryo-EM structures, residues 317-349 of GYG1 form a helix-turn-helix motif that interacts with GYS1 in a 1:1 ratio .

• This interaction complex forms a rectangular box shape with GYG1 fragments positioned at each corner of the GYS1 homotetramer .

Functional studies of patients with C-terminal truncating mutations in GYG1 (e.g., p.Arg324*) demonstrate that while autoglucosylation activity is preserved, the truncated protein cannot properly facilitate glycogen chain elongation by glycogen synthase . This provides strong evidence that the C-terminal binding between glycogenin-1 and glycogen synthase is essential for glycogen synthase function .

What are the common pathogenic variants in the GYG1 gene?

Several pathogenic variants in GYG1 have been identified in patients with polyglucosan body myopathy:

The most common variant is c.143+3G>C, which has been identified in patients from different ethnic backgrounds, either in homozygous or compound heterozygous states .

What are the molecular mechanisms underlying GYG1-associated myopathies?

GYG1-associated myopathies represent a specific type of glycogen storage disease characterized by the accumulation of polyglucosan bodies in muscle fibers. The molecular pathogenesis involves:

• Altered glycogenin-1 function: Most patients show either reduced or complete absence of glycogenin-1 protein, consistent with the deleterious effects of GYG1 mutations .

• Polyglucosan body formation: PAS-positive storage material is found in approximately 30-40% of muscle fibers in affected individuals . These inclusions have distinctive characteristics compared to other polyglucosan body myopathies:

  • Less alpha-amylase resistant

  • More frequently found in fibers with apparently normal glycogen content

  • Show morphological heterogeneity reflecting differences in primary gene defects

• Persistence of normal glycogen: Despite glycogenin-1 deficiency, various amounts of normal glycogen are still present in patients' muscle fibers, suggesting alternative mechanisms for glycogen formation when glycogenin-1 is absent or dysfunctional .

• No apparent dysregulation of related enzymes: Analysis of glycogen synthase and branching enzyme expression showed no changes in patients with GYG1 mutations, suggesting that the pathology is not due to secondary alterations in these enzymes .

The clinical phenotype typically manifests as a slowly progressive, adult-onset myopathy primarily affecting the hip girdle, shoulder girdle, and/or distal limb muscles .

How does the phenotype of GYG1-associated myopathy differ from other glycogen storage diseases?

GYG1-associated myopathy presents with distinctive features that differentiate it from other glycogen storage diseases:

• Clinical presentation:

  • Primarily a skeletal myopathy without cardiac involvement in most cases

  • Slowly progressive, typically adult-onset disease

  • Predominantly affects hip girdle, shoulder girdle, and/or hand and leg muscles

  • Variable age of onset (childhood to late adulthood)

• Histopathological features:

  • Polyglucosan bodies that are less alpha-amylase resistant compared to those in GBE1 or RBCK1 deficiency

  • Inclusions more frequently found in fibers with apparently normal glycogen content

• Biochemical characteristics:

  • Depletion of glycogenin-1 protein in most cases

  • Normal levels of glycogen synthase and branching enzyme

  • Serum CK levels normal in most patients (elevated in only one case in the study)

This differs from a previously reported patient with GYG1 mutations who presented with cardiomyopathy, glycogen storage in cardiomyocytes, and glycogen depletion (rather than polyglucosan body formation) in skeletal muscle . That patient had accumulation (rather than depletion) of glycogenin-1, suggesting that the type of pathogenic variant and other factors significantly influence the phenotype .

What experimental approaches can be used to study glycogenin-1 autoglucosylation?

Several experimental approaches can be employed to assess glycogenin-1 autoglucosylation:

• Western blot analysis with alpha-amylase treatment:

  • Protein samples are treated with alpha-amylase to cleave glucose residues from glycogen particles

  • Gel migration patterns are compared before and after treatment

  • Autoglucosylated glycogenin-1 weighs approximately 1kDa more than unglucosylated protein

  • A gel shift after alpha-amylase treatment indicates functional autoglucosylation

• In vitro autoglucosylation assays:

  • Recombinant glycogenin-1 (wild-type or mutant) is expressed

  • UDP-glucose is added as a substrate

  • Gel shift analysis is performed to detect glucose incorporation

  • Functional glycogenin-1 shows a shift after UDP-glucose addition, while non-functional variants (e.g., p.Trp250*, p.Asp102His, p.Ala16Pro) show no shift

• Comparative analysis with glycogen synthase-deficient samples:

  • Samples from patients with GYG1 mutations can be compared with those from patients with glycogen synthase deficiency

  • Similar gel shift patterns may indicate defects in the interaction between glycogenin-1 and glycogen synthase

  • This approach was used to demonstrate that glycogenin-1 lacking the C-terminal (p.Arg324*) shows a pattern similar to glycogen synthase deficiency

These methods provide complementary information about glycogenin-1 function and can help determine how specific mutations affect its autoglucosylation activity and interaction with other proteins in the glycogen synthesis pathway.

How do mutations in GYG1 affect its interaction with glycogen synthase?

The interaction between glycogenin-1 and glycogen synthase is critical for proper glycogen synthesis, and GYG1 mutations can disrupt this interaction in several ways:

• C-terminal truncating mutations: Mutations resulting in C-terminal truncation of glycogenin-1 (e.g., p.Arg324*) specifically impair the interaction with glycogen synthase while preserving autoglucosylation activity . This leads to free autoglucosylated glycogenin-1, similar to what is observed in glycogen synthase deficiency .

• Complete loss of glycogenin-1: Many GYG1 mutations result in complete absence or severe reduction of glycogenin-1 protein, eliminating the possibility of interaction with glycogen synthase .

• Structural perturbations: Missense mutations (e.g., p.Asp102His, p.Ala16Pro) may cause conformational changes in glycogenin-1 that affect both its autoglucosylation activity and its ability to interact with glycogen synthase .

Cryo-EM structural studies have revealed that glycogenin-1 (specifically residues 317-349) forms a helix-turn-helix motif that interacts with glycogen synthase in a 1:1 ratio . Each GYS1 monomer consists of two Rossmann domains and a tetramerization domain and interacts with GYG1 at a specific interface . Disruption of this interface through mutation can impair the functional coupling between these two enzymes.

What methods are effective for detecting GYG1 mutations in clinical samples?

Several complementary approaches can be used for detecting GYG1 mutations in clinical samples:

• DNA analysis:

  • Sanger sequencing of the GYG1 gene (NM_004130) from genomic DNA extracted from blood or frozen skeletal muscle

  • Focus on coding regions and splice sites, particularly the common c.143+3G>C variant site

• RNA analysis:

  • Isolation of total RNA from frozen skeletal muscle using specialized kits (e.g., RNeasy Fibrous Tissue Mini Kit)

  • Reverse transcription to cDNA (e.g., using QuantiTect reverse transcription kit)

  • Analysis of GYG1 cDNA by Sanger sequencing

  • This approach is particularly useful for detecting splicing abnormalities, such as exon 2 skipping (r.8_143del) in patients with the c.143+3G>C variant

• Protein analysis:

  • Western blot analysis of muscle tissue with anti-glycogenin-1 antibodies

  • Comparison of samples before and after alpha-amylase treatment to assess glycogenin-1 levels and autoglucosylation status

  • This can reveal reduced expression, complete absence, or abnormal molecular weight of glycogenin-1

• Histological analysis:

  • PAS staining of muscle biopsies to detect polyglucosan bodies

  • Alpha-amylase digestion to assess the resistance of glycogen deposits

  • These analyses provide supportive evidence of GYG1-related pathology

The combination of these approaches allows for comprehensive analysis of GYG1 mutations and their functional consequences at the DNA, RNA, and protein levels.

What expression systems are suitable for producing recombinant glycogenin-1 for structural studies?

Based on research experiences, several strategies have proven effective for expressing and purifying glycogenin-1 for structural studies:

• Insect cell expression systems:

  • Insect expression systems have been successfully used for producing human GYS1 in complex with GYG1

  • This approach appears advantageous for expressing human glycogen metabolism proteins

• Co-expression strategies:

  • Co-expression with binding partners improves solubility and stability

  • Human GYS1 has proven difficult to produce alone in soluble form, but co-expression with human GYG1 in insect cells enabled isolation of a stable complex

  • For glycogenin-1 expression, co-expression with interacting partners may similarly enhance solubility

• Truncated constructs:

  • Using the C-terminal domain of glycogenin-1 (aa 294-350) rather than the full-length protein can improve expression

  • Bicistronic constructs encoding untagged human GYS1 and His6-GST-tagged GYG1 C-terminus have been successful

• Affinity tagging:

  • His6-GST tags have been used successfully for the GYG1 C-terminus, enabling affinity purification

  • Selection of appropriate affinity tags can facilitate purification without compromising protein function

These approaches can be adapted for different structural biology techniques, including X-ray crystallography, cryo-EM, and NMR spectroscopy, depending on the specific research questions.

What are the characteristics of polyglucosan bodies in GYG1-associated myopathy compared to other glycogen storage diseases?

Polyglucosan bodies in GYG1-associated myopathy exhibit several distinctive characteristics compared to those found in other glycogen storage diseases:

These distinguishing features likely reflect the different primary defects in glycogen metabolism. While polyglucosan body formation in some cases has been attributed to an imbalance between glycogen synthase and branching enzyme activities, GYG1-deficient patients show no apparent upregulation or downregulation of these enzymes . This suggests that the mechanism of polyglucosan body formation in GYG1-associated myopathy may differ from that in other glycogen storage diseases.

How can researchers design experimental models to study GYG1-related disorders?

Researchers can employ several experimental models to study GYG1-related disorders:

• Patient-derived samples:

  • Muscle biopsies from patients with confirmed GYG1 mutations provide valuable material for histological, biochemical, and molecular analyses

  • Typical analyses include:

    • Histological examination with PAS staining

    • Western blot analysis of glycogenin-1 expression

    • Assessment of glycogenin-1 autoglucosylation via alpha-amylase treatment

    • Analysis of GYG1 mRNA splicing patterns

• Recombinant protein expression systems:

  • Expression of wild-type and mutant glycogenin-1 for functional studies

  • Co-expression with glycogen synthase to study protein-protein interactions

  • Insect cell expression systems have proven effective for human glycogen metabolism proteins

  • Appropriate constructs include:

    • Full-length proteins

    • Domain-specific constructs (e.g., GYG1 C-terminal domain)

    • Bicistronic vectors for co-expression

• In vitro enzymatic assays:

  • Autoglucosylation assays to assess the functional impact of GYG1 mutations

  • UDP-glucose incorporation studies

  • Analysis of glycogenin-1/glycogen synthase interactions

• Structural biology approaches:

  • Cryo-EM studies of glycogenin-1 in complex with interacting partners

  • Focus on key functional domains, such as the C-terminal region that interacts with glycogen synthase

When designing these experimental models, researchers should consider:

• The specific research question (e.g., protein function, pathogenic mechanisms, potential therapies)

• The type of GYG1 mutation being studied

• The relevant tissue context (primarily skeletal muscle)

• The potential for tissue-specific effects, as evidenced by the different phenotypes observed in cardiac versus skeletal muscle in some patients