Functional Analysis of a Novel Pathogenic Glycine Amidinotransferase Mutant in Hereditary Fanconi Syndrome

Abstract

The mutant glycine amidinotransferase (GATM) protein polymerizes into a characteristic crystal, resulting in mitochondrial dysfunction in proximal tubules and Fanconi syndrome with progressive kidney failure. We identified a novel hereditary Fanconi syndrome characterized by mutant GATM proteins in a Japanese family, the first in Asia. Kidney biopsies exhibited typical histological features, confirmed by immunofluorescent staining with anti-GATM and anti-ATP synthase β subunit (ATPB) antibodies and electron microscopy. Quantitative immunofluorescence analysis showed a significant correlation between ATPB and GATM–ATPB colocalization, suggesting mitochondrial pathogenicity of the mutant. Previously reported GATM mutations, clustered in the fourth of 5 β-sheets of the GATM protein, are assumed to have increased adhesive properties, changing the β-sheet from its original dimeric form to a unique polymerization. Targeted panel sequencing showed a novel mutation (Q268E) in the GATM gene (NM_001482.3: c.802C>G), distinct from this sheet. Because conventional methods failed to detect the significance, we employed an advanced molecular dynamics approach—hypersound-perturbed molecular dynamics simulations. This analysis effectively demonstrated that the Q268E mutation enhanced conformational flexibility of region-spanning residues that are spatially positioned between the mutated site and the sheet, leading to the pathological polymerization of the mutant protein. This simulation offers valuable insights into the pathological mechanisms of abnormal protein conformation.

Introduction

Fanconi syndrome (FS) is a disorder characterized by impaired reabsorption of amino acids, glucose, bicarbonate, and inorganic phosphorus in renal proximal tubular epithelial cells (PTECs), leading to metabolic acidosis, electrolyte imbalances, dehydration, developmental disorders, and rickets.¹ FS can be either congenital/hereditary or acquired, with various pathogenetic mechanisms identified.^1,2 Mitochondrial dysfunction, particularly relevant in PTECs, has been recognized as a contributing factor in hereditary FS.^3,4

Glycine amidinotransferase (GATM), a key enzyme in the biosynthesis of creatine, is abundantly expressed in the mitochondria of PTECs.5, 6, 7 Mutations in the nuclear-encoded GATM gene (eg, c.958C>T, p.P320S; c.1006A>G, p.T336A; c.1007C>T, p.T336I; and c.1022C>T, p.P341L), which produces a protein that localizes to mitochondria, have been associated with hereditary FS.^8,9 To date, 28 patients from 5 unrelated kindreds have been diagnosed worldwide outside of Asia, and the disease phenotype is consistently inherited in an autosomal dominant pattern.⁸ The pathogenesis of the disease is thought to involve the formation of a characteristic intracellular crystal-like linear aggregations, leading to mechanical damage of mitochondria that contributes to FS with progressive kidney failure.^8,9

Here, we report hereditary FS caused by a novel GATM mutation (NM_001482.3: c.802C>G, p.Q268E). After a microscopy-based experimental approach verified the mitochondrial damage caused by the GATM mutant proteins, we employed hypersound-perturbed molecular dynamics (HS-MD) simulation, which accelerates slow biomolecular processes by enhancing solvent motion without inducing protein denaturation and thereby enables efficient exploration of physiologically relevant protein recognition events and druggable sites. This technique effectively demonstrated that the mutant GATM can induce insoluble polymerization similar to that observed in previous reports.^8,10 We clarified that these GATM mutant proteins could structurally and functionally induce mitochondrial dysfunction, thereby causing congenital FS.

Case Report

A Japanese man in his 40s and his teenage daughter were referred to our hospital because of persistent proteinuria. Both exhibited declining kidney function with glucosuria, hypokalemia, hypophosphatemia, metabolic acidosis, and high urinary fractional excretion of uric acid and phosphate, suggesting hereditary FS (Table S1). Kidney biopsy showed normal glomeruli, except for some global sclerotic changes observed under light microscopy. Tubular atrophy and interstitial fibrosis were less pronounced in the daughter than in the father (Fig 1A). In both patients, numerous crystal-like linear aggregations of various sizes were observed within almost all PTECs (Fig 1B). Routine immunofluorescence staining was unremarkable. However, electron microscopy showed numerous electron-dense, crystal-like linear aggregations inside and adjacent to the mitochondria in PTECs (Fig 1C). Larger structures appeared to connect to the mitochondrial membrane and spread into the cytoplasm. Because this characteristic structure closely resembled that seen with the previously reported mutant GATM protein, we examined whether these structures represented polymerized GATM protein using indirect immunofluorescent microscopy with an anti-human GATM antibody.⁹ We also used anti-ATPB (β subunit of ATP synthase) and DAPI (4′,6-diamidino-2-phenylindole) to label mitochondria and nuclei in the PTECs (Item S1. Supplementary Methods). GATM staining was clearly evident in crystal-like linear aggregations, colocalized with ATPB staining in the PTECs (Fig 1D). Quantitative analysis of cytoplasmic GATM and ATPB (Fig 2 and Fig S4) showed that GATM was significantly increased (Fig 2G and Table S2), whereas ATPB was decreased (Fig 2H and Table S2), in the patients’ PTECs. Moreover, colocalization of GATM and ATPB was markedly reduced in the patients (Fig 2I and Table S2), showing a strong correlation with ATPB expression (Fig 2J). This suggests an association between the accumulation of mutant GATM near mitochondria and its functional impairment.

Figure 1.

Kidney biopsy findings of the daughter. (A) Light microscopic findings using periodic acid-methenamine-silver staining (scale bar: 100 μm). (B) Light microscopy showing crystal-like linear aggregations (arrow) within the PTECs. Masson trichrome staining (scale bar: 100 μm). The inset shows an enlarged image of the crystal-like linear aggregations (scale bar: 20 μm). (C) Electron microscopy revealing characteristic fibrous structures (arrow) inside and adjacent to mitochondria in PTECs (scale bar: 2 μm). (D) Indirect immunostaining with anti-GATM antibody (green), anti-ATPB antibody (red), and DAPI (blue). Numerous characteristic intracellular crystal-like linear aggregations were observed in almost all PTECs, confirmed as GATM by immunofluorescence (arrow). GATM protein accumulated both inside and adjacent to mitochondria, with incomplete merging of GATM and ATPB in the patient’s kidney compared with controls, likely because of variation in the size of the crystal-like linear aggregations. Reduced ATPB staining at mitochondrial GATM crystal sites suggests extramitochondrial localization or mitochondrial dysfunction (scale bar: 20 μm). ATPB, ATP synthase subunit β; DAPI, 4′,6-diamidino-2-phenylindole; GATM, glycine amidinotransferase; PTECs, proximal tubular epithelial cells.

Figure 2.

Quantitative analysis of GATM and ATPB expression and GATM–ATPB colocalization. (A-F) Representative indirect immunostaining of PTECs in kidney biopsy samples with anti-GATM antibody (green) and anti-ATPB antibody (red) from control (A and B) and the daughter (D and E). Merged images from both samples are shown in C and F, respectively (scale bar: 10 μm). Note that weak GATM staining is observed surrounding areas of strong granular ATPB staining of mitochondria in control PTECs (C), whereas weak ATPB staining is observed around strong GATM staining of fibrous structures in daughter’s PTECs (F). (G-H) Quantitative analyses of signal intensity of both GATM and ATPB in PTECs of control, daughter, and father. Twenty PTECs of high-magnification confocal images obtained from each sample were randomly selected, and the signal intensities of 5 independent fields were quantified with Fiji/ImageJ. We applied an intensity threshold of 105-255 for GATM and 95-255 for ATPB to exclude background noise. (G) Patient GATM ratio (expressed as GATM/cytoplasmic area) were significantly elevated compared with those of controls (P < 0.01). (H) ATPB ratio (expressed as ATPB-positive area/cytoplasmic area) were significantly reduced in both the father and daughter versus control (P < 0.01) and further decreased in the father (P < 0.01). (I) GATM–ATPB colocalization analysis. Colocalized signals of GATM and ATPB of control, daughter, and father were plotted. Colocalization was significantly lower in father and daughter compared with control (P < 0.01) and was lowest in the father (P < 0.01). Bars represent mean ± standard error; each dot represents 1 PTEC. ∗P < 0.01 (one-way analysis of variance followed by Tukey’s post hoc test). (J) Scatter plots of GATM–ATPB colocalization and ATPB ratio from control (blue circles), daughter (green triangles), and father (red rectangles). Note that GATM–ATPB colocalization and ATPB ratio show a strong positive correlation, indicating that crystal-like linear aggregates of GATM mutant proteins could impair mitochondrial function (Pearson’s r = 0.812, P < 0.01). ATPB, ATP synthase subunit β; GATM, glycine amidinotransferase; PTEC, proximal tubular epithelial cell.

Next, we performed targeted panel sequencing of genes associated with inherited kidney diseases, including FS, confirmed by Sanger sequencing. Both patients carried the same mutation in the GATM gene (NM_001482.3: c.802C>G), resulting in the amino acid substitution Q268E (Fig 3A). The identified variant (c.802C>G, p.Q268E) was absent from general population databases (Japanese Multi Omics Reference Panel [jMorp] and the Genome Aggregation Database [gnomAD]) and was classified as a variant of uncertain significance according to the American College of Medical Genetics and Genomics guidelines (Table S3).11, 12, 13

Figure 3.

Structural implications of the glycine amidinotransferase (GATM) Q268E mutation identified in the father and daughter. (A) Schematic representation of the GATM amino acid sequence highlighting the mutated residue at position 268 in both affected patients. Genetic analysis showed that the father and daughter carry the same heterozygous GATM mutation (NM_001482.3: c.802C>G), resulting in a glutamine-to-glutamate substitution at residue 268 (Q268E). (B) Structural model (PDB: 1JDW) indicating Q268 within the protein interior. The Q268E substitution is predicted to allosterically alter the B4 surface via coupling with residues 290-312.

The GATM molecule has 5 β-sheets, designated B1-B5, in its 3-dimensional structure.^5,8 Genetic abnormalities in GATM (P320S, T336A, T336I, and P341L) are clustered in an adjacent region on the B4 surface of the protein structure (Fig 3B).⁸ Wild-type GATM forms homodimers through interactions at the B2 surface, including the proximal residue 212, which plays a role in binding between B2 surfaces.⁵ Mutations on the B4 surface may cause changes in the electrical properties and shape of the B4 surface, converting the native dimer (simple/physiological B2–B2) into an abnormal linear polymer (sequential/pathological B2–B2 and B4–B4 interactions).⁸ Although conventional methods failed to detect significant conformational changes in the Q268E mutant GATM, we employed HS-MD simulations, an advanced molecular dynamics approach using a supercomputer¹⁰ (Fig S1 and Item S1. Supplementary Methods). We performed comparative binding simulations using 2 GATM molecules for the wild-type, the Q268E mutant, and the previously reported residue mutations. The results suggest that the Q268E mutant preferred to interact with both the B2–B2 and B4–B4 surfaces as well as the previously reported mutations, indicating the possibility that our mutant may form crystal-like linear aggregations (Fig 4A and Fig S2). Simultaneously, our HS-MD simulations reaffirmed the previous hypotheses of pathological polymerization of mutants clustered on the B4 surface.

Figure 4.

Conformational differences between wild-type GATM and the Q268E mutant obtained from HS-MD simulations. (A) The free energy landscapes for the binding of 2 GATM molecules, described as a function of the distance between the Cβ atoms of T212 (d_T212-T212) and that between the Cβ atoms of P320 (d_P320-P320). Each landscape was calculated from the probability distribution using the equation G(d_T212-T212, d_P320-P320) = ‒RT ln {P(d_T212-T212, d_P320-P320)}, where R is the gas constant, T is the absolute temperature, and P is the probability density estimated from 20 independent 1-μs HS-MD simulations. The unit of free energy is kcal/mol. For each landscape, the free energy corresponding to the global minimum was set to 0. The populations of GATM homodimers formed through B2–B2 interactions (dotted boxes) are significantly increased by the Q268E mutation, as well as by the known pathological mutations (Fig S2). (B-D) Conformational dynamics of the GATM monomer, estimated from 10 independent 200-ns HS-MD simulations. (B) Root-mean-square fluctuation (RMSF) of the backbone Cα atoms. RMSF values are represented as the mean ± standard deviation (SD) of the 10 HS-MD simulations. Two regions, comprising residues 220-250 and 290-312, are highlighted in blue. (C) Dynamic cross-correlations between GATM residues. After the backbone Cα atoms were structurally aligned to remove rotational and translational motion, the dynamic cross-correlation coefficients of side-chain heavy atoms were calculated for each residue pair. The maximum value is shown by a gradual color change from blue (−1) to red (1). Three regions, comprising residues 266-268, 290-312, and 318-346, are highlighted in blue. The emergence or enhancement of dynamic correlations by the Q268E mutation is indicated by arrows. (D) Mean structures from the 10 HS-MD simulations. The protein is represented as a surface model, and residues D298, P299, and T339, the solvent-accessible surface areas of which are significantly increased by the Q268E mutation (Table S4), are colored in red, green, and orange, respectively. GATM, glycine amidinotransferase; HS-MD, hypersound-perturbed molecular dynamics.

We further investigated the effect of the Q268E mutation on the protein structure of the GATM monomer. Unlike the mutated residues reported previously, which are located on the B4 surface, Q268E is situated away from this surface and buried within the protein’s interior (Fig 3B).⁸ Our simulations showed that Q268E significantly enhances the conformational flexibility of a region-spanning residues 290-312 (Fig 4B), which is spatially positioned between the mutated residue and the B4 surface. This alteration changes the interaction pattern between this region and the B4 surface (Fig 4C), leading to significant increases in the solvent exposure of several amino acids (D298, P299, and T339) on the B4 surface (Fig 4D; Table S4). These results suggest that the Q268E mutant could induce the formation of crystal-like linear aggregations and the insoluble intracellular deposition of polymerized GATM by indirectly altering the electrostatic properties and shape of the B4 surface. Taken together, we conclude that the novel GATM mutantation Q268E may cause hereditary FS not only by promoting the formation of large, insoluble crystal-like linear aggregations inside and next to the mitochondria, but also by leading to mitochondrial dysfunction in the affected PTECs.

Discussion

Although advances in sequencing have identified many causative genes in hereditary diseases, little is known about how these DNA mutations affect the 3-dimensional structure of the resulting protein products.¹⁴ AlphaFold, a deep learning-based structure prediction tool, has recently gained significant attention for its high accuracy in predicting static protein structures.¹⁵ However, it remains limited in capturing the dynamic behavior and conformational flexibility of proteins. In contrast, molecular dynamics simulations have been widely used to explore such dynamic properties.16, 17, 18 In this study, our HS-MD simulations effectively demonstrated that the Q268E mutation allosterically changes the electrostatic properties and shape of the B4 surface.¹⁰

Kidney biopsy showed characteristic crystal-like linear aggregations in the PTECs. These intracellular structures, located inside and adjacent to the mitochondria, were confirmed to contain GATM protein by indirect immunofluorescent microscopy. Notably, GATM and ATPB were not fully colocalized in the patient’s kidney compared with controls (Figs 1D, 2D-F). Mutant GATM proteins were proposed to cause mitochondrial dysfunction in a previous report.⁸ In this study, electron microscopy observations and our quantitative analysis of the colocalization of GATM and ATPB clearly demonstrated that crystal-like linear aggregations of GATM proteins inside and near mitochondria could indeed impair mitochondrial function (Fig 2J). The polymerized mutant GATM might directly or indirectly injure the mitochondria via a decrease of ATPB expression¹⁹ or impair mitochondrial fission, potentially leaving senescent mitochondria with reduced ATP production that cannot be degraded by mitophagy.⁸ Although the variant was classified as a variant of uncertain significance according to the American College of Medical Genetics and Genomics guidelines, our HS-MD simulations and immunofluorescent analyses clearly demonstrated the structural and functional pathogenicity of the Q268E GATM mutant proteins.

In our cases, the daughter, who developed symptoms at a younger age, exhibited a more severe phenotype than her father, showing more rapid decline of eGFR over the course of the disease (Fig S3). Other factors beyond the GATM mutation may be involved. We assessed for potential extrarenal disease manifestations that have been proposed to be associated with GATM variants; however, no such manifestations were observed in our patients.²⁰ In any case, there is currently no effective treatment for hereditary FS caused by GATM mutations.

In conclusion, we identified hereditary FS caused by a novel GATM gene mutation (NM_001482.3: c.802C>G, p.Q268E). We examined the pathogenicity of the mutant with quantitative immunofluorescent microscopy, and HS-MD simulations effectively demonstrated the possibility for abnormal polymerization through sequential β-sheet interactions (B2–B2 and B4–B4) of the mutant GATM protein. This study provides valuable insights not only into the pathological mechanisms of abnormal protein structure but also for developing potential therapeutic strategies in the future.

Supplementary Material

Supplementary File (PDF)

Item S1; Figure S1-S4; Table S1-S4