PGM1 Research Articles

Multiplexed detection of DOCK8, PGM3 and STAT3 proteins for the diagnosis of Hyper-Immunoglobulin E syndrome using gold nanoparticles-based immunosensor array platform

Multiplexed biosensors hold great promise for early diagnosis of diseases where the detection of multiple biomarkers is required. Hyper Immunoglobulin E syndromes (HIES) are rare primary immunodeficiency disorders associated with mutations either in the signal transducer and activator of transcription 3 (STAT3), dedicator of cytokinesis 8 DOCK8) or phosphoglucomutase 3 (PGM3) genes. Yet, the diagnosis of HIES is challenged by the complexity of the existing laboratory assays. Here, we report for the first time the development of a multiplexed electrochemical immunosensor for the simultaneous detection of DOCK8, STAT3 and PGM3 proteins. The immunosensor was constructed on carbon array electrodes that were first modified by electrodeposition of gold nanoparticles (AuNPs). The array electrodes were then used to immobilize specific antibodies for the three proteins after the functionalization of the electrodes with cysteamine/glutaraldehyde linkers. The simultaneous detection of the DOCK8, PGM3 and STAT3 proteins was successfully realized by the immunosensor with respective limits of detections of 3.1, 2.2 and 3.5 pg/ml. The immunosensor has shown good sensitivity as well as selectivity against other proteins such as cystic fibrosis transmembrane conductance regulator (CFTR) and Duchenne Muscular Dystrophy (DMD). Moreover, the immunosensor was successfully applied in human serum samples showing capability to distinguish the HIES from the control samples.

Clinical and molecular genetic characterization of two patients with mutations in the phosphoglucomutase 1 (PGM1) gene

Background The phosphoglucomutase 1 (PGM1) enzyme plays a central role in glucose homeostasis by catalyzing the inter-conversion of glucose 1-phosphate and glucose 6-phosphate. Recently, PGM1 deficiency has been recognized as a cause of the congenital disorders of glycosylation (CDGs). Methods Two Chinese Han pediatric patients with recurrent hypoglycemia, hepatopathy and growth retardation are described in this study. Targeted gene sequencing (TGS) was performed to screen for causal genetic variants in the genome of the patients and their parents to determine the genetic basis of the phenotype. Results DNA sequencing identified three variations of the PGM1 gene (NM_002633.2). Patient 1 had a novel homozygous mutation (c.119delT, p.Ile40Thrfs*28). In patient 2, we found a compound heterozygous mutation of c.1172G>T(p.Gly391Val) (novel) and c.1507C>T(p.Arg503*) (known pathogenic). Conclusions This report deepens our understanding of the clinical features of PGM1 mutation. The early molecular genetic analysis and multisystem assessment were here found to be essential to the diagnosis of PGM1-CDG and the provision of timely and proper treatment.

Intact transferrin and total plasma glycoprofiling for diagnosis and therapy monitoring in phosphoglucomutase-I deficiency

Phosphoglucomutase 1 (PGM1) deficiency results in a mixed phenotype of a Glycogen Storage Disorder and a Congenital Disorder of Glycosylation (CDG). Screening for abnormal glycosylation has identified more than 40 patients, manifesting with a broad clinical and biochemical spectrum which complicates diagnosis. Together with the availability of D-galactose as dietary therapy, there is an urgent need for specific glycomarkers for early diagnosis and treatment monitoring. We performed glycomics profiling by high-resolution QTOF mass spectrometry in a series of 19 PGM1-CDG patients, covering a broad range of biochemical and clinical severity. Bioinformatics and statistical analysis were used to select glycomarkers for diagnostics and define glycan-indexes for treatment monitoring. Using 3 transferrin glycobiomarkers, all PGM1-CDG patients were diagnosed with 100% specificity and sensitivity. Total plasma glycoprofiling showed an increase in high mannose glycans and fucosylation, while global galactosylation and sialylation were severely decreased. For treatment monitoring, we defined 3 glycan-indexes, reflecting normal glycosylation, a lack of complete glycans (LOCGI) and of galactose residues (LOGI). These indexes showed improved glycosylation upon D-galactose treatment with a fast and near-normalization of the galactose index (LOGI) in 6 out of 8 patients and a slower normalization of the LOCGI in all patients. Total plasma glycoprofiling showed improvement of the global high mannose glycans, fucosylation, sialylation, and galactosylation status on D-galactose treatment. Our study indicates specific glycomarkers for diagnosis of mildly and severely affected PGM1-CDG patients, and to monitor the glycan-specific effects of D-galactose therapy.

Disruption of the Responsible Gene in a Phosphoglucomutase 1 Deficiency Patient by Homozygous Chromosomal Inversion.

Phosphoglucomutase 1 (PGM1) deficiency is a recently defined disease characterized by glycogenosis and a congenital glycosylation disorder caused by recessive mutations in the PGM1 gene. We report a case of a 12-year-old boy with first-cousin parents who was diagnosed with a PGM1 deficiency due to significantly decreased PGM1 activity in his muscle. However, Sanger sequencing revealed no pathogenic mutation in the PGM1 gene in this patient. As this case presented with a cleft palate in addition to hypoglycemia and elevated transaminases and creatine kinase, karyotyping was performed and identified homozygous inv(1)(p31.1p32.3). Based on the chromosomal location of the PGM1 gene at 1p31, we analyzed the breakpoint of the inversion. Fluorescence in situ hybridization (FISH) combined with long PCR analysis revealed that the inversion disrupts the PGM1 gene within intron 1. Since the initiation codon in the PGM1 gene is located within exon 1, we speculated that this inversion inactivates the PGM1 gene and was therefore responsible for the patient's phenotype. When standard molecular testing fails to reveal a mutation despite a positive clinical and biochemical diagnosis, the presence of a gross structural variant that requires karyotypic examination must be considered.

Nutritional Therapies in Congenital Disorders of Glycosylation (CDG).

Congenital disorders of glycosylation (CDG) are a group of more than 130 inborn errors of metabolism affecting N-linked, O-linked protein and lipid-linked glycosylation. The phenotype in CDG patients includes frequent liver involvement, especially the disorders belonging to the N-linked protein glycosylation group. There are only a few treatable CDG. Mannose-Phosphate Isomerase (MPI)-CDG was the first treatable CDG by high dose mannose supplements. Recently, with the successful use of d-galactose in Phosphoglucomutase 1 (PGM1)-CDG, other CDG types have been trialed on galactose and with an increasing number of potential nutritional therapies. Current mini review focuses on therapies in glycosylation disorders affecting liver function and dietary intervention in general in N-linked glycosylation disorders. We also emphasize now the importance of early screening for CDG in patients with mild hepatopathy but also in cholestasis.

Sequence-structure relationships, expression profiles, and disease-associated mutations in the paralogs of phosphoglucomutase 1.

The key metabolic enzyme phosphoglucomutase 1 (PGM1) controls glucose homeostasis in most human cells. Four proteins related to PGM1, known as PGM2, PGM2L1, PGM3 and PGM5, and referred to herein as paralogs, are encoded in the human genome. Although all members of the same enzyme superfamily, these proteins have distinct substrate preferences and different functional roles. The recent association of PGM1 and PGM3 with inherited enzyme deficiencies prompts us to revisit sequence-structure and other relationships among the PGM1 paralogs, which are understudied despite their importance in human biology. Using currently available sequence, structure, and expression data, we investigated evolutionary relationships, tissue-specific expression profiles, and the amino acid preferences of key active site motifs. Phylogenetic analyses indicate both ancient and more recent divergence between the different enzyme sub-groups comprising the human paralogs. Tissue-specific protein and RNA expression profiles show widely varying patterns for each paralog, providing insight into function and disease pathology. Multiple sequence alignments confirm high conservation of key active site regions, but also reveal differences related to substrate specificity. In addition, we find that sequence variants of PGM2, PGM2L1, and PGM5 verified in the human population affect residues associated with disease-related mutants in PGM1 or PGM3. This suggests that inherited diseases related to dysfunction of these paralogs will likely occur in humans.

The Mechanism of Starch Over-Accumulation in Chlamydomonas reinhardtii High-Starch Mutants Identified by Comparative Transcriptome Analysis.

The focus of this study was the mechanism of starch accumulation in Chlamydomonas reinhardtii high-starch mutants. Three C. reinhardtii mutants showing high-starch content were generated using gamma irradiation. When grown under nitrogen-deficient conditions, these mutants had more than twice as much starch than a wild-type control. The mechanism of starch over-accumulation in these mutants was studied with comparative transcriptome analysis. In all mutants, induction of phosphoglucomutase 1 (PGM1) expression was detected; PGM1 catalyzes the inter-conversion of glucose 1-phosphate and glucose 6-phosphate in both starch biosynthetic and glycolytic pathway. Interestingly, transcript levels of phosphoglucose isomerase 1 (PGI1), fructose 1,6-bisphosphate aldolase 1 and 2 (FBA1 and FBA2) were down-regulated in all mutants; PGI1, FBA1, and FBA2 act on downstream of glucose 6-phosphate conversion in glycolytic pathway. Therefore, down-regulations of PGI1, FBA1, and FBA2 may lead to accumulation of upstream metabolites, notably glucose 6-phosphate, resulting in induction of PGM1 expression through feed-forward regulation and that PGM1 overexpression caused starch over-accumulation in mutants. These results suggest that PGI1, FBA1, FBA2, and PGM1 correlate with each other in terms of coordinated transcriptional regulation and play central roles for starch over-accumulation in C. reinhardtii.

Identification of a Novel Pathomechanism Underlying a Congenital Disorder of Glycosylation: Crosstalk between Hexosamine Biosynthetic Pathway, PI3K/AKT Signaling, and Protein Glycosylation

Phosphoglucomutase 1 (PGM1) deficiency is a recently identified subtype of congenital disorder of glycosylation that can be treated by dietary modification. PGM1 is required for the interconversion of glucose 1‐phosphate and glucose 6‐phosphate, and is a key enzyme in glycogen metabolism. Although the exact mechanism remains unclear, PGM1 deficiency has been associated with abnormal intracellular glucose and galactose metabolites, as well as reduced N‐linked glycosylation in the endoplasmic reticulum and Golgi. These metabolic disturbances are thought to be responsible for both missing and truncated glycans that have been detected in patient serum transferrin. A recent landmark study demonstrated that oral D‐galactose supplementation improved cellular glycosylation in six patients and resolved hypogonadotropic hypogonadism in two patients. While the clinical benefits of galactose supplementation in PGM1 deficiency are clear, its biological effect on the biochemical and cellular level has not been characterized in detail. It was assumed that dietary galactose supplementation improves galactosylation by increasing intracellular UDP‐galactose relative to UDP‐glucose; however, this has not been confirmed experimentally, nor has an alternative mechanism of action been explored. Here, we report no difference in the ratio of intracellular UDP‐glucose to UDP‐galactose between patient and control cells, despite significant improvement in N‐linked and O‐linked glycosylation in patient cells following galactose supplementation. We found no evidence to support galactose acting as a pharmacological chaperone for PGM1 missense variants. Surprisingly, we discovered disruptions in the hexosamine biosynthetic pathway (HBP) at the metabolic and transcriptional level, as well as downregulation of PI3K/AKT signaling in PGM1 deficient patient cells. Interestingly, galactose supplementation appears to rescue HBP and PI3K/AKT signaling. Collectively, our preliminary studies suggest a hitherto unexplored crosstalk between HBP, PI3K/AKT signaling, and protein glycosylation.Support or Funding InformationThis study is supported in part by the Hayward Foundation and by 1 U54 GM104940 from the National Institute of General Medical Sciences of the National Institutes of Health, which funds the Louisiana Clinical and Translational Science Center.

Asp263 missense variants perturb the active site of human phosphoglucomutase 1.

Structural data are available in the PDB under the accession numbers 5JN5 and 5TR2.

Partial correction of neutrophil dysfunction by oral galactose therapy in glycogen storage disease type Ib

Glycogen storage disease type Ib (GSD-Ib) is characterized by impaired glucose homeostasis, neutropenia and neutrophil dysfunction.Mass spectrometric glycomic profiling of GSD-Ib neutrophils showed severely truncated N-glycans, lacking galactose. Experiments indicated the hypoglycosylation of the electron transporting subunit of NADPH oxidase, which is crucial for the defense against bacterial infections. In phosphoglucomutase 1 (PGM1) deficiency, an inherited disorder with an enzymatic defect just one metabolic step ahead, hypogalactosylation can be successfully treated by dietary galactose. We hypothesized the same pathomechanism in GSD-Ib and started a therapeutic trial with oral galactose and uridine. The aim was to improve neutrophil dysfunction through the correction of hypoglycosylation in neutrophils. The GSD-Ib patient was treated for 29weeks. Monitoring included glycomics analysis of the patient's neutrophils and neutrophil function tests including respiratory burst activity, phagocytosis and migration. Although no substantial restoration of neutrophil glycosylation was found, there was partial improvement of respiratory burst activity.

Detection of phosphoglucomutase-3 deficiency by lectin-based flow cytometry

Detection of phosphoglucomutase-3 deficiency by lectin-based flow cytometry

Differential Neuroproteomic and Systems Biology Analysis of Spinal Cord Injury

Acute spinal cord injury (SCI) is a devastating condition with many consequences and no known effective treatment. Although it is quite easy to diagnose traumatic SCI, the assessment of injury severity and projection of disease progression or recovery are often challenging, as no consensus biomarkers have been clearly identified. Here rats were subjected to experimental moderate or severe thoracic SCI. At 24h and 7d postinjury, spinal cord segment caudal to injury center versus sham samples was harvested and subjected to differential proteomic analysis. Cationic/anionic-exchange chromatography, followed by 1D polyacrylamide gel electrophoresis, was used to reduce protein complexity. A reverse phase liquid chromatography-tandem mass spectrometry proteomic platform was then utilized to identify proteome changes associated with SCI. Twenty-two and 22 proteins were up-regulated at 24 h and 7 day after SCI, respectively; whereas 19 and 16 proteins are down-regulated at 24 h and 7 day after SCI, respectively, when compared with sham control. A subset of 12 proteins were identified as candidate SCI biomarkers - TF (Transferrin), FASN (Fatty acid synthase), NME1 (Nucleoside diphosphate kinase 1), STMN1 (Stathmin 1), EEF2 (Eukaryotic translation elongation factor 2), CTSD (Cathepsin D), ANXA1 (Annexin A1), ANXA2 (Annexin A2), PGM1 (Phosphoglucomutase 1), PEA15 (Phosphoprotein enriched in astrocytes 15), GOT2 (Glutamic-oxaloacetic transaminase 2), and TPI-1 (Triosephosphate isomerase 1), data are available via ProteomeXchange with identifier PXD003473. In addition, Transferrin, Cathepsin D, and TPI-1 and PEA15 were further verified in rat spinal cord tissue and/or CSF samples after SCI and in human CSF samples from moderate/severe SCI patients. Lastly, a systems biology approach was utilized to determine the critical biochemical pathways and interactome in the pathogenesis of SCI. Thus, SCI candidate biomarkers identified can be used to correlate with disease progression or to identify potential SCI therapeutic targets.

PGM1 deficiency diagnosed during an endocrine work-up of low IGF-1 mediated growth failure

Objective and importance: Phosphoglucomutase 1 (PGM1) deficiency, first described as a glycogenosis (type XIV) is also a congenital disorder of glycosylation (CDG). We want to illustrate the wide clinical spectrum of PGM1 deficiency and in particular the associated disturbance in glucose metabolism and the endocrine dysfunction. Treatment with d-galactose is experimental. Case presentation: PGM1 deficiency was diagnosed in an 8-year-old boy, who was referred because of an unexplained complex syndrome, including recurrent hypoglycaemia and low IGF-1 mediated growth failure. Conclusion: The timely diagnosis of this disorder is particularly important, because d-galactose treatment can improve the latter symptoms.

Induced Structural Disorder as a Molecular Mechanism for Enzyme Dysfunction in Phosphoglucomutase 1 Deficiency

Human phosphoglucomutase 1 (PGM1) plays a central role in cellular glucose homeostasis, mediating the switch between glycolysis and gluconeogenesis through the conversion of glucose 1-phosphate and glucose 6-phosphate. Recent clinical studies have identified mutations in this enzyme as the cause of PGM1 deficiency, an inborn error of metabolism classified as both a glycogen storage disease and a congenital disorder of glycosylation. Reported here are the first crystal structures of two disease-related missense variants of PGM1, along with the structure of the wild-type enzyme. Two independent glycine-to-arginine substitutions (G121R and G291R), both affecting key active site loops of PGM1, are found to induce regions of structural disorder, as evidenced by a nearly complete loss of electron density for as many as 23 aa. The disordered regions are not contiguous in sequence to the site of mutation, and even cross domain boundaries. Other structural rearrangements include changes in the conformations of loops and side chains, some of which occur nearly 20Å away from the site of mutation. The induced structural disorder is correlated with increased sensitivity to proteolysis and lower-resolution diffraction, particularly for the G291R variant. Examination of the multi-domain effects of these G➔R mutations establishes a correlation between interdomain interfaces of the enzyme and missense variants of PGM1 associated with disease. These crystal structures provide the first insights into the structural basis of enzyme dysfunction in PGM1 deficiency and highlight a growing role for biophysical characterization of proteins in the field of precision medicine.

Phosphorylation on TRPV4 Serine Residue 824 Enhances Its Association with PGM1

The TRPV4 cation channel is expressed in a broad range of tissues and participates in the generation of a Ca2+ signal and/or depolarization of membrane potential. Here, human phosphoglucomutase- 1 (PGM1), an enzyme that converts glucose-6 phosphate to glucose-1 phosphate in the glycolysis pathway, as the first auxiliary protein of TRPV4 Ca2+ channels, is identified with yeast two hybrid system, coimmunoprecipitation, confocal microscopy, and GST pull-down assays. TRPV4 forms a complex with PGM1 through its C-terminal cytoplasmic domain. Because it is demonstrated that TRPV4 serine residue 824 (S824) is phosphorylated by serum/glucocorticoid regulated kinase 1, we elucidate the effect of TRPV4 S824 phosphorylation on TRPV association with PGM1. Even an inactivated mutant version of TRPV4, S824A, exhibited a decreased ability to bind PGM1, an activated phosphomimetic mutant version of TRPV4, S824D, exhibited enhanced binding to PGM1. Thus, formation of the TRPV4/PGM1 complex and localization of this complex to the plasma membrane appear to be regulated by the phosphorylation status of residue S824 in TRPV4. The newly identified interactor of TRPV4 may help the molecular pathways modulating transport activity or glucose metabolism, respectively.

Susceptibility to infections, without concomitant hyper-IgE, reported in 1976, is caused by hypomorphic mutation in the phosphoglucomutase 3 (PGM3) gene

Phosphoglucomutase 3 (PGM3) is an enzyme converting N-acetyl-glucosamine-6-phosphate to N-acetyl-glucosamine-1-phosphate, a precursor important for glycosylation. Mutations in the PGM3 gene have recently been identified as the cause of novel primary immunodeficiency with a hyper-IgE like syndrome. Here we report the occurrence of a homozygous mutation in the PGM3 gene in a family with immunodeficient children, described already in 1976. DNA from two of the immunodeficient siblings was sequenced and shown to encode the same homozygous missense mutation, causing a destabilized protein with reduced enzymatic capacity. Affected individuals were highly prone to infections, but lack the developmental defects in the nervous and skeletal systems, reported in other families. Moreover, normal IgE levels were found. Thus, belonging to the expanding group of congenital glycosylation defects, PGM3 deficiency is characterized by immunodeficiency, with or without increased IgE levels, and with variable forms of developmental defects affecting other organ systems.

The mechanism of improved pullulan production by nitrogen limitation in batch culture of Aureobasidium pullulans

Batch culture of Aureobasidium pullulans CCTCC M 2012259 for pullulan production at different concentrations of ammonium sulfate and yeast extract was investigated. Increased pullulan production was obtained under nitrogen-limiting conditions, as compared to that without nitrogen limitation. The mechanism of nitrogen limitation favoring to pullulan overproduction was revealed by determining the activity as well as gene expression of key enzymes, and energy supply for pullulan biosynthesis. Results indicated that nitrogen limitation increased the activities of α-phosphoglucose mutase and glucosyltransferase, up-regulated the transcriptional levels of pgm1 and fks genes, and supplied more ATP intracellularly, which were propitious to further pullulan biosynthesis. The economic analysis of batch pullulan production indicated that nitrogen limitation could reduce more than one third of the cost of raw materials when glucose was supplemented to a total concentration of 70g/L. This study also helps to understand the mechanism of other polysaccharide overproduction by nitrogen limitation.

Molecular characterization of porcine PGM1 gene associated with meat quality traits

Hwang, J. H., Kwon, S. G., Park, D. H., Kim, T. W., Kang, D. G., Ha, J., Kim, S. W. and Kim, C. W. 2015. Molecular characterization of porcine PGM1 gene associated with meat quality traits. Can. J. Anim. Sci. 95: 31-36. The PGM1 gene from four porcine breeds (Berkshire, Duroc, Landrace, and Yorkshire) is highly expressed in liver tissue at the transcriptional level. Single nucleotide polymorphisms (SNPs) of PGM1 were examined to analyze association with increased expression of PGM1 gene in the Berkshire liver. A Leu525 synonymous SNP of Chr6:137174682A>G (c.1575A>G) was identified and showed significant (P<0.05) differences to backfat thickness, drip loss, protein content, fat content, Warner-Bratzler shear force, and post-mortem pH24h. Therefore, it is concluded that PGM1 synonymous SNP is an important factor regulating meat quality.

Molecular characterization of porcine PGM1 gene associated with meat quality traits

Hwang, J. H., Kwon, S. G., Park, D. H., Kim, T. W., Kang, D. G., Ha, J., Kim, S. W. and Kim, C. W. 2015. Molecular characterization of porcine PGM1 gene associated with meat quality traits. Can. J. Anim. Sci. 95: 31–36. The PGM1 gene from four porcine breeds (Berkshire, Duroc, Landrace, and Yorkshire) is highly expressed in liver tissue at the transcriptional level. Single nucleotide polymorphisms (SNPs) of PGM1 were examined to analyze association with increased expression of PGM1 gene in the Berkshire liver. A Leu525 synonymous SNP of Chr6:137174682A&gt;G (c.1575A&gt;G) was identified and showed significant (P&lt;0.05) differences to backfat thickness, drip loss, protein content, fat content, Warner–Bratzler shear force, and post-mortem pH24h. Therefore, it is concluded that PGM1 synonymous SNP is an important factor regulating meat quality.

Molecular characterization, expression patterns, and promoter activity analysis of PGM1 in pigs

The phosphoglucomutase 1 (PGM1) gene was differentially expressed in tissues of Chinese Meishan and Large White pigs. In this study, the promoter region, expression profile, and genetic mutations of the gene were determined. Expression of a 5'-deletion in both C2C12 and PK-15 cells showed that a negative regulatory element was at -1871 to +185 bp and a positive regulatory element was at -1158 to +185 bp. Among the different types of muscle fibers, PGM1 had the highest expression in both longissimus dorsi and biceps femoris. The expression was concentrated in the muscle fibers at different growth stages of Meishan and Large White pigs. The synonymous mutation C462T in the coding sequence was confirmed by polymerase chain reaction-restriction fragment length polymorphism, and the frequency of the C allele was dominant in Chinese indigenous breeds. Association analysis with lean meat showed that the C462T site was different.