Characterization of a novel biochemical abnormality in galactosemia: Deficiency of glycolipids containing galactose or N-acetylgalactosamine and accumulation of precursors in brain and lymphocytes

doi:10.1016/0885-4505(91)90054-O

Biochemical Medicine and Metabolic Biology

Volume 46, Issue 1, August 1991, Pages 93-104

Biochemical Medicine...

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Abstract

Classic galactosemia, an inborn error of human galactose metabolism, is characterized by a deficiency of the enzyme galactose-1-phosphate uridyltransferase (GALT). The current model for the pathophysiology of this disease ascribes most of its symptoms to the toxicity of intracellular galactose-1-phosphate (Gal-1-P), one of the substrates of GALT which accumulates in the untreated disease state. Recently, a reduction in the intracellular concentration of UDP-Gal (uridine diphosphogalactose), one of the products of GALT, has been described in treated galactosemic patients. We investigated whether galactosemic patients might also have reduced amounts of those macromolecules that depend on UDP-Gal for their biosynthesis. We report a reduction in glycolipids that contain either galactose or its derivative N-acetylgalactosamine and an accumulation of the precursors to these compounds in the brain of a neonate with galactosemia. We also found an imbalance in glycolipids in galactosemic lymphoblasts. This novel biochemical abnormality observed in galactosemic patients is not addressed by dietary galactose-restriction therapy and could explain some of the chronic neurologic and other complications of galactosemia.

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Cited by (68)

Gray and white matter are both affected in classical galactosemia: An explorative study on the association between neuroimaging and clinical outcome
2020, Molecular Genetics and Metabolism
Citation Excerpt :
As previous studies have suggested that myelin is affected in CG patients [2,5,14,15] we investigated R1 values indicative of myelin content. The formation of myelin depends on galactose containing glycolipids, such as galactocerebrosides [46,47] and the deficient GALT enzyme in CG patients causes a reduction in UDP-sugars which are essential for the formation of glycoproteins and glycolipids [8]. Therefore, the observed WM abnormalities on MRI in CG patients, such as the abnormal WM intensity signal, have been attributed to abnormal myelination.
Classical Galactosemia (CG) is an inherited disorder of galactose metabolism caused by a deficiency of the galactose-1-phosphate uridylyltransferase (GALT) enzyme resulting in neurocognitive complications. As in many Inborn Errors of Metabolism, the metabolic pathway of CG is well-defined, but the pathophysiology and high variability in clinical outcome are poorly understood. The aim of this study was to investigate structural changes of the brain of CG patients on MRI and their association with clinical outcome.
In this prospective cohort study an MRI protocol was developed to evaluate gray matter (GM) and white matter (WM) volume of the cerebrum and cerebellum, WM hyperintensity volume, WM microstructure and myelin content with the use of conventional MRI techniques, diffusion tensor imaging (DTI) and quantitative T1 mapping. The association between several neuroimaging parameters and both neurological and intellectual outcome was investigated.
Twenty-one patients with CG (median age 22 years, range 8–47) and 24 controls (median age 30, range 16–52) were included. Compared to controls, the WM of CG patients was lower in volume and the microstructure of WM was impaired both in the whole brain and corticospinal tract (CST) and the lower R1 values of WM, GM and the CST were indicative of less myelin. The volume of WM lesions were comparable between patients and controls. The 9/16 patients with a poor neurological outcome (defined as the presence of a tremor and/or dystonia), demonstrated a lower WM volume, an impaired WM microstructure and lower R1 values of the WM indicative of less myelin content compared to 7/16 patients without movement disorders. In 15/21 patients with a poor intellectual outcome (defined as an IQ < 85) both GM and WM were affected with a lower cerebral and cerebellar WM and GM volume compared to 6/21 patients with an IQ ≥ 85. Both the severity of the tremor (as indicated by the Tremor Rating Scale) and IQ (as continuous measure) were associated with several neuroimaging parameters such as GM volume, WM volume, CSF volume, WM microstructure parameters and R1 values of GM and WM.
In this explorative study performed in patients with Classical Galactosemia, not only WM but also GM pathology was found, with more severe brain abnormalities on MRI in patients with a poor neurological and intellectual outcome. The finding that structural changes of the brain were associated with the severity of long-term complications indicates that quantitative MRI techniques could be of use to explain neurological and cognitive dysfunction as part of the disease spectrum. Based on the clinical outcome of patients, the absence of widespread WM lesions and the finding that both GM and WM are affected, CG could be primarily a GM disease with secondary damage to the WM as a result of neuronal degeneration. To investigate this further the course of GM and WM should be evaluated in longitudinal research, which could also clarify if CG is a neurodegenerative disease.
Hereditary galactosemia
2018, Metabolism: Clinical and Experimental
Citation Excerpt :
UDP-hexose deficiency and/or imbalance have been pointed out as an important pathogenic mechanism [98–102]. Abnormal pattern of glycoproteins [53] and deficiency of glycolipids containing galactose or N-acetylgalactosamine [103] have been observed in postmortem galactosemic brain. Studies with patients' fibroblasts have described defective galactosylation [104,105].
Hereditary galactosemia is an inborn error of carbohydrate metabolism. Galactose is metabolized by Leloir pathway enzymes; galactokinase (GALK), galactose-1-phosphate uridylyltransferase (GALT) and UDP-galactose 4-epimerase (GALE). The defects in these enzymes cause galactosemia in an autosomal recessive manner. The severe GALT deficiency, or classic galactosemia, is life-threatening in the newborn period. The treatment for classic galactosemia is dietary restriction of lactose. Although implementation of lactose restricted diet is efficient in resolving the acute complications, it is not sufficient to prevent long-term complications affecting the brain and female gonads, the two main target organs of damage. Implementation of molecular genetics diagnostic tools and GALT enzyme assays are instrumental in distinguishing classic galactosemia from clinical and biochemical variant forms of GALT deficiency. Better understanding of mechanisms responsible for the phenotypic variation even within the same genotype is essential to provide appropriate counseling for families. Utilization of a lactose restricted diet is also recommended for GALK deficiency and some rare forms of GALE deficiency. Novel modes of therapies are being explored; they may be beneficial if access issues to the affected tissues are circumvented and optimum use of therapeutic window is achieved.
Reversal of aberrant PI3K/Akt signaling by Salubrinal in a GalT-deficient mouse model
2017, Biochimica et Biophysica Acta - Molecular Basis of Disease
Citation Excerpt :
The molecular mechanisms of these acute and long-term complications are still poorly understood despite several hypotheses that have been presented in the literature [7–11]. Among the biochemical abnormalities detected in patients, glycosylation defects of glycoproteins and glycolipids have received significant attention [8,12–15]. It has been suggested that the observed glycosylation defects are caused by the accumulation of toxic galactose metabolites of the blocked GALT reaction, as well as deficiency of UDP-galactose (Fig. 1) [15,16].
Classic Galactosemia is an autosomal recessive disorder caused by deleterious mutations in the GALT gene, which encodes galactose-1 phosphate uridylyltransferase enzyme (GALT: EC 2.7.7.12). Recent studies of primary skin fibroblasts isolated from the GalT-deficient mice demonstrated a slower growth rate, a higher level of endoplasmic reticulum (ER) stress, and down-regulation of the Phosphoinositide 3 kinase/Protein kinase B (PI3K/Akt) signaling pathway. In this study, we compared the expression levels of the PI3K/Akt signaling pathway in normal and GalT-deficient mouse tissues. In mutant mouse ovaries, phospho-Akt [pAkt (Ser473)] and pGsk3β were reduced by 62.5% and 93.5%, respectively (p < 0.05 versus normal controls). In mutant cerebella, pAkt (Ser473) and pGsk3β were reduced by 62%, 50%, respectively (p < 0.05). To assess the role of ER stress in the down-regulation of PI3K/Akt signaling, we examined if administration of Salubrinal, a chemical compound that alleviates ER stress, to GalT-deficient fibroblasts and animals could normalize the pathway. Our results demonstrated that Salubrinal effectively reversed the down-regulated PI3K/Akt signaling pathway in the mutant cells and animals to levels close to those of their normal counterparts. Moreover, we revealed that Salubrinal can significantly slow down the loss of Purkinje cells in the cerebella, as well as the premature loss of primordial ovarian follicles in young mutant mice. These results open the door for a new therapeutic approach for the patients with Classic Galactosemia.
The molecular basis of galactosemia — Past, present and future
2016, Gene
Citation Excerpt :
In addition to decreased galactosylation, increased inappropriate incorporation of other monosaccharide moieties such as fucose has been observed (Sturiale et al., 2005). Similarly, glycolipids from galactosemic patients were shown to have reduced levels of galactose and N-acetylgalactosamine compared to healthy patients; this effect was not reversed by a low galactose diet (Petry et al., 1991). In galactosemic patients, N-linked protein glycosylation is associated with increased amounts of mannose and increased numbers of truncated oligosaccharide chains (Liu et al., 2012; Staubach et al., 2012).
Galactosemia, an inborn error of galactose metabolism, was first described in the 1900s by von Ruess. The subsequent 100 years has seen considerable progress in understanding the underlying genetics and biochemistry of this condition. Initial studies concentrated on increasing the understanding of the clinical manifestations of the disease. However, Leloir's discovery of the pathway of galactose catabolism in the 1940s and 1950s enabled other scientists, notably Kalckar, to link the disease to a specific enzymatic step in the pathway. Kalckar's work established that defects in galactose 1-phosphate uridylyltransferase (GALT) were responsible for the majority of cases of galactosemia. However, over the next three decades it became clear that there were two other forms of galactosemia: type II resulting from deficiencies in galactokinase (GALK1) and type III where the affected enzyme is UDP-galactose 4′-epimerase (GALE). From the 1970s, molecular biology approaches were applied to galactosemia. The chromosomal locations and DNA sequences of the three genes were determined. These studies enabled modern biochemical studies. Structures of the proteins have been determined and biochemical studies have shown that enzymatic impairment often results from misfolding and consequent protein instability. Cellular and model organism studies have demonstrated that reduced GALT or GALE activity results in increased oxidative stress. Thus, after a century of progress, it is possible to conceive of improved therapies including drugs to manipulate the pathway to reduce potentially toxic intermediates, antioxidants to reduce the oxidative stress of cells or use of “pharmacological chaperones” to stabilise the affected proteins.
Systemic gene dysregulation in classical Galactosaemia: Is there a central mechanism?
2014, Molecular Genetics and Metabolism
Citation Excerpt :
If galactose intoxication disrupts the localisation of ALG9, even to a minor degree, this in combination with decreased UDP-galactose levels, and the dysregulation of other glycosyltransferases, could have a significant role to play in systemic glycosylation defects. Severe galactose intoxication during the neonatal stage results in gross assembly defects in patients [8,12–15,59], which supports our findings here of a susceptibility of the assembly stage of glycosylation during exposure to galactose. The abnormal expression of ALG9 gene, and potential defects in protein level and localisation in cells, may be a marker for individuals at risk of aberrant glycosylation during increased galactose intake.
Classical Galactosaemia is a rare disorder of carbohydrate metabolism caused by a deficiency of galactose-1-phosphate uridyltransferase (GALT). The disease is life-threatening in the neonate, and the only treatment option is life-long dietary restriction of galactose. However, long-term complications persist in treated patients including cognitive impairments, speech and language abnormalities and premature ovarian insufficiency in females. Microarray analysis of T-lymphocytes from treated adult patients identified systemic dysregulation of numerous gene pathways, including the glycosylation, inflammatory and inositol pathways. Analysis of gene expression in patient-derived dermal fibroblasts of patients exposed to toxic levels of galactose, with immunostaining, has further identified the susceptibility of the glycosylation gene alpha-1,2-mannosyltransferase (ALG9) and the inflammatory gene annexin A1 (ANXA1) to increased galactose concentrations. These data suggest that Galactosaemia is a multi-system disorder affecting numerous signalling pathways.
N- and O-linked glycosylation of total plasma glycoproteins in galactosemia
2012, Molecular Genetics and Metabolism
Citation Excerpt :
In the early 1970s, Haberland and colleagues [3,4] demonstrated an abnormal pattern of glycoproteins in the postmortem brain of a galactosemic patient. Twenty years later, Petry and colleagues [5] described a deficiency of glycolipids containing galactose or N-acetylgalactosamine, and an accumulation of the precursors of these moieties in the postmortem brain of a neonate with galactosemia. Also in the early 1990s, Jaeken and colleagues described abnormalities in the glycosylated serum lysosomal enzymes from patients with galactosemia [6], and Dobbie and colleagues [7] and Ornstein and colleagues [8] reported defective galactosylation of complex carbohydrates and glycoproteins in fibroblasts derived from patients with galactosemia.
Classic galactosemia is a potentially lethal metabolic disorder that results from profound impairment of the enzyme galactose-1-phosphate uridylyltransferase (GALT); despite decades of research, the underlying mechanism of pathophysiology remains unclear. Previous studies of plasma and tissue samples from patients with classic galactosemia have revealed defects of protein and lipid glycosylation, however, the underlying bases for these defects and their clinical significance, if any, has remained unclear. As a step toward addressing these questions we characterized both the N- and O-linked glycomes of plasma proteins from neonates, infants, children, and adults with galactosemia using mass spectrometry and asked (1) whether similar or disparate defects exist for N-linked and O-linked modifications, (2) what factors correlate with the severity of these defects in different patients, and perhaps most important, (3) whether there is any apparent relationship between chronic glycosylation defects and long-term outcome in patients. We found that some but not all of the galactosemic neonates tested exhibited abnormal N- and O-linked glycosylation of plasma proteins. The types of abnormalities seen were similar between N- and O-linked moieties, but the extent of the defects varied between patients. Age, gender, GALT genotype, and predicted residual GALT activity all failed to explain the extent of the glycosylation defect in the samples studied. Dietary galactose restriction markedly normalized both the N- and O-linked glycosylation patterns for all infants tested; however, any remaining glycosylation defects evident in the plasma of older children or adults on galactose-restricted diets showed no correlation with clinical outcome. These data cannot rule out the possibility that subtle or localized glycosylation defects, not detectable by our methods or not reflected in plasma, may contribute to acute or long-term outcome severity.

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¹: Present address: Université de Bordeaux II, Department d' Immunologie et de Biologie Parasitaire, F-33076 Bordeaux, France.

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Biochemical Medicine and Metabolic Biology

Abstract

References (20)

Newborn screening for galactosemia and other galactose metabolic defects

J Pediat

Sulfated lipids represent common antigens on both the surface of Trypanosoma cruzi and mammalian tissue

Mol Biochem Parasitol

Separation of gluco- and galactocerebrosides by means of thin-layer chromatography

J Lipid Res

Ganglioside-mediated modulation of cell growth

J Biol Chem

Determination of gangliosides, glycoproteins and glycosaminoglycans in brain tissue

Clin Chim Acta

Disorders of galactose metabolism

Long-term prognosis in galactosaemia: Results from a survey of 350 cases

J Inher Metabol Dis

Deficit of uridine diphosphate galactose in galactosaemia

J Inher Metab Dis

Bifunctional role of glycosphingolipids

J Biol Chem

Functions of shingolipids and shingolipid breakdown products in cellular regulation

Science

Cited by (68)

Gray and white matter are both affected in classical galactosemia: An explorative study on the association between neuroimaging and clinical outcome

Hereditary galactosemia

Reversal of aberrant PI3K/Akt signaling by Salubrinal in a GalT-deficient mouse model

The molecular basis of galactosemia — Past, present and future

Systemic gene dysregulation in classical Galactosaemia: Is there a central mechanism?

N- and O-linked glycosylation of total plasma glycoproteins in galactosemia