Artificial Sweeteners — Enhancing Glycosylation to Treat Muscular Dystrophies
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《新英格兰医药杂志》
A large number of genetic mutations that cause muscular dystrophies have been identified. Most forms of muscular dystrophy are caused by mutations that result in protein deficiencies. This knowledge has led to the hope that a cure will be just around the corner in the form of gene therapy. Unfortunately, gene therapy has proved to be a much larger challenge than generally appreciated even a decade ago. There has thus been an increased focus on alternative approaches to the treatment of muscular dystrophies in recent years. Specifically, several groups have evaluated ways to bypass, rather than correct, the genetic defects and have reported therapeutic benefits in animal models.1
A recent report by Barresi et al. is one example.2 These investigators showed that overexpression of a glycosyltransferase, a type of enzyme that adds sugar residues to cellular proteins, resulted in the functional rescue of dystrophic muscle cells. This study is based on the recent finding that a subgroup of the muscular dystrophies, the congenital muscular dystrophies, are caused by mutations in genes involved in protein glycosylation.3 Defects in different genes in the glycosylation pathway are associated with distinct clinical phenotypes. One glycosyltransferase, LARGE, is deficient in a dystrophic mouse strain and a form of human congenital muscular dystrophy. Barresi et al. found, not surprisingly, that overexpression of LARGE was an effective treatment in this dystrophic mouse.2 More interesting, however, was their finding that overexpression of LARGE ameliorated, at least partially, the glycosylation defect in cells from patients with congenital muscular dystrophies due to mutations in other genes (Figure 1). Even though LARGE was normal in these cells, increasing its expression had a beneficial effect by somehow compensating for a different glycosylation defect. Whether these results with cultured cells will translate into therapeutic benefits in animal models, and ultimately in humans, remains to be seen. Nevertheless, these data suggest that enhancing protein glycosylation, even if somewhat nonspecifically, may be advantageous in various forms of congenital muscular dystrophy.
Figure 1. Functional Bypass of Genetic Defect by Enhanced Glycosylation.
Under normal conditions, glycoproteins gain complex carbohydrate moieties during processing as they are transported to the cell membrane (Panel A). There, the carbohydrate components help bind to ligands in the extracellular milieu. In the case of skeletal muscle, the glycosylation of -dystroglycan is critical to its binding to laminin, agrin, and perlecan. Defects in glycosylation lead to truncated carbohydrate chains (Panel B). Abnormal glycosylation interferes with the interactions between normal -dystroglycan and matrix proteins and may be the reason for muscle cell degeneration in this group of muscular dystrophies. Barresi et al.2 showed that overexpression of a glycosyltransferase (LARGE) can hyperglycosylate -dystroglycan and thereby enhance its binding to matrix proteins, even though the pattern of glycosylation may be abnormal (Panel C). This restoration of function as a result of enhanced glycosylation may be an effective treatment for a variety of muscular dystrophies.
The idea of enhancing glycosylation as a treatment for muscular dystrophies has a precedent. Nguyen et al.4 showed that overexpression of a glycosyltransferase — cytotoxic T-cell N-acetylgalactosaminyltransferase — was beneficial in a mouse model of muscular dystrophy in which the genetic defect was not even in a glycosylation pathway. The strain these investigators used was the mdx mouse, which has a mutation in the dystrophin gene and thus serves as a model for the human disease Duchenne's muscular dystrophy. Dystrophin is part of a multifunctional transmembrane protein complex, the dystrophin–glycoprotein complex, which serves as a scaffold for adhesion, signaling, and cytoskeletal proteins; disruption of this complex leads to the death of muscle cells from apoptosis and necrosis.5 So, why would enhancing glycosylation benefit muscle deficient in dystrophin? Nguyen et al.4 found that increasing the glycosylation of dystrophin-associated proteins led to a stabilization of the complex even in the absence of dystrophin. Apparently, this effect was due, at least in part, to increased binding of the hyperglycosylated proteins to utrophin, which is a homologue of dystrophin. Utrophin can substitute for dystrophin in the dystrophin–glycoprotein complex, but it is not normally present at the muscle membrane at levels sufficient to prevent cellular degeneration when dystrophin is absent. Thus, increased expression of a glycosyltransferase resulted in increased glycosylation of dystrophin-associated proteins; up-regulation of utrophin; stabilization of the complex, with utrophin substituting for dystrophin; and a therapeutic benefit in the mdx mouse.4
Both LARGE and cytotoxic T-cell N-acetylgalactosaminyltransferase, when overexpressed, can increase the glycosylation of -dystroglycan,2,4 a key component of the dystrophin–glycoprotein complex that is normally heavily glycosylated and that links the transmembrane proteins to components of the extracellular matrix. It may be the increased binding of hyperglycosylated -dystroglycan to matrix proteins that stabilizes the complex and produces the therapeutic benefit. In that sense, enhancing the glycosylation of -dystroglycan may be the final common pathway by which these treatments work. A little bit of extra sugar, in the right place at the right time, may be good for what ails you.
The approach used by Barresi et al. is part of a growing list of "bypass treatments" that are beneficial in animal models of muscular dystrophies. It is surprising and encouraging how many different proteins with different functions can, when overexpressed, confer protection against the pathogenetic processes.1 Although conventional gene therapy, whereby a mutated gene is replaced with its normal counterpart, continues to be at the forefront of experimental therapeutics for muscular dystrophy, the use of bypass strategies is gaining support.
Formidable challenges remain. In all the studies to date, overexpression of the "bypass protein" has been accomplished either by creating a transgenic mouse or by using a viral vector to introduce the gene into the relevant tissue. The former approach is not applicable to humans, and the latter approach involves all of the hurdles of viral-mediated gene transfer. Rather, the real promise lies in the hope that a drug will be able to up-regulate the patient's own gene for the bypass protein, as in, for example, the treatment of sickle cell anemia by the pharmacologic induction of the expression of fetal hemoglobin. Ideally, such a drug would induce a sustained and selective increase in the therapeutic protein, and overexpression of the protein would not have any unwanted side effects. These are tall orders. The identification of such drugs has become a major goal of research in therapy for genetic diseases. As new approaches to compensate for genetic deficiencies in the muscular dystrophies continue to be devised, each new finding will represent a potential therapeutic target and provide hope that pharmacologic treatment for genetic diseases may indeed become a reality.
Source Information
From the Department of Neurology and Neurological Sciences, Stanford University, and the Neurology Service, Veterans Affairs Palo Alto Health Care Systems, Palo Alto — both in California.
References
Engvall E, Wewer UM. The new frontier in muscular dystrophy research: booster genes. FASEB J 2003;17:1579-1584.
Barresi R, Michele DE, Kanagawa M, et al. LARGE can functionally bypass alpha-dystroglycan glycosylation defects in distinct congenital muscular dystrophies. Nat Med 2004;117:953-964.
Muntoni F, Brockington M, Torelli S, Brown SC. Defective glycosylation in congenital muscular dystrophies. Curr Opin Neurol 2004;17:205-209.
Nguyen HH, Jayasinha V, Xia B, Hoyte K, Martin PT. Overexpression of the cytotoxic T cell GalNAc transferase in skeletal muscle inhibits muscular dystrophy in mdx mice. Proc Natl Acad Sci U S A 2002;99:5616-5621.
Rando TA. The dystrophin-glycoprotein complex, cellular signaling, and the regulation of cell survival in the muscular dystrophies. Muscle Nerve 2001;24:1575-1594.(Thomas A. Rando, M.D., Ph)
A recent report by Barresi et al. is one example.2 These investigators showed that overexpression of a glycosyltransferase, a type of enzyme that adds sugar residues to cellular proteins, resulted in the functional rescue of dystrophic muscle cells. This study is based on the recent finding that a subgroup of the muscular dystrophies, the congenital muscular dystrophies, are caused by mutations in genes involved in protein glycosylation.3 Defects in different genes in the glycosylation pathway are associated with distinct clinical phenotypes. One glycosyltransferase, LARGE, is deficient in a dystrophic mouse strain and a form of human congenital muscular dystrophy. Barresi et al. found, not surprisingly, that overexpression of LARGE was an effective treatment in this dystrophic mouse.2 More interesting, however, was their finding that overexpression of LARGE ameliorated, at least partially, the glycosylation defect in cells from patients with congenital muscular dystrophies due to mutations in other genes (Figure 1). Even though LARGE was normal in these cells, increasing its expression had a beneficial effect by somehow compensating for a different glycosylation defect. Whether these results with cultured cells will translate into therapeutic benefits in animal models, and ultimately in humans, remains to be seen. Nevertheless, these data suggest that enhancing protein glycosylation, even if somewhat nonspecifically, may be advantageous in various forms of congenital muscular dystrophy.
Figure 1. Functional Bypass of Genetic Defect by Enhanced Glycosylation.
Under normal conditions, glycoproteins gain complex carbohydrate moieties during processing as they are transported to the cell membrane (Panel A). There, the carbohydrate components help bind to ligands in the extracellular milieu. In the case of skeletal muscle, the glycosylation of -dystroglycan is critical to its binding to laminin, agrin, and perlecan. Defects in glycosylation lead to truncated carbohydrate chains (Panel B). Abnormal glycosylation interferes with the interactions between normal -dystroglycan and matrix proteins and may be the reason for muscle cell degeneration in this group of muscular dystrophies. Barresi et al.2 showed that overexpression of a glycosyltransferase (LARGE) can hyperglycosylate -dystroglycan and thereby enhance its binding to matrix proteins, even though the pattern of glycosylation may be abnormal (Panel C). This restoration of function as a result of enhanced glycosylation may be an effective treatment for a variety of muscular dystrophies.
The idea of enhancing glycosylation as a treatment for muscular dystrophies has a precedent. Nguyen et al.4 showed that overexpression of a glycosyltransferase — cytotoxic T-cell N-acetylgalactosaminyltransferase — was beneficial in a mouse model of muscular dystrophy in which the genetic defect was not even in a glycosylation pathway. The strain these investigators used was the mdx mouse, which has a mutation in the dystrophin gene and thus serves as a model for the human disease Duchenne's muscular dystrophy. Dystrophin is part of a multifunctional transmembrane protein complex, the dystrophin–glycoprotein complex, which serves as a scaffold for adhesion, signaling, and cytoskeletal proteins; disruption of this complex leads to the death of muscle cells from apoptosis and necrosis.5 So, why would enhancing glycosylation benefit muscle deficient in dystrophin? Nguyen et al.4 found that increasing the glycosylation of dystrophin-associated proteins led to a stabilization of the complex even in the absence of dystrophin. Apparently, this effect was due, at least in part, to increased binding of the hyperglycosylated proteins to utrophin, which is a homologue of dystrophin. Utrophin can substitute for dystrophin in the dystrophin–glycoprotein complex, but it is not normally present at the muscle membrane at levels sufficient to prevent cellular degeneration when dystrophin is absent. Thus, increased expression of a glycosyltransferase resulted in increased glycosylation of dystrophin-associated proteins; up-regulation of utrophin; stabilization of the complex, with utrophin substituting for dystrophin; and a therapeutic benefit in the mdx mouse.4
Both LARGE and cytotoxic T-cell N-acetylgalactosaminyltransferase, when overexpressed, can increase the glycosylation of -dystroglycan,2,4 a key component of the dystrophin–glycoprotein complex that is normally heavily glycosylated and that links the transmembrane proteins to components of the extracellular matrix. It may be the increased binding of hyperglycosylated -dystroglycan to matrix proteins that stabilizes the complex and produces the therapeutic benefit. In that sense, enhancing the glycosylation of -dystroglycan may be the final common pathway by which these treatments work. A little bit of extra sugar, in the right place at the right time, may be good for what ails you.
The approach used by Barresi et al. is part of a growing list of "bypass treatments" that are beneficial in animal models of muscular dystrophies. It is surprising and encouraging how many different proteins with different functions can, when overexpressed, confer protection against the pathogenetic processes.1 Although conventional gene therapy, whereby a mutated gene is replaced with its normal counterpart, continues to be at the forefront of experimental therapeutics for muscular dystrophy, the use of bypass strategies is gaining support.
Formidable challenges remain. In all the studies to date, overexpression of the "bypass protein" has been accomplished either by creating a transgenic mouse or by using a viral vector to introduce the gene into the relevant tissue. The former approach is not applicable to humans, and the latter approach involves all of the hurdles of viral-mediated gene transfer. Rather, the real promise lies in the hope that a drug will be able to up-regulate the patient's own gene for the bypass protein, as in, for example, the treatment of sickle cell anemia by the pharmacologic induction of the expression of fetal hemoglobin. Ideally, such a drug would induce a sustained and selective increase in the therapeutic protein, and overexpression of the protein would not have any unwanted side effects. These are tall orders. The identification of such drugs has become a major goal of research in therapy for genetic diseases. As new approaches to compensate for genetic deficiencies in the muscular dystrophies continue to be devised, each new finding will represent a potential therapeutic target and provide hope that pharmacologic treatment for genetic diseases may indeed become a reality.
Source Information
From the Department of Neurology and Neurological Sciences, Stanford University, and the Neurology Service, Veterans Affairs Palo Alto Health Care Systems, Palo Alto — both in California.
References
Engvall E, Wewer UM. The new frontier in muscular dystrophy research: booster genes. FASEB J 2003;17:1579-1584.
Barresi R, Michele DE, Kanagawa M, et al. LARGE can functionally bypass alpha-dystroglycan glycosylation defects in distinct congenital muscular dystrophies. Nat Med 2004;117:953-964.
Muntoni F, Brockington M, Torelli S, Brown SC. Defective glycosylation in congenital muscular dystrophies. Curr Opin Neurol 2004;17:205-209.
Nguyen HH, Jayasinha V, Xia B, Hoyte K, Martin PT. Overexpression of the cytotoxic T cell GalNAc transferase in skeletal muscle inhibits muscular dystrophy in mdx mice. Proc Natl Acad Sci U S A 2002;99:5616-5621.
Rando TA. The dystrophin-glycoprotein complex, cellular signaling, and the regulation of cell survival in the muscular dystrophies. Muscle Nerve 2001;24:1575-1594.(Thomas A. Rando, M.D., Ph)