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Understanding Human Glycosylation Disorders: Biochemistry Leads the Charge *

Understanding Human Glycosylation Disorders: Biochemistry Leads the Charge * MINIREVIEW THE JOURNAL OF BIOLOGICAL CHEMISTRY VOL. 288, NO. 10, pp. 6936 –6945, March 8, 2013 © 2013 by The American Society for Biochemistry and Molecular Biology, Inc. Published in the U.S.A. rently known glycosylation reactions (7). The bottom line is Understanding Human that many glycosylation disorders are known, many more will Glycosylation Disorders: be found, and gene sequencing technology will deliver diverse * medical specialties to glycobiology in the search for biochemi- Biochemistry Leads the Charge cal validation and a deeper understanding of therapeutic Published, JBC Papers in Press, January 17, 2013, DOI 10.1074/jbc.R112.429274 options. This minireview focuses on how clinical medicine and Hudson H. Freeze basic science, now more than ever, generate an immediate, From the Genetic Disease Program, Sanford-Burnham Medical Research symbiotic, cross-fertilizing partnership. Several recently dis- Institute, La Jolla, California 92037 covered glycosylation disorders will also raise important ques- Nearly 70 inherited human glycosylation disorders span a tions for further biochemical investigation. breathtaking clinical spectrum, impacting nearly every organ The collection of glycosylation disorders causes abnormali- system and launching a family-driven diagnostic odyssey. ties in nearly every organ system (7, 8). This means that physi- Advances in genetics, especially next generation sequencing, cians from every specialty will likely encounter patients who propelled discovery of many glycosylation disorders in single carry glycosylation defects. Although near-term exome (or and multiple pathways. Interpretation of whole exome sequenc- genome) sequencing will undoubtedly indicate either known or ing results, insights into pathological mechanisms, and possible predictable glycosylation genes within their clinical arena, therapies will hinge on biochemical analysis of patient-derived other genes may appear on the “fringes” of the current under- materials and animal models. Biochemical diagnostic markers standing of what constitutes a “glycosylation gene.” Examples and readouts offer a physiological context to confirm candidate follow. Essentially all of the known glycosylation biosynthetic genes. Recent discoveries suggest novel perspectives for text- pathways are included in these disorders. Rather than cover all book biochemistry and novel research opportunities. Basic sci- of them, this minireview focuses on recent discoveries that gen- ence and patients are the immediate beneficiaries of this bidi- erate novel perspectives and orient future areas for research. A rectional collaboration. general point to remember is that although many of the disor- ders are restricted to a specific biosynthetic pathway, such as the assembly of the precursor glycan for N-glycosylation, oth- ers, such as those that generate metabolic precursors or Golgi Orientation trafficking complexes, can impact multiple pathways. Recent progress in exome sequencing (1, 2) places biochem- istry in the enviable position of having to explain how patients Glycosylation Pathways with inherited glycosylation disorders develop their symptoms Mammals have eight major glycosylation pathways in the and to suggest therapies to treat them. The technology to endoplasmic reticulum (ER) -Golgi (7–9). Three of these will identify defective genes is no longer rate-limiting; it is available, be highlighted here because they house most of the newly dis- inexpensive, and in demand. Informatics effectively sifts covered glycosylation disorders. N-Glycosylation (Fig. 1) through predictions of damaging mutations in hundreds of occurs in the ER during or soon after the synthesis of nascent cases of patients with unknown genetic disorders (3, 4). Now, proteins. UDP-GlcNAc, GDP-Man, dolichol-phosphate (Dol- biochemistry must step in to provide context and functional P)-Man, and Dol-P-Glc provide the activated precursors to information for genes, some known only by letters and construct a glycan composed of Glc Man GlcNAc , which is 3 9 2 numbers. built stepwise onto a dolichol acceptor embedded in the mem- Each of these developments increases biochemical momen- brane. The glycan from this lipid-linked oligosaccharide (LLO) tum. The recent report from the National Academy of Sciences precursor is transferred en bloc to asparagine within an National Research Council on the future of glycosciences (5) NX(T/S) context of the protein acceptors. Remodeling (pro- alerts the scientific community, funding agencies, and politi- cessing) of the protein-bound chain excises glucose in the ER cians to this often overlooked field. The creation of the National and a variable number of mannose units (ER and Golgi), and Center for Advancing Translational Sciences marks a fuller this can be followed by the addition of variable amounts of commitment of the National Institutes of Health to transla- GlcNAc, Gal, Fuc, and sialic acid (Golgi). Some chains are dec- tional medicine. The goal of the newly formed Centers for Men- orated with sulfate or phosphate, and the glycoproteins are sent delian Genomics is to solve the genetic basis of 3500 rare to destinations within the cell, on its surface, or beyond (10). disorders. About 70 known genetic disorders affect glycan syn- Glycosylphosphatidylinositol (GPI) anchors are assembled thesis, and it is estimated that 2% of the genome encodes cur- stepwise on phosphatidylinositol in the ER membrane (11), starting with transfer of GlcNAc by a protein complex on the * This work was supported, in whole or in part, by National Institutes of Health Grant R01 DK55615. This work was also supported by The Rocket Fund and The Bertrand Might Research Fund. This is the fifth article in the Thematic The abbreviations used are: ER, endoplasmic reticulum; Dol-P, dolichol- Minireview Series on Glycobiology and Extracellular Matrices: Glycan Func- phosphate; LLO, lipid-linked oligosaccharide; GPI, glycosylphosphatidyl- tions Pervade Biology at All Levels. inositol; -DG, -dystroglycan; GlcUA, glucuronic acid; CDG, congenital To whom correspondence should be addressed. E-mail: hudson@ disorders of glycosylation; Tf, transferrin; CMD, congenital muscular dys- sanfordburnham.org. trophy; WWS, Walker-Warburg syndrome; ALP, alkaline phosphatase. 6936 JOURNAL OF BIOLOGICAL CHEMISTRY VOLUME 288 • NUMBER 10 •MARCH 8, 2013 This is an Open Access article under the CC BY license. MINIREVIEW: Understanding Human Glycosylation Disorders FIGURE 1. Schematic of the N-glycosylation pathway. The upper oval rep- resents the ER. The early steps involve GlcNAc (blue squares), Glc (blue circles), and Man (green circles) addition to an LLO assembly on Dol-P (red circles with wavy green lines) using UDP-GlcNAc (orange star with blue square) and GDP- Man (orange star with green circle) donors on the cytoplasmic face. The par- tially completed glycan flips to the luminal side, where it is completed using the donors Dol-P-Man (large green circle with small red circle and wavy green line) and Dol-P-Glc (large blue circle with small red circle and wavy green line)to FIGURE 2. GPI anchor synthesis. This schematic shows the stepwise pathway form Glc Man GlcNAc -P-P-Dol, which is then transferred to proteins in the 3 9 2 for GPI anchor assembly, including the reaction and the names of the ER. The lower oval denotes processing of the N-glycans using a series of gly- enzymes and genes involved. Known glycosylation disorders are highlighted cosidases and glycosyltransferases that require UDP-GlcNAc (orange star with in red. White and blue squares, glucosamine; EtNP (N connected to P with bent blue square), UDP-Gal (orange star with yellow circle), and CMP-Sia (orange star lines), ethanolamine phosphate; GlcNAc-PI, N-acetylglucosaminylphosphati- with purple diamond) transported into the Golgi. Purple diamonds, sialic acid; dylinositol. This figure was adapted from Ref. 11. yellow circles, galactose. cytoplasmic face (PIG-A), followed by de-N-acetylation (PIG- tionally important chains are phosphorylated to generate Man- L). Flip to the luminal side for addition of an extra acyl chain to 6-P and receive a glycosaminoglycan-like polymer containing inositol (PIG-W). This is followed by the addition of 2 mannose alternating 1,3-Xyl and 1,3-GlcUA residues (14). Man-6-P is units (PIG-M and PIG-V), ethanolamine phosphate (PIG-N), converted to a diester of unknown composition (15). The pre- another mannose (PIG-B), and two ethanolamine phosphates requisite glycan structures, order of addition, and donor sub- (PIG-O and PIG-F). The entire sugar-lipid unit is transferred to strates are undefined. Human glycosylation disorders, the proteins with the appropriate C-terminal amino acid sequence -dystroglycanopathies, have been key to solving this pathway. using a multisubunit transamidase complex. A deacylase Mutations in the fukutin (FKTN) and FKRP genes that define removes the acyl chain generated by PIG-W (Fig. 2). glycosylation-related muscular dystrophies encode putative O-Mannose-based glycosylation structures and biosynthetic glycosyltransferases, but both are enzymes in search of donor pathways are incomplete, and studies are ongoing. Selected and acceptor substrates (13). Ser/Thr residues on target proteins (primarily -dystroglycan Clinical and Genetic Nomenclature (-DG)) use Dol-P-Man and a POMT1-POMT2 complex to begin the assembly with mannose (12). The addition of GlcNAc Many human glycosylation disorders were first described by (POMGnT1) and Gal (1,4 Galactosyltransferase) and sialic physicians and based on their patients’ clinical presentations acid makes simple structures. More complex branched glycans because the genetic basis was unknown. A good example is the add GlcNAc and sulfated glucuronic acid (GlcUA) (13). Beyond severity-based categories of -dystroglycanopathies that affect this point, the biosynthetic pathways are unclear. Some func- the addition of O-mannose-based glycans on the -DG com- MARCH 8, 2013• VOLUME 288 • NUMBER 10 JOURNAL OF BIOLOGICAL CHEMISTRY 6937 MINIREVIEW: Understanding Human Glycosylation Disorders plex in muscle cells (12). A revised nomenclature was proposed tion (30–33). Another is to complement yeast or mammalian (16). However, many cases still lack specific diagnoses as the cells with defects in the candidate gene with the normal human search for new gene defects continues. allele but not the mutated version. Restoration of cell growth, One group of glycosylation disorders was called carbohy- lectin binding, or normal glycosylation of target proteins is typ- drate-deficient glycoprotein syndromes and then congenital ically done. In more complex systems (flies, fish, worms, and disorders of glycosylation (CDG) (17), subdivided into two mice), functional knockdown of the gene that generates a sim- groups based on whether the mutated genes affected the addi- ilar gross phenotype is taken as evidence that the mutations in tion of N-glycans (type I) or their processing (type II). The latest the gene are damaging (34–36). simplified version (18) employs a non-italicized gene name fol- Rediscovering Sugar Metabolism lowed by CDG, e.g. CDG-Ia is now PMM2-CDG. Both systems will likely co-exist for some time. The well established metabolic pathways connecting mono- saccharide metabolism to protein glycosylation are incomplete Biochemical Markers for Glycosylation Disorders (Fig. 3). They show potential reactions but do not reflect con- Potential glycosylation disorders can be assessed with bio- tributions of different sources to convergent pathways, cell and chemical biomarkers (19–21). However, markers do not iden- organ preferences, or allowance for metabolic states. Glycosyl- tify the genetic defect. Serum transferrin (Tf) is the best marker ation disorders and models of these diseases are adding unex- for detecting most disorders affecting the N-glycosylation path- pected dimensions to the two-dimensional pathways (10, way (8, 22). Tf has two N-linked glycans, each containing two 37–39). sialic acids. Mass spectrometry, HPLC, or isoelectric focusing Patients deficient in mannose-6-phosphate isomerase (Fru- easily identifies disorders in which one or both of the sites are 6-P 7 Man-6-P) lack sufficient Man-6-P for full N-glycosyla- unoccupied. The same methods yield different abnormal pat- tion. Clearly, glucose is a vital source of this precursor. How- terns when the defects alter N-glycan processing. Defects in ever, modest daily supplements of mannose correct most of the GPI anchor synthesis can often be identified using antibodies patients’ glycosylation deficiencies (40, 41). On the other hand, (20, 23) against the GPI anchor itself or more commonly GPI- hypomorphic PMM2-deficient patients (Man-6-P3 Man-1-P) anchored proteins, such as CD59 on leukocytes. The -dystro- do not benefit from mannose treatment. In mice, null alleles of glycanopathies caused by defects in O-mannose-based glycans either gene are embryonic lethal (embryonic days 2–10.5) (42, can be recognized in muscle biopsies using monoclonal anti- 43). Compound heterozygous mice carrying patient-equivalent bodies (IIH6 and VIA4) directed against the glycan itself (24). mutations in Pmm2 with10% residual enzymatic activity also Obviously, obtaining muscle is more invasive than a simple die in midgestation. Surprisingly, providing mannose to the blood test and would not be routinely done as an inexpensive dams in their drinking water (intake of25 mg/day) bypasses a first step. There are no simple markers for defects in glycosami- critical block and produces viable full-term embryos that con- noglycan chain synthesis. tinue to thrive beyond weaning without mannose (38). This result suggests that the critical period is a gestational glycosyl- Revolution in Gene Discovery ation insufficiency. It is premature to advise at-risk parents to Previously, the genes responsible for most glycosylation dis- consider mannose supplements in an attempt to ameliorate orders were identified by biochemical analysis of fibroblasts potential defects in their glycosylation-deficient pathways (7). and serum glycans (8, 25). Now, genetic mapping techniques Zebrafish morphants deficient in pmm2 or mpi have signifi- have largely replaced this approach (1, 7, 8). This is especially cant morphological abnormalities in many ways comparable to important for finding defects in consanguineous families. the patients (35, 44). The mpi morphants could be substantially Plummeting sequencing costs, lightning speed, improved infor- rescued with 50 mM mannose in the water, but supplementa- matic analysis, and widespread availability of technology put tion was only required in the first 24 h. Removal of mannose or these approaches in hand for solving glycosylation and other continued treatment beyond that time did not alter the out- rare disorders. However, employing biomarkers as a first step come. The pmm2 morphant fish were not given mannose, but quickly focuses the search on glycosylation, and more impor- reducing the metabolic flux through the glycosylation pathway tantly, it provides a metabolic context for candidate genes. In by generating double morphants deficient in both pmm2 and the past 2 years, three defects were solved with traditional bio- mpi actually improved the pmm2 phenotype. A curious aspect chemical analysis and five by autozygosity mapping, linkage is that pmm2 morphants have increased amounts of Man-6-P. analysis, or targeted genomic arrays. Ten defects were solved The accumulation correlates with the loss of LLO and appear- with state-of-the-art whole exome analysis (Table 1). ance of free oligosaccharides, presumably released from the The mutated gene must be shown to impair the function of LLO (45, 46). These results recall important in vitro studies the protein or cause pathology. In many cases, the effects of the showing that increased Man-6-P levels lead to degradation of mutation on RNA splicing or protein structure are predicted mature LLO precursor and release of the intact glycan. This based on multiple programs, such as SIFT (26), PolyPhen (27), suggests that Man-6-P may function as an intracellular sensor and Condel (28), along with evolutionary conservation (phyloP) or signaling molecule. The mechanism is unknown. (29) and frequency of the occurrence of the non-synonymous Congenital myasthenic syndromes result from impaired sig- SNP. A physiologically relevant approach is to complement nal transmission at the neuromuscular synapse (47). Using the patient’s fibroblasts with the normal allele of the mutated genetic linkage, one study (48) identified 13 unrelated families gene and show it corrects abnormal insufficient glycosyla- with mutations in GFPT1 (glutamine:fructose-6-phosphate 6938 JOURNAL OF BIOLOGICAL CHEMISTRY VOLUME 288 • NUMBER 10 •MARCH 8, 2013 MINIREVIEW: Understanding Human Glycosylation Disorders TABLE 1 Glycosylation disorders identified in 2011–2012 MARCH 8, 2013• VOLUME 288 • NUMBER 10 JOURNAL OF BIOLOGICAL CHEMISTRY 6939 MINIREVIEW: Understanding Human Glycosylation Disorders FIGURE 3. Monosaccharide metabolism in mammals and human glycosylation disorders. The gray oval indicates the ER, and all other reactions are thought to occur in the cytoplasm. The purple asterisks indicate known points of metabolic regulations. This figure was adapted from Ref. 10. transaminase 1), used for UDP-GlcNAc synthesis supplying found effects on neutrophils: neutropenia, ER stress, and most glycosylation pathways. No specific pathway was shown abnormal N- and O-linked glycans. Abnormal glycosylation in phox to cause pathology, but knockdown of the zebrafish ortholog particular affects gp91 , the electron-transporting compo- gfpt1 altered muscle fiber morphology and impaired neuro- nent of the NADPH oxidase that is critical for the oxidative muscular junction development in embryos. A surprising fea- burst. This leads to a poor oxidative response, and increased ER ture of this study was that some GFPT1 mutations had no effect stress causes excessive apoptosis. Both N- and O-glycan chains on enzymatic activity, suggesting that the organization or local- were truncated, with many lacking galactose and sialic acid. ization of the enzyme in the cytoplasm is important for normal However, it was unclear whether N-glycosylation sites were function. fully occupied. This is important because tunicamycin causes Another whole exome study (49) describes five patients with similar ER stress by generating unoccupied sites. a limb-girdle myasthenia syndrome and mutations in DPAGT1, How these two genes exert their effects on glycosylation is the first enzyme in LLO synthesis and well known cause of CDG unknown, but deficiency of galactose and sialic acid on both N- (7, 8). The deficiency may be due to a failure to export acetyl- and O-linked glycans suggests that it involves insufficient sup- choline receptors to the end plate. No enzymatic assay was per- ply of UDP-Gal. Exome sequencing showed that mutations in formed, but patients had abnormal Tf and a much milder phe- PGM1 (Glc-1-P 7 Glc-6-P) in two patients cause hypoglyce- notype than seen in previous CDG patients (50). mia and liver abnormalities and result in both absence of N-gly- Whole exome and other genetic mapping studies showed can chains and insufficient galactosylation/sialylation (52, 53). glycosylation abnormalities due to mutations in G6PT1, Incomplete glycan chains could be due to PGM1 effects on the G6PG3, and PGM1. All of these involve Glc-6-P metabolism UDP-Gal pool through the well known metabolic steps (Fig. 3). (Fig. 3). G6PT1 encodes the Glc-6-P (51) translocator, which There is no explanation for how PGM1 deficiency causes the causes glycogen storage disease Ib; and G6PC3 encodes glu- absence of entire glycan chains from proteins. However, uncon- cose-6-phosphatase catabolic-3. These disorders have pro- trolled galactosemia that leads to Gal-1-P accumulation (54) 6940 JOURNAL OF BIOLOGICAL CHEMISTRY VOLUME 288 • NUMBER 10 •MARCH 8, 2013 MINIREVIEW: Understanding Human Glycosylation Disorders and hereditary fructose intolerance and Fru-1-P accumulation laminin-binding glycans contain Man-6-P residues in an acid- produce similar glycosylation abnormalities (55). Intracellular resistant diester linkage. Details of biosynthesis are nebulous, accumulation of various sugar phosphates or other metabolic but Man-6-P addition is unrelated to the lysosomal enzyme- byproducts appears to prevent full N-glycosylation in selected targeting pathway. LARGE is a protein with two putative glyco- cells and tissues. Only Man-6-P has been shown to have an syltransferase domains and functions as a co-polymerase that effect on LLO levels (45). It is clear that mutations in fundamen- adds a variable number of alternating units of 1,3-Xyl and tal glucose utilization pathways must now consider additional 1,3-GlcUA to the protein (14). The acceptor sugar and struc- cell-specific effects on glycosylation. ture of the glycan are unknown. Solving this long-standing Although Tf analysis led to the identification of 35 glycosyl- enigma relied on compositional analysis of expressed -DG and ation disorders spanning a large clinical spectrum, it focused testing a series of glycosides as acceptors for expressed LARGE. most of the attention on the lack of N-glycans. Accumulation of Walker-Warburg syndrome (WWS) is a clinically defined, toxic incomplete or unnatural products (as seen in uncon- severe CMD. Only about 50% of these cases result from muta- trolled galactosemia) is seldom considered. Disorders involving tions in POMT1 or POMT2. One study showed that a putative dolichol biosynthesis or activation of mannose and its down- glycosyltransferase, GTDC2, is mutated in some WWS patients stream donors could also impair synthesis of GPI anchors, based on whole exome analysis and homozygosity mapping of O-mannose glycans, and C-mannosylation (56), but few studies consanguineous families (63). Morpholino knockdown of gtdc2 have probed other types of glycans (57). Synthesis of Dol-P- in zebrafish duplicated the WWS phenotype. Two additional Man requires a complex containing DPM1–3, each encoded by studies using a combination of linkage analysis and exome a different gene. DPM1 is the catalytic subunit, and DPM3 teth- sequencing identified a large number of patients with recessive ers the complex to the ER membrane (58), whereas DPM2 sta- mutations in ISPD (isoprenoid synthase domain-containing). bilizes DPM1 and enhances binding of Dol-P-Man (59). A sin- One study complemented patients’ fibroblasts with wild-type gle patient with mutations in DPM3 showed a mild and late ISPD (62), and the other used zebrafish morphants to recapit- onset muscular dystrophy but did not have any of the typical ulate the human phenotype, including hydrocephalus, smaller symptoms seen in Dol-P-Man-deficient patients. The muta- eye size, muscle degeneration, and reduced -DG glycosylation tions disrupt the DPM1/DPM3 binding interface (57). Two (34). The gene ISPD is the human homolog of a series of genes families with much more severe pathology typical of CDG found in plants and prokaryotes in the non-mevalonate (2-C- patients had mutations in DPM2 with muscular dystrophy, methyl-D-erythritol 4-phosphate) pathway of isoprenoid syn- with reduced O-mannose staining in muscle (60). thesis (64). However, this pathway does not exist in chordates, so its biochemical function, presumably in the biosynthesis of -Dystroglycanopathies the O-mannosyl glycan, is unknown. Two homologs in bacteria An entire set of congenital muscular dystrophies (CMDs) have cytidylyltransferase activity and are used in the synthesis with variable severity results from defects in the biosynthetic of CDP-methylerythritol and CDP-ribitol. One suggestion (34) pathway that adds O-mannose-linked glycans to -DG. This is that the gene is involved in CTP-driven substrate activation peripheral membrane component of the dystrophin-glycopro- to a precursor in the pathway, but this idea awaits more struc- tein complex is located in muscle, nerve, heart, and brain. -DG tural information on the glycan. is one of the two subunits of the dystrophin-glycoprotein com- Human glycosylation defects in the isoprenoid-derived plex, bridging the extracellular matrix to the cytoskeleton. dolichol pathway are well established. Mutations in SRD5A3, -DG and -DG are derived from a single gene, DAG1. In mus- the long-sought polyprenol reductase, show the existence of cle, cytoskeletal actin is linked to -DG, which spans the cell another unknown route of dolichol biosynthesis (65, 66). Muta- membrane. The extracellular domain of -DG binds to -DG, tions in DHDDS,a cis-isoprenyltransferase, cause retinitis pig- which in turn binds to laminin in the extracellular matrix via its mentosa but no other clinical deficits (67, 68). Knockdown of glycan-containing domain. The degree and types of -DG gly- NUS1, the Nogo-B receptor that contains a cis-isoprenyltrans- cosylation vary in different tissues. Monoclonal antibodies ferase domain, reduces the LLO level and decreases N-glycosyl- against the glycans have been key to identifying glycosylation- ation (69). To date, no defects are known in this likely CDG related defects that affect -DG. target gene. Collectively, these disorders, called -dystroglycanopathies, Defects in GPI Anchor Synthesis result from mutations in seven genes, and more will likely be found. Other proteins probably contain these glycans and Whole sequencing and autozygosity mapping identified six might contribute to CMD pathology in the brain because brain- disorders in GPI anchor biosynthesis: PIG-A, PIG-L, PIG-M, specific deletion of -DG does not reduce the amount of those PIG-N, PIG-O, and PIG-V. PIG-A catalyzes the first step in GPI glycans. Recently, Dwyer et al. (61) reported that these glycans anchor synthesis, and somatic mutations in the X-linked gene occur on a receptor protein-tyrosine phosphatase and the cause the well known hematological disorder paroxysmal noc- secreted form, phosphacan, and that mutations in POMGnT1, turnal hemoglobinuria, which results in erythrocyte lysis (70). the second enzyme in the pathway, in a mutant mouse strain A lethal germ-line mutation in PIGA was found in one patient result in a lower molecular weight and loss of the glycan who appeared to retain residual activity (71). antigen. PIG-L carries out the second step of the pathway, de-N- Defining the structure of the critical glycan(s) and their loca- acetylation of N-acetylglucosaminylphosphatidylinositol, and tion on the protein has been challenging (34, 62). The key mutations in it cause CHIME syndrome, with ocular coloboma, MARCH 8, 2013• VOLUME 288 • NUMBER 10 JOURNAL OF BIOLOGICAL CHEMISTRY 6941 MINIREVIEW: Understanding Human Glycosylation Disorders heart defects, ichthyosis, mental retardation, and ear anoma- but first, the N-glycan chains must be stripped. The gene lies. PIG-M is a mannosyltransferase that adds the first man- NGLY1 encodes a stripper enzyme that clears the glycan, and nose to the core GPI (72). Its decrease causes venous thrombo- exome mapping identified a patient with mutations that elimi- sis and seizures (73). However, the mutation occurs at an SP1 nated the protein (81). This may be considered the first “con- transcription factor-binding site. Butyrate restores normal genital disorder of deglycosylation” and is predicted to cause PIGM transcription and cell surface GPI expression in patients’ accumulation of N-glycosylated proteins in the cytoplasm and lymphoblasts. Two-week treatment with phenylbutyrate elim- possibly ER stress. Accumulation of the undegraded material in inates seizures and improves motor skills (74). PIGN encodes the cytoplasm could have separate toxic effects. the ethanolamine phosphate transferase, which adds the etha- Conclusions and Perspectives nolamine phosphate to the first mannose on the GPI anchor. Mutations in PIGN cause multiple congenital anomalies, A thorough whole exome sequencing study (3) focused on including hypotonia and seizures (23). 100 patients with intellectual disability and solved about half of Mutations in the second mannosyltransferase, PIGV, were the cases by employing sophisticated informatics to identify the first identified as the cause of hyperphosphatasia and mental gene and Sanger sequencing for confirmation. Causality was retardation syndrome (75). Patients with a similar phenotype based on programs used to predict the effects of mutations on who lacked mutations in PIGV had mutations in PIGO, an eth- the protein structure or analogy to known genes in the same or anolamine phosphate transferase (76). Both had reduced level related pathway. Surprisingly, the study identified 22 patients of GPI-anchored substrates at the cell surface. Alkaline phos- with potentially causative de novo mutations in novel candidate phatase (ALP) is normally GPI-anchored, but it is secreted into genes. Sixteen patients had mutations in known intellectual dis- the blood, accounting for the characteristic of these two disor- ability genes. ders. The other GPI disorders do not result in ALP secretion. It Whole exome/genome sequencing will continue to identify appears that secretion of ALP depends on GPI transamidase new glycosylation disorders. Biochemical markers will help removal of the C-terminal GPI attachment signal peptide and focus the search, but these approaches will also identify glyco- GPI addition. Defects in which shorter, non-mannosylated GPI sylation-related genes that the serum biomarkers missed or chains accumulate result in ALP degradation, whereas it is patients who were never tested for the markers. Both secreted from cells with incomplete mannose-containing approaches will continue to focus on new genes that impact chains. Transamidase appears to recognize the presence of glycosylation, and biochemical analysis will continue its prom- incomplete mannose-containing chains and cleaves a hydro- inent role to provide a physiological context and basis for phobic signal peptide, resulting in secretion (77). therapies. Fringes of Glycosylation Disorders Acknowledgments—I thank Bobby Ng for help in preparing the figures Most glycosylation disorders are caused by defects in genes and Amy Zimmon for administrative assistance. that conform to our current concepts. Others suggest that an expanded view might be better. One case in point is a glycosyl- REFERENCES ation disorder caused by mutations in the gene TMEM165 1. Matthijs, G., Rymen, D., Millo´n, M. B., Souche, E., and Race, V. (2013) (TPARL) (78). Tf and total serum N- and O-glycans from Approaches to homozygosity mapping and exome sequencing for the patients are deficient in sialic acid and galactose, suggesting a identification of novel types of CDG. Glycoconj. J. 30, 67–76 defect in the Golgi. Staining with Golgi markers TGN46 and 2. Saunders, C. J., Miller, N. A., Soden, S. E., Dinwiddie, D. L., Noll, A., Alnadi, GM130 showed a dilated morphology and fragmented trans- N. A., Andraws, N., Patterson, M. L., Krivohlavek, L. 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Biol. 75. Krawitz, P. M., Schweiger, M. R., Ro¨delsperger, C., Marcelis, C., Ko¨lsch, 22, R622–R626 U., Meisel, C., Stephani, F., Kinoshita, T., Murakami, Y., Bauer, S., Isau, M., 80. Shang, J., Ko¨rner, C., Freeze, H., and Lehrman, M. A. (2002) Extension of Fischer, A., Dahl, A., Kerick, M., Hecht, J., Ko¨hler, S., Ja¨ger, M., Gru¨nha- lipid-linked oligosaccharides is a high-priority aspect of the unfolded pro- gen, J., de Condor, B. J., Doelken, S., Brunner, H. G., Meinecke, P., Pas- tein response: endoplasmic reticulum stress in type I congenital disorder sarge, E., Thompson, M. D., Cole, D. E., Horn, D., Roscioli, T., Mundlos, S., of glycosylation fibroblasts. Glycobiology 12, 307–317 and Robinson, P. N. (2010) Identity-by-descent filtering of exome se- 81. Need, A. C., Shashi, V., Hitomi, Y., Schoch, K., Shianna, K. V., McDonald, quence data identifies PIGV mutations in hyperphosphatasia mental re- M. T., Meisler, M. H., and Goldstein, D. B. (2012) Clinical application of tardation syndrome. Nat. Genet. 42, 827–829 exome sequencing in undiagnosed genetic conditions. J. Med. Genet. 49, 76. Krawitz, P. M., Murakami, Y., Hecht, J., Kru¨ger, U., Holder, S. E., Mortier, 353–361 G. R., Delle Chiaie, B., De Baere, E., Thompson, M. D., Roscioli, T., Kiel- 82. Rafiq, M. A., Kuss, A. W., Puettmann, L., Noor, A., Ramiah, A., Ali, G., Hu, basa, S., Kinoshita, T., Mundlos, S., Robinson, P. N., and Horn, D. (2012) H., Kerio, N. A., Xiang, Y., Garshasbi, M., Khan, M. A., Ishak, G. E., Weks- Mutations in PIGO, a member of the GPI-anchor-synthesis pathway, berg, R., Ullmann, R., Tzschach, A., Kahrizi, K., Mahmood, K., Naeem, F., cause hyperphosphatasia with mental retardation. Am. J. Hum. Genet. 91, Ayub, M., Moremen, K. W., Vincent, J. B., Ropers, H. H., Ansar, M., and 146–151 Najmabadi, H. (2011) Mutations in the 1,2-mannosidase gene, 77. Murakami, Y., Kanzawa, N., Saito, K., Krawitz, P. M., Mundlos, S., Robin- MAN1B1, cause autosomal-recessive intellectual disability. Am. J. Hum. son, P. N., Karadimitris, A., Maeda, Y., and Kinoshita, T. (2012) Mecha- Genet. 89, 176–182 MARCH 8, 2013• VOLUME 288 • NUMBER 10 JOURNAL OF BIOLOGICAL CHEMISTRY 6945 http://www.deepdyve.com/assets/images/DeepDyve-Logo-lg.png Journal of Biological Chemistry American Society for Biochemistry and Molecular Biology

Understanding Human Glycosylation Disorders: Biochemistry Leads the Charge *

Journal of Biological Chemistry , Volume 288 (10) – Mar 8, 2013

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American Society for Biochemistry and Molecular Biology
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Copyright © 2013 Elsevier Inc.
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0021-9258
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1083-351X
DOI
10.1074/jbc.r112.429274
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Abstract

MINIREVIEW THE JOURNAL OF BIOLOGICAL CHEMISTRY VOL. 288, NO. 10, pp. 6936 –6945, March 8, 2013 © 2013 by The American Society for Biochemistry and Molecular Biology, Inc. Published in the U.S.A. rently known glycosylation reactions (7). The bottom line is Understanding Human that many glycosylation disorders are known, many more will Glycosylation Disorders: be found, and gene sequencing technology will deliver diverse * medical specialties to glycobiology in the search for biochemi- Biochemistry Leads the Charge cal validation and a deeper understanding of therapeutic Published, JBC Papers in Press, January 17, 2013, DOI 10.1074/jbc.R112.429274 options. This minireview focuses on how clinical medicine and Hudson H. Freeze basic science, now more than ever, generate an immediate, From the Genetic Disease Program, Sanford-Burnham Medical Research symbiotic, cross-fertilizing partnership. Several recently dis- Institute, La Jolla, California 92037 covered glycosylation disorders will also raise important ques- Nearly 70 inherited human glycosylation disorders span a tions for further biochemical investigation. breathtaking clinical spectrum, impacting nearly every organ The collection of glycosylation disorders causes abnormali- system and launching a family-driven diagnostic odyssey. ties in nearly every organ system (7, 8). This means that physi- Advances in genetics, especially next generation sequencing, cians from every specialty will likely encounter patients who propelled discovery of many glycosylation disorders in single carry glycosylation defects. Although near-term exome (or and multiple pathways. Interpretation of whole exome sequenc- genome) sequencing will undoubtedly indicate either known or ing results, insights into pathological mechanisms, and possible predictable glycosylation genes within their clinical arena, therapies will hinge on biochemical analysis of patient-derived other genes may appear on the “fringes” of the current under- materials and animal models. Biochemical diagnostic markers standing of what constitutes a “glycosylation gene.” Examples and readouts offer a physiological context to confirm candidate follow. Essentially all of the known glycosylation biosynthetic genes. Recent discoveries suggest novel perspectives for text- pathways are included in these disorders. Rather than cover all book biochemistry and novel research opportunities. Basic sci- of them, this minireview focuses on recent discoveries that gen- ence and patients are the immediate beneficiaries of this bidi- erate novel perspectives and orient future areas for research. A rectional collaboration. general point to remember is that although many of the disor- ders are restricted to a specific biosynthetic pathway, such as the assembly of the precursor glycan for N-glycosylation, oth- ers, such as those that generate metabolic precursors or Golgi Orientation trafficking complexes, can impact multiple pathways. Recent progress in exome sequencing (1, 2) places biochem- istry in the enviable position of having to explain how patients Glycosylation Pathways with inherited glycosylation disorders develop their symptoms Mammals have eight major glycosylation pathways in the and to suggest therapies to treat them. The technology to endoplasmic reticulum (ER) -Golgi (7–9). Three of these will identify defective genes is no longer rate-limiting; it is available, be highlighted here because they house most of the newly dis- inexpensive, and in demand. Informatics effectively sifts covered glycosylation disorders. N-Glycosylation (Fig. 1) through predictions of damaging mutations in hundreds of occurs in the ER during or soon after the synthesis of nascent cases of patients with unknown genetic disorders (3, 4). Now, proteins. UDP-GlcNAc, GDP-Man, dolichol-phosphate (Dol- biochemistry must step in to provide context and functional P)-Man, and Dol-P-Glc provide the activated precursors to information for genes, some known only by letters and construct a glycan composed of Glc Man GlcNAc , which is 3 9 2 numbers. built stepwise onto a dolichol acceptor embedded in the mem- Each of these developments increases biochemical momen- brane. The glycan from this lipid-linked oligosaccharide (LLO) tum. The recent report from the National Academy of Sciences precursor is transferred en bloc to asparagine within an National Research Council on the future of glycosciences (5) NX(T/S) context of the protein acceptors. Remodeling (pro- alerts the scientific community, funding agencies, and politi- cessing) of the protein-bound chain excises glucose in the ER cians to this often overlooked field. The creation of the National and a variable number of mannose units (ER and Golgi), and Center for Advancing Translational Sciences marks a fuller this can be followed by the addition of variable amounts of commitment of the National Institutes of Health to transla- GlcNAc, Gal, Fuc, and sialic acid (Golgi). Some chains are dec- tional medicine. The goal of the newly formed Centers for Men- orated with sulfate or phosphate, and the glycoproteins are sent delian Genomics is to solve the genetic basis of 3500 rare to destinations within the cell, on its surface, or beyond (10). disorders. About 70 known genetic disorders affect glycan syn- Glycosylphosphatidylinositol (GPI) anchors are assembled thesis, and it is estimated that 2% of the genome encodes cur- stepwise on phosphatidylinositol in the ER membrane (11), starting with transfer of GlcNAc by a protein complex on the * This work was supported, in whole or in part, by National Institutes of Health Grant R01 DK55615. This work was also supported by The Rocket Fund and The Bertrand Might Research Fund. This is the fifth article in the Thematic The abbreviations used are: ER, endoplasmic reticulum; Dol-P, dolichol- Minireview Series on Glycobiology and Extracellular Matrices: Glycan Func- phosphate; LLO, lipid-linked oligosaccharide; GPI, glycosylphosphatidyl- tions Pervade Biology at All Levels. inositol; -DG, -dystroglycan; GlcUA, glucuronic acid; CDG, congenital To whom correspondence should be addressed. E-mail: hudson@ disorders of glycosylation; Tf, transferrin; CMD, congenital muscular dys- sanfordburnham.org. trophy; WWS, Walker-Warburg syndrome; ALP, alkaline phosphatase. 6936 JOURNAL OF BIOLOGICAL CHEMISTRY VOLUME 288 • NUMBER 10 •MARCH 8, 2013 This is an Open Access article under the CC BY license. MINIREVIEW: Understanding Human Glycosylation Disorders FIGURE 1. Schematic of the N-glycosylation pathway. The upper oval rep- resents the ER. The early steps involve GlcNAc (blue squares), Glc (blue circles), and Man (green circles) addition to an LLO assembly on Dol-P (red circles with wavy green lines) using UDP-GlcNAc (orange star with blue square) and GDP- Man (orange star with green circle) donors on the cytoplasmic face. The par- tially completed glycan flips to the luminal side, where it is completed using the donors Dol-P-Man (large green circle with small red circle and wavy green line) and Dol-P-Glc (large blue circle with small red circle and wavy green line)to FIGURE 2. GPI anchor synthesis. This schematic shows the stepwise pathway form Glc Man GlcNAc -P-P-Dol, which is then transferred to proteins in the 3 9 2 for GPI anchor assembly, including the reaction and the names of the ER. The lower oval denotes processing of the N-glycans using a series of gly- enzymes and genes involved. Known glycosylation disorders are highlighted cosidases and glycosyltransferases that require UDP-GlcNAc (orange star with in red. White and blue squares, glucosamine; EtNP (N connected to P with bent blue square), UDP-Gal (orange star with yellow circle), and CMP-Sia (orange star lines), ethanolamine phosphate; GlcNAc-PI, N-acetylglucosaminylphosphati- with purple diamond) transported into the Golgi. Purple diamonds, sialic acid; dylinositol. This figure was adapted from Ref. 11. yellow circles, galactose. cytoplasmic face (PIG-A), followed by de-N-acetylation (PIG- tionally important chains are phosphorylated to generate Man- L). Flip to the luminal side for addition of an extra acyl chain to 6-P and receive a glycosaminoglycan-like polymer containing inositol (PIG-W). This is followed by the addition of 2 mannose alternating 1,3-Xyl and 1,3-GlcUA residues (14). Man-6-P is units (PIG-M and PIG-V), ethanolamine phosphate (PIG-N), converted to a diester of unknown composition (15). The pre- another mannose (PIG-B), and two ethanolamine phosphates requisite glycan structures, order of addition, and donor sub- (PIG-O and PIG-F). The entire sugar-lipid unit is transferred to strates are undefined. Human glycosylation disorders, the proteins with the appropriate C-terminal amino acid sequence -dystroglycanopathies, have been key to solving this pathway. using a multisubunit transamidase complex. A deacylase Mutations in the fukutin (FKTN) and FKRP genes that define removes the acyl chain generated by PIG-W (Fig. 2). glycosylation-related muscular dystrophies encode putative O-Mannose-based glycosylation structures and biosynthetic glycosyltransferases, but both are enzymes in search of donor pathways are incomplete, and studies are ongoing. Selected and acceptor substrates (13). Ser/Thr residues on target proteins (primarily -dystroglycan Clinical and Genetic Nomenclature (-DG)) use Dol-P-Man and a POMT1-POMT2 complex to begin the assembly with mannose (12). The addition of GlcNAc Many human glycosylation disorders were first described by (POMGnT1) and Gal (1,4 Galactosyltransferase) and sialic physicians and based on their patients’ clinical presentations acid makes simple structures. More complex branched glycans because the genetic basis was unknown. A good example is the add GlcNAc and sulfated glucuronic acid (GlcUA) (13). Beyond severity-based categories of -dystroglycanopathies that affect this point, the biosynthetic pathways are unclear. Some func- the addition of O-mannose-based glycans on the -DG com- MARCH 8, 2013• VOLUME 288 • NUMBER 10 JOURNAL OF BIOLOGICAL CHEMISTRY 6937 MINIREVIEW: Understanding Human Glycosylation Disorders plex in muscle cells (12). A revised nomenclature was proposed tion (30–33). Another is to complement yeast or mammalian (16). However, many cases still lack specific diagnoses as the cells with defects in the candidate gene with the normal human search for new gene defects continues. allele but not the mutated version. Restoration of cell growth, One group of glycosylation disorders was called carbohy- lectin binding, or normal glycosylation of target proteins is typ- drate-deficient glycoprotein syndromes and then congenital ically done. In more complex systems (flies, fish, worms, and disorders of glycosylation (CDG) (17), subdivided into two mice), functional knockdown of the gene that generates a sim- groups based on whether the mutated genes affected the addi- ilar gross phenotype is taken as evidence that the mutations in tion of N-glycans (type I) or their processing (type II). The latest the gene are damaging (34–36). simplified version (18) employs a non-italicized gene name fol- Rediscovering Sugar Metabolism lowed by CDG, e.g. CDG-Ia is now PMM2-CDG. Both systems will likely co-exist for some time. The well established metabolic pathways connecting mono- saccharide metabolism to protein glycosylation are incomplete Biochemical Markers for Glycosylation Disorders (Fig. 3). They show potential reactions but do not reflect con- Potential glycosylation disorders can be assessed with bio- tributions of different sources to convergent pathways, cell and chemical biomarkers (19–21). However, markers do not iden- organ preferences, or allowance for metabolic states. Glycosyl- tify the genetic defect. Serum transferrin (Tf) is the best marker ation disorders and models of these diseases are adding unex- for detecting most disorders affecting the N-glycosylation path- pected dimensions to the two-dimensional pathways (10, way (8, 22). Tf has two N-linked glycans, each containing two 37–39). sialic acids. Mass spectrometry, HPLC, or isoelectric focusing Patients deficient in mannose-6-phosphate isomerase (Fru- easily identifies disorders in which one or both of the sites are 6-P 7 Man-6-P) lack sufficient Man-6-P for full N-glycosyla- unoccupied. The same methods yield different abnormal pat- tion. Clearly, glucose is a vital source of this precursor. How- terns when the defects alter N-glycan processing. Defects in ever, modest daily supplements of mannose correct most of the GPI anchor synthesis can often be identified using antibodies patients’ glycosylation deficiencies (40, 41). On the other hand, (20, 23) against the GPI anchor itself or more commonly GPI- hypomorphic PMM2-deficient patients (Man-6-P3 Man-1-P) anchored proteins, such as CD59 on leukocytes. The -dystro- do not benefit from mannose treatment. In mice, null alleles of glycanopathies caused by defects in O-mannose-based glycans either gene are embryonic lethal (embryonic days 2–10.5) (42, can be recognized in muscle biopsies using monoclonal anti- 43). Compound heterozygous mice carrying patient-equivalent bodies (IIH6 and VIA4) directed against the glycan itself (24). mutations in Pmm2 with10% residual enzymatic activity also Obviously, obtaining muscle is more invasive than a simple die in midgestation. Surprisingly, providing mannose to the blood test and would not be routinely done as an inexpensive dams in their drinking water (intake of25 mg/day) bypasses a first step. There are no simple markers for defects in glycosami- critical block and produces viable full-term embryos that con- noglycan chain synthesis. tinue to thrive beyond weaning without mannose (38). This result suggests that the critical period is a gestational glycosyl- Revolution in Gene Discovery ation insufficiency. It is premature to advise at-risk parents to Previously, the genes responsible for most glycosylation dis- consider mannose supplements in an attempt to ameliorate orders were identified by biochemical analysis of fibroblasts potential defects in their glycosylation-deficient pathways (7). and serum glycans (8, 25). Now, genetic mapping techniques Zebrafish morphants deficient in pmm2 or mpi have signifi- have largely replaced this approach (1, 7, 8). This is especially cant morphological abnormalities in many ways comparable to important for finding defects in consanguineous families. the patients (35, 44). The mpi morphants could be substantially Plummeting sequencing costs, lightning speed, improved infor- rescued with 50 mM mannose in the water, but supplementa- matic analysis, and widespread availability of technology put tion was only required in the first 24 h. Removal of mannose or these approaches in hand for solving glycosylation and other continued treatment beyond that time did not alter the out- rare disorders. However, employing biomarkers as a first step come. The pmm2 morphant fish were not given mannose, but quickly focuses the search on glycosylation, and more impor- reducing the metabolic flux through the glycosylation pathway tantly, it provides a metabolic context for candidate genes. In by generating double morphants deficient in both pmm2 and the past 2 years, three defects were solved with traditional bio- mpi actually improved the pmm2 phenotype. A curious aspect chemical analysis and five by autozygosity mapping, linkage is that pmm2 morphants have increased amounts of Man-6-P. analysis, or targeted genomic arrays. Ten defects were solved The accumulation correlates with the loss of LLO and appear- with state-of-the-art whole exome analysis (Table 1). ance of free oligosaccharides, presumably released from the The mutated gene must be shown to impair the function of LLO (45, 46). These results recall important in vitro studies the protein or cause pathology. In many cases, the effects of the showing that increased Man-6-P levels lead to degradation of mutation on RNA splicing or protein structure are predicted mature LLO precursor and release of the intact glycan. This based on multiple programs, such as SIFT (26), PolyPhen (27), suggests that Man-6-P may function as an intracellular sensor and Condel (28), along with evolutionary conservation (phyloP) or signaling molecule. The mechanism is unknown. (29) and frequency of the occurrence of the non-synonymous Congenital myasthenic syndromes result from impaired sig- SNP. A physiologically relevant approach is to complement nal transmission at the neuromuscular synapse (47). Using the patient’s fibroblasts with the normal allele of the mutated genetic linkage, one study (48) identified 13 unrelated families gene and show it corrects abnormal insufficient glycosyla- with mutations in GFPT1 (glutamine:fructose-6-phosphate 6938 JOURNAL OF BIOLOGICAL CHEMISTRY VOLUME 288 • NUMBER 10 •MARCH 8, 2013 MINIREVIEW: Understanding Human Glycosylation Disorders TABLE 1 Glycosylation disorders identified in 2011–2012 MARCH 8, 2013• VOLUME 288 • NUMBER 10 JOURNAL OF BIOLOGICAL CHEMISTRY 6939 MINIREVIEW: Understanding Human Glycosylation Disorders FIGURE 3. Monosaccharide metabolism in mammals and human glycosylation disorders. The gray oval indicates the ER, and all other reactions are thought to occur in the cytoplasm. The purple asterisks indicate known points of metabolic regulations. This figure was adapted from Ref. 10. transaminase 1), used for UDP-GlcNAc synthesis supplying found effects on neutrophils: neutropenia, ER stress, and most glycosylation pathways. No specific pathway was shown abnormal N- and O-linked glycans. Abnormal glycosylation in phox to cause pathology, but knockdown of the zebrafish ortholog particular affects gp91 , the electron-transporting compo- gfpt1 altered muscle fiber morphology and impaired neuro- nent of the NADPH oxidase that is critical for the oxidative muscular junction development in embryos. A surprising fea- burst. This leads to a poor oxidative response, and increased ER ture of this study was that some GFPT1 mutations had no effect stress causes excessive apoptosis. Both N- and O-glycan chains on enzymatic activity, suggesting that the organization or local- were truncated, with many lacking galactose and sialic acid. ization of the enzyme in the cytoplasm is important for normal However, it was unclear whether N-glycosylation sites were function. fully occupied. This is important because tunicamycin causes Another whole exome study (49) describes five patients with similar ER stress by generating unoccupied sites. a limb-girdle myasthenia syndrome and mutations in DPAGT1, How these two genes exert their effects on glycosylation is the first enzyme in LLO synthesis and well known cause of CDG unknown, but deficiency of galactose and sialic acid on both N- (7, 8). The deficiency may be due to a failure to export acetyl- and O-linked glycans suggests that it involves insufficient sup- choline receptors to the end plate. No enzymatic assay was per- ply of UDP-Gal. Exome sequencing showed that mutations in formed, but patients had abnormal Tf and a much milder phe- PGM1 (Glc-1-P 7 Glc-6-P) in two patients cause hypoglyce- notype than seen in previous CDG patients (50). mia and liver abnormalities and result in both absence of N-gly- Whole exome and other genetic mapping studies showed can chains and insufficient galactosylation/sialylation (52, 53). glycosylation abnormalities due to mutations in G6PT1, Incomplete glycan chains could be due to PGM1 effects on the G6PG3, and PGM1. All of these involve Glc-6-P metabolism UDP-Gal pool through the well known metabolic steps (Fig. 3). (Fig. 3). G6PT1 encodes the Glc-6-P (51) translocator, which There is no explanation for how PGM1 deficiency causes the causes glycogen storage disease Ib; and G6PC3 encodes glu- absence of entire glycan chains from proteins. However, uncon- cose-6-phosphatase catabolic-3. These disorders have pro- trolled galactosemia that leads to Gal-1-P accumulation (54) 6940 JOURNAL OF BIOLOGICAL CHEMISTRY VOLUME 288 • NUMBER 10 •MARCH 8, 2013 MINIREVIEW: Understanding Human Glycosylation Disorders and hereditary fructose intolerance and Fru-1-P accumulation laminin-binding glycans contain Man-6-P residues in an acid- produce similar glycosylation abnormalities (55). Intracellular resistant diester linkage. Details of biosynthesis are nebulous, accumulation of various sugar phosphates or other metabolic but Man-6-P addition is unrelated to the lysosomal enzyme- byproducts appears to prevent full N-glycosylation in selected targeting pathway. LARGE is a protein with two putative glyco- cells and tissues. Only Man-6-P has been shown to have an syltransferase domains and functions as a co-polymerase that effect on LLO levels (45). It is clear that mutations in fundamen- adds a variable number of alternating units of 1,3-Xyl and tal glucose utilization pathways must now consider additional 1,3-GlcUA to the protein (14). The acceptor sugar and struc- cell-specific effects on glycosylation. ture of the glycan are unknown. Solving this long-standing Although Tf analysis led to the identification of 35 glycosyl- enigma relied on compositional analysis of expressed -DG and ation disorders spanning a large clinical spectrum, it focused testing a series of glycosides as acceptors for expressed LARGE. most of the attention on the lack of N-glycans. Accumulation of Walker-Warburg syndrome (WWS) is a clinically defined, toxic incomplete or unnatural products (as seen in uncon- severe CMD. Only about 50% of these cases result from muta- trolled galactosemia) is seldom considered. Disorders involving tions in POMT1 or POMT2. One study showed that a putative dolichol biosynthesis or activation of mannose and its down- glycosyltransferase, GTDC2, is mutated in some WWS patients stream donors could also impair synthesis of GPI anchors, based on whole exome analysis and homozygosity mapping of O-mannose glycans, and C-mannosylation (56), but few studies consanguineous families (63). Morpholino knockdown of gtdc2 have probed other types of glycans (57). Synthesis of Dol-P- in zebrafish duplicated the WWS phenotype. Two additional Man requires a complex containing DPM1–3, each encoded by studies using a combination of linkage analysis and exome a different gene. DPM1 is the catalytic subunit, and DPM3 teth- sequencing identified a large number of patients with recessive ers the complex to the ER membrane (58), whereas DPM2 sta- mutations in ISPD (isoprenoid synthase domain-containing). bilizes DPM1 and enhances binding of Dol-P-Man (59). A sin- One study complemented patients’ fibroblasts with wild-type gle patient with mutations in DPM3 showed a mild and late ISPD (62), and the other used zebrafish morphants to recapit- onset muscular dystrophy but did not have any of the typical ulate the human phenotype, including hydrocephalus, smaller symptoms seen in Dol-P-Man-deficient patients. The muta- eye size, muscle degeneration, and reduced -DG glycosylation tions disrupt the DPM1/DPM3 binding interface (57). Two (34). The gene ISPD is the human homolog of a series of genes families with much more severe pathology typical of CDG found in plants and prokaryotes in the non-mevalonate (2-C- patients had mutations in DPM2 with muscular dystrophy, methyl-D-erythritol 4-phosphate) pathway of isoprenoid syn- with reduced O-mannose staining in muscle (60). thesis (64). However, this pathway does not exist in chordates, so its biochemical function, presumably in the biosynthesis of -Dystroglycanopathies the O-mannosyl glycan, is unknown. Two homologs in bacteria An entire set of congenital muscular dystrophies (CMDs) have cytidylyltransferase activity and are used in the synthesis with variable severity results from defects in the biosynthetic of CDP-methylerythritol and CDP-ribitol. One suggestion (34) pathway that adds O-mannose-linked glycans to -DG. This is that the gene is involved in CTP-driven substrate activation peripheral membrane component of the dystrophin-glycopro- to a precursor in the pathway, but this idea awaits more struc- tein complex is located in muscle, nerve, heart, and brain. -DG tural information on the glycan. is one of the two subunits of the dystrophin-glycoprotein com- Human glycosylation defects in the isoprenoid-derived plex, bridging the extracellular matrix to the cytoskeleton. dolichol pathway are well established. Mutations in SRD5A3, -DG and -DG are derived from a single gene, DAG1. In mus- the long-sought polyprenol reductase, show the existence of cle, cytoskeletal actin is linked to -DG, which spans the cell another unknown route of dolichol biosynthesis (65, 66). Muta- membrane. The extracellular domain of -DG binds to -DG, tions in DHDDS,a cis-isoprenyltransferase, cause retinitis pig- which in turn binds to laminin in the extracellular matrix via its mentosa but no other clinical deficits (67, 68). Knockdown of glycan-containing domain. The degree and types of -DG gly- NUS1, the Nogo-B receptor that contains a cis-isoprenyltrans- cosylation vary in different tissues. Monoclonal antibodies ferase domain, reduces the LLO level and decreases N-glycosyl- against the glycans have been key to identifying glycosylation- ation (69). To date, no defects are known in this likely CDG related defects that affect -DG. target gene. Collectively, these disorders, called -dystroglycanopathies, Defects in GPI Anchor Synthesis result from mutations in seven genes, and more will likely be found. Other proteins probably contain these glycans and Whole sequencing and autozygosity mapping identified six might contribute to CMD pathology in the brain because brain- disorders in GPI anchor biosynthesis: PIG-A, PIG-L, PIG-M, specific deletion of -DG does not reduce the amount of those PIG-N, PIG-O, and PIG-V. PIG-A catalyzes the first step in GPI glycans. Recently, Dwyer et al. (61) reported that these glycans anchor synthesis, and somatic mutations in the X-linked gene occur on a receptor protein-tyrosine phosphatase and the cause the well known hematological disorder paroxysmal noc- secreted form, phosphacan, and that mutations in POMGnT1, turnal hemoglobinuria, which results in erythrocyte lysis (70). the second enzyme in the pathway, in a mutant mouse strain A lethal germ-line mutation in PIGA was found in one patient result in a lower molecular weight and loss of the glycan who appeared to retain residual activity (71). antigen. PIG-L carries out the second step of the pathway, de-N- Defining the structure of the critical glycan(s) and their loca- acetylation of N-acetylglucosaminylphosphatidylinositol, and tion on the protein has been challenging (34, 62). The key mutations in it cause CHIME syndrome, with ocular coloboma, MARCH 8, 2013• VOLUME 288 • NUMBER 10 JOURNAL OF BIOLOGICAL CHEMISTRY 6941 MINIREVIEW: Understanding Human Glycosylation Disorders heart defects, ichthyosis, mental retardation, and ear anoma- but first, the N-glycan chains must be stripped. The gene lies. PIG-M is a mannosyltransferase that adds the first man- NGLY1 encodes a stripper enzyme that clears the glycan, and nose to the core GPI (72). Its decrease causes venous thrombo- exome mapping identified a patient with mutations that elimi- sis and seizures (73). However, the mutation occurs at an SP1 nated the protein (81). This may be considered the first “con- transcription factor-binding site. Butyrate restores normal genital disorder of deglycosylation” and is predicted to cause PIGM transcription and cell surface GPI expression in patients’ accumulation of N-glycosylated proteins in the cytoplasm and lymphoblasts. Two-week treatment with phenylbutyrate elim- possibly ER stress. Accumulation of the undegraded material in inates seizures and improves motor skills (74). PIGN encodes the cytoplasm could have separate toxic effects. the ethanolamine phosphate transferase, which adds the etha- Conclusions and Perspectives nolamine phosphate to the first mannose on the GPI anchor. Mutations in PIGN cause multiple congenital anomalies, A thorough whole exome sequencing study (3) focused on including hypotonia and seizures (23). 100 patients with intellectual disability and solved about half of Mutations in the second mannosyltransferase, PIGV, were the cases by employing sophisticated informatics to identify the first identified as the cause of hyperphosphatasia and mental gene and Sanger sequencing for confirmation. Causality was retardation syndrome (75). Patients with a similar phenotype based on programs used to predict the effects of mutations on who lacked mutations in PIGV had mutations in PIGO, an eth- the protein structure or analogy to known genes in the same or anolamine phosphate transferase (76). Both had reduced level related pathway. Surprisingly, the study identified 22 patients of GPI-anchored substrates at the cell surface. Alkaline phos- with potentially causative de novo mutations in novel candidate phatase (ALP) is normally GPI-anchored, but it is secreted into genes. Sixteen patients had mutations in known intellectual dis- the blood, accounting for the characteristic of these two disor- ability genes. ders. The other GPI disorders do not result in ALP secretion. It Whole exome/genome sequencing will continue to identify appears that secretion of ALP depends on GPI transamidase new glycosylation disorders. Biochemical markers will help removal of the C-terminal GPI attachment signal peptide and focus the search, but these approaches will also identify glyco- GPI addition. Defects in which shorter, non-mannosylated GPI sylation-related genes that the serum biomarkers missed or chains accumulate result in ALP degradation, whereas it is patients who were never tested for the markers. Both secreted from cells with incomplete mannose-containing approaches will continue to focus on new genes that impact chains. Transamidase appears to recognize the presence of glycosylation, and biochemical analysis will continue its prom- incomplete mannose-containing chains and cleaves a hydro- inent role to provide a physiological context and basis for phobic signal peptide, resulting in secretion (77). therapies. 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M., Mundlos, S., Robin- MAN1B1, cause autosomal-recessive intellectual disability. Am. J. Hum. son, P. N., Karadimitris, A., Maeda, Y., and Kinoshita, T. (2012) Mecha- Genet. 89, 176–182 MARCH 8, 2013• VOLUME 288 • NUMBER 10 JOURNAL OF BIOLOGICAL CHEMISTRY 6945

Journal

Journal of Biological ChemistryAmerican Society for Biochemistry and Molecular Biology

Published: Mar 8, 2013

Keywords: Glycobiology; Glycosylation; Glycosylation Inhibitors; Glycosyltransferases; Golgi; Congenital Disorders of Glycosylation; GPI Anchor; N-Glycosylation; Dystroglycanopathy; Whole Exome Sequencing

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