Mass spectrometry for congenital disorders of glycosylation, CDG

Congenital disorders of glycosylation (CDG) constitute a group of diseases affecting N-linked glycosylation pathways. The classical type of CDG, now called CDG-I, results from deficiencies in the early glycosylation pathway for biosynthesis of lipid-linked oligosaccharide and its transfer to proteins in endoplasmic reticulum, while the CDG-II diseases are caused by defects in the subsequent processing steps. Mass spectrometry (MS) produced a milestone in CDG research, by localizing the CDG-I defect to the early glycosylation pathway in 1992. Currently, MS of transferrin, either by electrospray ionization or matrix-assisted laser desorption/ionization, plays the central role in laboratory screening of CDG-I. On the other hand, the glycopeptide analysis recently developed for site-specific glycans of glycoproteins allows detailed glycan analysis in a high throughput manner and will solve problems in CDG-II diagnosis. These techniques will facilitate studying CDG, a field now expanding to O-linked glycosylation and to acquired as well as inherited conditions that can affect protein glycosylation.     

Mutation of the COG complex subunit gene COG7 causes a lethal congenital disorder

The congenital disorders of glycosylation (CDG) are characterized by defects in N-linked glycan biosynthesis that result from mutations in genes encoding proteins directly involved in the glycosylation pathway. Here we describe two siblings with a fatal form of CDG caused by a mutation in the gene encoding COG-7, a subunit of the conserved oligomeric Golgi (COG) complex. The mutation impairs integrity of the COG complex and alters Golgi trafficking, resulting in disruption of multiple glycosylation pathways. These cases represent a new type of CDG in which the molecular defect lies in a protein that affects the trafficking and function of the glycosylation machinery.     

Mass spectrometry of transferrin and apolipoprotein C-III for diagnosis and screening of congenital disorder of glycosylation

Congenital disorder of glycosylation (CDG), formerly representing a group of diseases due to defects in the biosynthetic pathway of protein N-glycosylation, currently covers a wide range of disorders affecting glycoconjugates. Since its first application to serum transferrin from a CDG patient with phosphomannomutase-2 deficiency in 1992, mass spectrometry (MS) has been playing a key role in identification and characterization of glycosylation defects affecting glycoproteins. MS of native transferrin detects a lack of glycans characteristic to the classical CDG-I type of molecular abnormality. Electrospray ionization MS of native transferrin, especially, allows glycoforms to be analyzed precisely but requires basic knowledge regarding deconvolution of multiply-charged ions which may generate ghost signals upon transformation into a singly-charged form. MS of glycopeptides from tryptic digestion of transferrin delineates site-specific glycoforms and reveals a delicate balance of donor/acceptor substrates or the conformational effect of nascent proteins in cells. Matrix-assisted laser desorption ionization MS of apolipoprotein C-III is a simple method of elucidating the profiles of mucin-type core 1 O-glycans including site occupancy and glycoforms. In this technological review, the principle and pitfalls of MS for CDG are discussed and mass spectra of various types of CDG are presented.     

COG defects,birth and rise!

The COG complex is a cytosolic heteromeric Golgi complex constituted of 8 subunits (Cog1 to Cog8) and involved in retrograde vesicular Golgi trafficking. The involvement of this complex in glycosylation and more specifically in Golgi glycosyltransferases localization has been highlighted with the discovery of COG subunit deficiencies leading to CDG (Congenital Disorders of Glycosylation), a group of inherited disorders of glycosylation. To date, many COG deficient CDG patients have been discovered and this article reviews the birth and rise of this group of defects. The architecture of the COG complex and its cellular functions in Golgi trafficking and Golgi glycosylation are discussed.     

Congenital Disorders of Glycosylation: CDG-I, CDG-II, and beyond

The Congenital Disorders of Glycosylation (CDG) are a collection of over 20 inherited diseases that impair protein N-glycosylation. The clinical appearance of CDG patients is quite diverse making it difficult for physicians to recognize them. A simple blood test of transferrin glycosylation status signals a glycosylation abnormality, but not the specific defect. An abnormal trasferrin glycosylation pattern suggests that the defect is in either genes that synthesize and add the precursor glycan (Glc(3)Man(9)GlcNAc(2)) to proteins (Type I) or genes that process the protein-bound N-glycans (Type II). Type I defects create unoccupied glycosylation sites, while Type II defects give fully occupied sites with abnormally processed glycans. These types are expected to be mutually exclusive, but a group of patients is now emerging who have variable coagulopathy and hypoglycemia together with a combination of Type I and Type II transferrin features. This surprising finding makes identifying their defects more challenging, but the defects and associated clinical manifestations of these patients suggest that the N-glycosylation pathway has some secrets left to share.     

The evolving genetic landscape of congenital disorders of glycosylation

Congenital Disorders of Glycosylation (CDG) are an expanding and complex group of rare genetic disorders caused by defects in the glycosylation of proteins and lipids. The genetic spectrum of CDG is extremely broad with mutations in over 140 genes leading to a wide variety of symptoms ranging from mild to severe and life-threatening. There has been an expansion in the genetic complexity of CDG in recent years. More specifically several examples of alternate phenotypes in recessive forms of CDG and new types of CDG following an autosomal dominant inheritance pattern have been identified. In addition, novel genetic mechanisms such as expansion repeats have been reported and several already known disorders have been classified as CDG as their pathophysiology was better elucidated. Furthermore, we consider the future and outlook of CDG genetics, with a focus on exploration of the non-coding genome using whole genome sequencing, RNA-seq and multi-omics technology.     

Laboratory diagnosis of congenital disorders of glycosylation type I by analysis of transferrin glycoforms

Congenital disorders of glycosylation (CDG) are being recognized as a rapidly growing and complex group of disorders. The pathophysiology results from depressed synthesis or remodeling of oligosaccharide moieties of glycoproteins. The ultimate result is the formation of abnormal glycoproteins affecting their structure and metabolic functions. The most thoroughly studied subset of CDG are the type I defects affecting N-glycosylation. Causal mutations occur in at least 12 different genes which encode primarily monosaccharide transferases necessary for N-glycosylation in the endoplasmic reticulum. The broad clinical presentation of these glycosylation defects challenge clinicians to test for these defects in a variety of clinical settings. The first described CDG was a phosphomannomutase deficiency (CDG-Ia). The original method used to define the glycosylation defect was isoelectric focusing (IEF) of transferrin. More recently, the use of other charge separation methods and electrospray-mass spectrometry (ESI-MS) has proven valuable in detecting type I CDG defects. By mass resolution, the under-glycosylation of transferrin is characterized as the total absence of one or both N-linked oligosaccharide. Beyond providing a new understanding of the structure of transferrin in type I CDG patients, it is adaptable to high throughput serum analysis. The use of transferrin under-glycosylation to detect the type I CDG provides limited insight into the specific site of the defect in oligosaccharide assembly since its value is constrained to observation of the final product of glycoprotein synthesis. New analytical targets and tools are converging with the clinical need for diagnosis of CDG. Defining the biosynthetic sites responsible for specific CDG phenotypes is in progress, and ten more type I defects have been putatively identified. This review discusses current methods, such as IEF and targeted proteomics using mass spectrometry, that are used routinely to test for type I CDG disorders, along with some newer approaches to define the defective synthetic sites responsible for the type I CDG defects. All diagnostic endeavors are followed by the quest for a reliable treatment. The isolated success of CDG-Ib treatment will be described with the hope that this may expand to other type I CDG disorders.     

Hypoglycosylation with increased fucosylation and branching of serum transferrin N-glycans in untreated galactosemia

Untreated classic galactosemia (galactose-1-phosphate uridyltransferase [GALT] deficiency) is known as a secondary congenital disorders of glycosylation (CDG) characterized by galactose deficiency of glycoproteins and glycolipids (processing defect or CDG-II). The mechanism of this undergalactosylation has not been established. Here we show that in untreated galactosemia, there is also a partial deficiency of whole glycans of serum transferrin associated with increased fucosylation and branching as seen in genetic glycosylation assembly defects (CDG-I). Thus galactosemia seems to be a secondary "dual" CDG causing a processing as well as an assembly N-glycosylation defect. We also demonstrated that in galactosemia patients, transferrin N-glycan biosynthesis is restored upon dietary treatment.     

Improvement of dolichol-linked oligosaccharide biosynthesis by the squalene synthase inhibitor zaragozic acid

The majority of congenital disorders of glycosylation (CDG) are caused by defects of dolichol (Dol)-linked oligosaccharide assembly, which lead to under-occupancy of N-glycosylation sites. Most mutations encountered in CDG are hypomorphic, thus leaving residual activity to the affected biosynthetic enzymes. We hypothesized that increased cellular levels of Dol-linked substrates might compensate for the low biosynthetic activity and thereby improve the output of protein N-glycosylation in CDG. To this end, we investigated the potential of the squalene synthase inhibitor zaragozic acid A to redirect the flow of the polyisoprene pathway toward Dol by lowering cholesterol biosynthesis. The addition of zaragozic acid A to CDG fibroblasts with a Dol-P-Man synthase defect led to the formation of longer Dol-P species and to increased Dol-P-Man levels. This treatment was shown to decrease the pathologic accumulation of incomplete Dol pyrophosphate-GlcNAc(2)Man(5) in Dol-P-Man synthase-deficient fibroblasts. Zaragozic acid A treatment also decreased the amount of truncated protein N-linked oligosaccharides in these CDG fibroblasts. The increased cellular levels of Dol-P-Man and possibly the decreased cholesterol levels in zaragozic acid A-treated cells also led to increased availability of the glycosylphosphatidylinositol anchor as shown by the elevated cell-surface expression of the CD59 protein. This study shows that manipulation of the cellular Dol pool, as achieved by zaragozic acid A addition, may represent a valuable approach to improve N-linked glycosylation in CDG cells.     

Hypoglycosylation due to dolichol metabolism defects

Dolichol phosphate is a lipid carrier embedded in the endoplasmic reticulum (ER) membrane essential for the synthesis of N-glycans, GPI-anchors and protein C- and O-mannosylation. The availability of dolichol phosphate on the cytosolic site of the ER is rate-limiting for N-glycosylation. The abundance of dolichol phosphate is influenced by its de novo synthesis and the recycling of dolichol phosphate from the luminal leaflet to the cytosolic leaflet of the ER. Enzymatic defects affecting the de novo synthesis and the recycling of dolichol phosphate result in glycosylation defects in yeast or cell culture models, and are expected to cause glycosylation disorders in humans termed congenital disorders of glycosylation (CDG). Currently only one disorder affecting the dolichol phosphate metabolism has been described. In CDG-Im, the final step of the de novo synthesis of dolichol phosphate catalyzed by the enzyme dolichol kinase is affected. The defect causes a severe phenotype with death in early infancy. The present review summarizes the biosynthesis of dolichol-phosphate and the recycling pathway with respect to possible defects of the dolichol phosphate metabolism causing glycosylation defects in humans.     

Identification and functional analysis of a defect in the human ALG9 gene: definition of congenital disorder of glycosylation type IL

Defects of lipid-linked oligosaccharide assembly lead to alterations of N-linked glycosylation known as "type I congenital disorders of glycosylation" (CDG). Dysfunctions along this stepwise assembly pathway are characterized by intracellular accumulation of intermediate lipid-linked oligosaccharides, the detection of which contributes to the identification of underlying enzymatic defects. Using this approach, we have found, in a patient with CDG, a deficiency of the ALG9 alpha 1,2 mannosyltransferase enzyme, which causes an accumulation of lipid-linked-GlcNAc(2)Man(6) and -GlcNAc(2)Man(8) structures, which was paralleled by the transfer of incomplete oligosaccharides precursors to protein. A homozygous point-mutation 1567G-->A (amino acid substitution E523K) was detected in the ALG9 gene. The functional homology between the human ALG9 and Saccharomyces cerevisiae ALG9, as well as the deleterious effect of the E523K mutation detected in the patient with CDG, were confirmed by a yeast complementation assay lacking the ALG9 gene. The ALG9 defect found in the patient with CDG--who presented with developmental delay, hypotonia, seizures, and hepatomegaly--shows that efficient lipid-linked oligosaccharide synthesis is required for proper human development and physiology. The ALG9 defect presented here defines a novel form of CDG named "CDG-IL."     

How Golgi glycosylation meets and needs trafficking: the case of the COG complex

Protein glycosylation is one of the major biosynthetic functions occurring in the endoplasmic reticulum and Golgi compartments. It requires an amazing number of enzymes, chaperones, lectins and transporters whose actions delicately secure the fidelity of glycan structures. Over the past 30 years, glycobiologists hammered that glycan structures are not mere decorative elements but serve crucial cellular functions. This becomes dramatically illustrated by a group of mostly severe, inherited human disorders named congenital disorders of glycosylation (CDG). To date, many types of CDG have been defined genetically and most of the time the defects impair the biosynthesis, transfer and remodeling of N-glycans. Recently, the identification of the several types of CDG caused by deficiencies in the conserved oligomeric Golgi (COG) complex, a complex involved in vesicular Golgi trafficking, expanded the field of CDG but also brought novel insights in glycosylation. The molecular mechanisms underlying the complex pathway of N-glycosylation in the Golgi are far from understood. The availability of COG-deficient CDG patients and patients' cells offered a new way to study how COG, and its different subunits, could influence the Golgi N-glycosylation machinery and localization. This review summarizes the recent findings on the implication of COG in Golgi glycosylation. It highlights the need for a dynamic, finely tuned balance between anterograde and retrograde trafficking for the correct localization of Golgi enzymes to assure the stepwise maturation of N-glycan chains.     

A combined defect in the biosynthesis of N- and O-glycans in patients with cutis laxa and neurological involvement: the biochemical characteristics

Based on our preliminary observation of abnormal glycosylation in a cutis laxa patient, nine cutis laxa patients were analyzed for congenital defects of glycosylation (CDG). Isoelectric focusing of plasma transferrin and apolipoproteinC-III showed that three out of nine patients had a defect in the biosynthesis of N-glycans and core 1 mucin type O-glycans, respectively. Mass spectrometric N-glycan analyses revealed a relative increase of glycans lacking sialic acid and glycans lacking sialic acid and galactose residues. Mutation analysis of the fibulin-5 gene (FBLN5), which has been reported in cases of autosomal recessive cutis laxa, revealed no mutations in the patients' DNA. Evidence is presented that extracellular matrix (ECM) proteins of skin are likely to be highly glycosylated with N- and/or mucin type O-glycans by using algorithms for predicting glycosylation. The conclusions in this study were that the clinical phenotype of autosomal recessive cutis laxa seen in three patients is not caused by mutations in the FBLN5 gene. Our findings define a novel form of CDG with cutis laxa and neurological involvement due to a defect in the sialylation and/or galactosylation of N- and O-glycans. Improper glycosylation of ECM proteins of skin may form the pathophysiological basis for the cutis laxa phenotype.     

Neural and synaptic defects in slytherin, a zebrafish model for human congenital disorders of glycosylation

Congenital disorder of glycosylation type IIc (CDG IIc) is characterized by mental retardation, slowed growth and severe immunodeficiency, attributed to the lack of fucosylated glycoproteins. While impaired Notch signaling has been implicated in some aspects of CDG IIc pathogenesis, the molecular and cellular mechanisms remain poorly understood. We have identified a zebrafish mutant slytherin (srn), which harbors a missense point mutation in GDP-mannose 4,6 dehydratase (GMDS), the rate-limiting enzyme in protein fucosylation, including that of Notch. Here we report that some of the mechanisms underlying the neural phenotypes in srn and in CGD IIc are Notch-dependent, while others are Notch-independent. We show, for the first time in a vertebrate in vivo, that defects in protein fucosylation leads to defects in neuronal differentiation, maintenance, axon branching, and synapse formation. Srn is thus a useful and important vertebrate model for human CDG IIc that has provided new insights into the neural phenotypes that are hallmarks of the human disorder and has also highlighted the role of protein fucosylation in neural development.     

DDOST mutations identified by whole-exome sequencing are implicated in congenital disorders of glycosylation

Congenital disorders of glycosylation (CDG) are inherited autosomal-recessive diseases that impair N-glycosylation. Approximately 20% of patients do not survive beyond the age of 5 years old as a result of widespread organ dysfunction. Although most patients receive a CDG diagnosis based on abnormal glycosylation of transferrin, this test cannot provide a genetic diagnosis; indeed, many patients with abnormal transferrin do not have mutations in any known CDG genes. Here, we combined biochemical analysis with whole-exome sequencing (WES) to identify the genetic defect in an untyped CDG patient, and we found a 22 bp deletion and a missense mutation in DDOST, whose product is a component of the oligosaccharyltransferase complex that transfers the glycan chain from a lipid carrier to nascent proteins in the endoplasmic reticulum lumen. Biochemical analysis with three biomarkers revealed that N-glycosylation was decreased in the patient's fibroblasts. Complementation with wild-type-DDOST cDNA in patient fibroblasts restored glycosylation, indicating that the mutations were pathological. Our results highlight the power of combining WES and biochemical studies, including a glyco-complementation system, for identifying and confirming the defective gene in an untyped CDG patient. This approach will be very useful for uncovering other types of CDG as well.     

Differential effects of lobe A and lobe B of the Conserved Oligomeric Golgi complex on the stability of {beta}1,4-galactosyltransferase 1 and {alpha}2,6-sialyltransferase 1

Initially described by Jaeken et al. in 1980, congenital disorders of glycosylation (CDG) is a rapidly expanding group of human multisystemic disorders. To date, many CDG patients have been identified with deficiencies in the conserved oligomeric Golgi (COG) complex which is a complex involved in the vesicular intra-Golgi retrograde trafficking. Composed of eight subunits that are organized in two lobes, COG subunit deficiencies have been associated with Golgi glycosylation abnormalities. Analysis of the total serum N-glycans of COG-deficient CDG patients demonstrated an overall decrease in terminal sialylation and galactosylation. According to the mutated COG subunits, differences in late Golgi glycosylation were observed and led us to address the question of an independent role and requirement for each of the two lobes of the COG complex in the stability and localization of late terminal Golgi glycosylation enzymes. For this, we used a small-interfering RNAs strategy in HeLa cells stably expressing green fluorescent protein (GFP)-tagged β1,4-galactosyltransferase 1 (B4GALT1) and α2,6-sialyltransferase 1 (ST6GAL1), two major Golgi glycosyltransferases involved in late Golgi N-glycosylation. Using fluorescent lectins and flow cytometry analysis, we clearly demonstrated that depletion of both lobes was associated with deficiencies in terminal Golgi N-glycosylation. Lobe A depletion resulted in dramatic changes in the Golgi structure, whereas lobe B depletion severely altered the stability of B4GALT1 and ST6GAL1. Only MG132 was able to rescue their steady-state levels, suggesting that B4GALT1- and ST6GAL1-induced degradation are likely the consequence of an accumulation in the endoplasmic reticulum (ER), followed by a retrotranslocation into the cytosol and proteasomal degradation. All together, our results suggest differential effects of lobe A and lobe B for the localization/stability of B4GALT1 and ST6GAL1. Lobe B would be crucial in preventing these two Golgi glycosyltransferases from inappropriate retrograde trafficking to the ER, whereas lobe A appears to be essential for maintaining the overall Golgi structure.     

Deficiency of Dol-P-Man Synthase Subunit DPM3 Bridges the Congenital Disorders of Glycosylation with the Dystroglycanopathies

Alpha-dystroglycanopathies such as Walker Warburg syndrome represent an important subgroup of the muscular dystrophies that have been related to defective O-mannosylation of alpha-dystroglycan. In many patients, the underlying genetic etiology remains unsolved. Isolated muscular dystrophy has not been described in the congenital disorders of glycosylation (CDG) caused by N-linked protein glycosylation defects. Here, we present a genetic N-glycosylation disorder with muscular dystrophy in the group of CDG type I. Extensive biochemical investigations revealed a strongly reduced dolichol-phosphate-mannose (Dol-P-Man) synthase activity. Sequencing of the three DPM subunits and complementation of DPM3-deficient CHO2.38 cells showed a pathogenic p.L85S missense mutation in the strongly conserved coiled-coil domain of DPM3 that tethers catalytic DPM1 to the ER membrane. Cotransfection experiments in CHO cells showed a reduced binding capacity of DPM3(L85S) for DPM1. Investigation of the four Dol-P-Man-dependent glycosylation pathways in the ER revealed strongly reduced O-mannosylation of alpha-dystroglycan in a muscle biopsy, thereby explaining the clinical phenotype of muscular dystrophy. This mild Dol-P-Man biosynthesis defect due to DPM3 mutations is a cause for alpha-dystroglycanopathy, thereby bridging the congenital disorders of glycosylation with the dystroglycanopathies.     

Update and perspectives on congenital disorders of glycosylation.

Defects in nine genes of the N-linked glycosylation pathway cause congenital disorders of glycosylation (CDGs) and serious medical consequences. Although glycobiology is seldom featured in a general medical education, an increasing number of physicians are becoming acquainted with the field because it directly impacts patient diagnosis and care. Medical practice and attitudes will change in the postgenomic era, and glycobiology has an opportunity to be a cornerstone of part of that new perspective. This review of recent developments in the CDG field describes the biochemical and molecular basis of these disorders, describes successful experimental approaches, and points out a few perspectives on current problems. The broad, multisystemic presentations of these patients emphasize that glycobiology is very much a general medical science, cutting across many traditional medical specialties. The glycobiology community is well poised to provide novel perspectives for the dedicated clinicians treating both well-known and emerging human diseases.     

Glycosylation disorders of membrane trafficking

During evolution from prokaryotic to eukaryotic cells, compartmentalization of cellular functions has been achieved with a high degree of complexity. Notably, all secreted and transmembrane proteins travel through endoplasmic reticulum (ER) and Golgi apparatus, where they are synthesized, folded and subjected to covalent modifications, most particularly glycosylation. N-glycosylation begins in the ER with synthesis and transfer of glycan onto nascent protein and proceeds in Golgi apparatus where maturation occurs. This process not only requires the precise localization of glycosyltransferases, glycosidases and substrates but also an efficient, finely regulated and bidirectional vesicular trafficking among membrane-enclosed organelles. Basically, it is no surprise that alterations in membrane transport or related pathways can lead to glycosylation abnormalities. During the last few years, this has particularly been highlighted in genetic diseases called CDG (Congenital Disorders of Glycosylation). Alterations in mechanisms of vesicle formation due to COPII coat component SEC23B deficiency, or in vesicles tethering, caused by defects of the COG complex, but also impaired Golgi pH homeostasis due to ATP6V0A2 defects have been discovered in CDG patients. This mini review will summarize these fascinating discoveries.