Clinical diagnostics and therapy monitoring in the congenital disorders of glycosylation

Abnormal protein glycosylation is observed in many common disorders like cancer, inflammation, Alzheimer’s disease and diabetes. However, the actual use of this information in clinical diagnostics is still very limited. Information is usually derived from analysis of total serum N-glycan profiling methods, whereas the current use of glycoprotein biomarkers in the clinical setting is commonly based on protein levels. It can be envisioned that combining protein levels and their glycan isoforms would increase specificity for early diagnosis and therapy monitoring. To establish diagnostic assays, based on the mass spectrometric analysis of protein-specific glycosylation abnormalities, still many technical improvements have to be made. In addition, clinical validation is equally important as well as an understanding of the genetic and environmental factors that determine the protein-specific glycosylation abnormalities. Important lessons can be learned from the group of monogenic disorders in the glycosylation pathway, the Congenital Disorders of Glycosylation (CDG). Now that more and more genetic defects are being unraveled, we start to learn how genetic factors influence glycomics profiles of individual and total serum proteins. Although only in its initial stages, such studies suggest the importance to establish diagnostic assays for protein-specific glycosylation profiling, and the need to look beyond the single glycoprotein diagnostic test. Here, we review progress in and lessons from genetic disease, and review the increasing opportunities of mass spectrometry to analyze protein glycosylation in the clinical diagnostic setting. Furthermore, we will discuss the possibilities to expand current CDG diagnostics and how this can be used to approach glycoprotein biomarkers for more common diseases.     

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.     

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.     

CDG biochemical screening: Where do we stand?

BackgroundGlycosylation is one of the most complex post-translational modifications of proteins and lipids, notably requiring many glycosyltransferases, glycosidases and sugar transporters encoded by about 1–2% of all human genes. Deleterious variants in any of them may result in improper protein or lipid glycosylation, thus yielding the so-called ‘congenital disorders of glycosylation’ or CDG.Scope of reviewWe first review the current state of knowledge on the common blood and cellular glycoproteins used in the biochemical screening of CDG, as well as the emerging ones for an improved diagnosis. We then provide an overview of the current state-of-the-art methodologies ranging from gel electrophoresis to mass spectrometry to measure improper glycosylation. Finally, we discuss how additional tools such as metabolomics and microfluidics can be added to the current toolbox to better diagnose and delineate CDG.Major ConclusionsCombining several biochemical indicators and related methods is often required to cope with the large clinical heterogeneity of CDG and establish a definitive diagnosis.General significanceThis review aims to critically present current available CDG biochemical biomarkers and dedicated methods in the context of highly diverse glycosylation pathways and related inherited diseases.     

Glycosylation site prediction using ensembles of Support Vector Machine classifiers

Background  

Glycosylation is one of the most complex post-translational modifications (PTMs) of proteins in eukaryotic cells. Glycosylation plays an important role in biological processes ranging from protein folding and subcellular localization, to ligand recognition and cell-cell interactions. Experimental identification of glycosylation sites is expensive and laborious. Hence, there is significant interest in the development of computational methods for reliable prediction of glycosylation sites from amino acid sequences.     

Therapies and therapeutic approaches in Congenital Disorders of Glycosylation

Inborn errors in glycoconjugate biosynthesis termed ‘Congenital Disorders of Glycosylation’ (CDG) comprise a rapidly expanding group of metabolic diseases in man. Up till now more than 60 different inherited disorders in N- and O-glycosylation pathways have been identified. They affect the biosynthesis of glycan moieties linked to proteins as well as lipids. Due to failures in protein glycosylation, CDG patients suffer from multi systemic disorders, which mostly present with severe psychomotor and mental retardations, muscular impairment, ataxia, failure to thrive and developmental delay. Although improved biochemical and genetic investigations led to identification of a variety of new molecular defects in glycoconjugate biosynthesis, effective therapies for most types of the CDG are so far not available. Therefore, intensive investigations on treatment options for this group of diseases have been carried out in recent years.     

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.     

A rapid mass spectrometric strategy for the characterization of N- and O-glycan chains in the diagnosis of defects in glycan biosynthesis

Glycosylation of proteins is a very complex process which involves numerous factors such as enzymes or transporters. A defect in one of these factors in glycan biosynthetic pathways leads to dramatic disorders named congenital disorders of glycosylation (CDG). CDG can affect the biosynthesis of not only protein N-glycans but also O-glycans. The structural analysis of glycans on serum glycoproteins is essential to solving the defect. For this reason, we propose in this paper a strategy for the simultaneous characterization of both N- and O-glycan chains isolated from the serum glycoproteins. The serum (20 microL) is used for the characterization of N-glycans which are released by enzymatic digestion with PNGase F. O-glycans are chemically released by reductive elimination from whole serum glycoproteins using 10 microL of the serum. Using strategies based on mass spectrometric analysis, the structures of N- and O-glycan chains are defined. These strategies were applied on the sera from one patient with CDG type IIa, and one patient with a mild form of congenital disorder of glycosylation type II (CDG-II) that is caused by a deficiency in the Cog1 subunit of the complex.     

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.     

More than just sugars: Conserved oligomeric Golgi complex deficiency causes glycosylation‐independent cellular defects

The conserved oligomeric Golgi (COG) complex controls membrane trafficking and ensures Golgi homeostasis by orchestrating retrograde vesicle trafficking within the Golgi. Human COG defects lead to severe multisystemic diseases known as COG‐congenital disorders of glycosylation (COG‐CDG). To gain better understanding of COG‐CDGs, we compared COG knockout cells with cells deficient to 2 key enzymes, Alpha‐1,3‐mannosyl‐glycoprotein 2‐beta‐N‐acetylglucosaminyltransferase and uridine diphosphate‐glucose 4‐epimerase (GALE), which contribute to proper N‐ and O‐glycosylation. While all knockout cells share similar defects in glycosylation, these defects only account for a small fraction of observed COG knockout phenotypes. Glycosylation deficiencies were not associated with the fragmented Golgi, abnormal endolysosomes, defective sorting and secretion or delayed retrograde trafficking, indicating that these phenotypes are probably not due to hypoglycosylation, but to other specific interactions or roles of the COG complex. Importantly, these COG deficiency specific phenotypes were also apparent in COG7‐CDG patient fibroblasts, proving the human disease relevance of our CRISPR knockout findings. The knowledge gained from this study has important implications, both for understanding the physiological role of COG complex in Golgi homeostasis in eukaryotic cells, and for better understanding human diseases associated with COG/Golgi impairment.      

Towards automation of glycomic profiling of complex biological materials

Glycosylation is considered one of the most complex and structurally diverse post-translational modifications of proteins. Glycans play important roles in many biological processes such as protein folding, regulation of protein stability, solubility and serum half-life. One of the ways to study glycosylation is systematic structural characterizations of protein glycosylation utilizing glycomics methodology based around mass spectrometry (MS). The most prevalent bottleneck stages for glycomic analyses is laborious sample preparation steps. Therefore, in this study, we aim to improve sample preparations by automation. We recently demonstrated the successful application of an automated high-throughput (HT), glycan permethylation protocol based on 96-well microplates, in the analysis of purified glycoproteins. Therefore, we wanted to test if these developed HT methodologies could be applied to more complex biological starting materials. Our automated 96-well-plate based permethylation method showed very comparable results with established glycomic methodology. Very similar glycomic profiles were obtained for complex glycoprotein/protein mixtures derived from heterogeneous mouse tissues. Automated N-glycan release, enrichment and automated permethylation of samples proved to be convenient, robust and reliable. Therefore we conclude that these automated procedures are a step forward towards the development of a fully automated, fast and reliable glycomic profiling system for analysis of complex biological materials.     

Protein glycosylation in bacterial mucosal pathogens

In eukaryotes, glycosylated proteins are ubiquitous components of extracellular matrices and cellular surfaces. Their oligosaccharide moieties are implicated in a wide range of cell-cell and cell-matrix recognition events that are required for biological processes ranging from immune recognition to cancer development. Glycosylation was previously considered to be restricted to eukaryotes; however, through advances in analytical methods and genome sequencing, there have been increasing reports of both O-linked and N-linked protein glycosylation pathways in bacteria, particularly amongst mucosal-associated pathogens. Studying glycosylation in relatively less-complicated bacterial systems provides the opportunity to elucidate and exploit glycoprotein biosynthetic pathways. We will review the genetic organization, glycan structures and function of glycosylation systems in mucosal bacterial pathogens, and speculate on how this knowledge may help us to understand glycosylation processes in more complex eukaryotic systems and how it can be used for glycoengineering.     

The cellular and molecular events of central nervous system remyelination

Central nervous system (CNS)* regeneration is a subject of great interest, particularly in diseases causing a dramatic loss of neurons. However, some CNS diseases do not affect neurons but damage other cells, such as the myelin-forming cells--called oligodendrocytes--which are also crucial to the harmonious function of the nervous system. Diseases in which oligodendrocytes and myelin are attacked can cause devastating neurological dysfunction which is sometimes followed by recovery and myelin repair or remyelination. The question of the regeneration potential of oligodendrocytes in experimental and human demyelinating diseases such as multiple sclerosis has been debated for a long time. Present evidence suggests that oligodendrocyte precursor cells persist in the adult CNS and that oligodendrocyte regeneration can occur but may be limited by ongoing disease processes. Here we will briefly review recent advances which have broadened our understanding of the cellular and molecular events of CNS remyelination.     

Detailed glycan analysis of serum glycoproteins of patients with congenital disorders of glycosylation indicates the specific defective glycan processing step and provides an insight into pathogenesis

The fundamental importance of correct protein glycosylation is abundantly clear in a group of diseases known as congenital disorders of glycosylation (CDGs). In these diseases, many biological functions are compromised, giving rise to a wide range of severe clinical conditions. By performing detailed analyses of the total serum glycoproteins as well as isolated transferrin and IgG, we have directly correlated aberrant glycosylation with a faulty glycosylation processing step. In one patient the complete absence of complex type sugars was consistent with ablation of GlcNAcTase II activity. In another CDG type II patient, the identification of specific hybrid sugars suggested that the defective processing step was cell type-specific and involved the mannosidase III pathway. In each case, complementary serum proteome analyses revealed significant changes in some 31 glycoproteins, including components of the complement system. This biochemical approach to charting diseases that involve alterations in glycan processing provides a rapid indicator of the nature, severity, and cell type specificity of the suboptimal glycan processing steps; allows links to genetic mutations; indicates the expression levels of proteins; and gives insight into the pathways affected in the disease process.     

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.     

The use of mass spectrometry for the proteomic analysis of glycosylation

Of all protein PTMs, glycosylation is by far the most common, and is a target for proteomic research. Glycosylation plays key roles in controlling various cellular processes and the modifications of the glycan structures in diseases highlight the clinical importance of this PTM. Glycosylation analysis remains a difficult task. MS, in combination with modern separation methodologies, is one of the most powerful and versatile techniques for the structural analysis of glycoconjugates. This review describes methodologies based on MS for detailed characterization of glycoconjugates in complex biological samples at the sensitivity required for proteomic work.     

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.     

Glycobiology of neuromuscular disorders

There has been a recent explosion in the identification of neuromuscular diseases caused by mutations in genes that affect carbohydrate metabolism or protein glycosylation. A number of these findings relate to defects in the glycosylation of alpha dystroglycan. Alpha dystroglycan is an essential component of the dystrophin-glycoprotein complex, and aberrant glycosylation of alpha dystroglycan is associated with multiple forms of muscular dystrophy in mice and humans. We review the evidence that defects in dystroglycan glycosylation cause muscular dystrophy. In addition, we review evidence that glycobiology is important in other disorders that affect muscle, including hereditary inclusion body myopathy type II and congenital disorders of glycosylation. Finally, we discuss the long-term potential of glycotherapies for muscle disorders.     

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.     

Altered glycan structures: the molecular basis of congenital disorders of glycosylation

Congenital disorders of glycosylation (CDG) are a group of diseases that affect glycoprotein biogenesis. Eighteen different types of CDG have been defined genetically. They result from deficiencies in either the biosynthesis of oligosaccharide precursors or specific steps of N-glycan assembly, resulting in the absence or structural alteration of N-glycan chains. These diseases have a broad range of clinical phenotypes and affect nearly every organ system, with special emphasis on normal brain development and the multiple functions of the nervous, hepatic, gastrointestinal and immune systems. Although most of the deficiencies observed in CDG patients are only partial, the severity of the clinical manifestations signifies the relevance of protein N-glycosylation and shows the importance of defined glycan structures.