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.     

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.     

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.     

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.     

Diagnosis of congenital disorders of glycosylation type-I using protein chip technology

A method for the diagnosis of the congenital disorders of glycosylation type I (CDG-I) by SELDI-TOF-MS of serum transferrin immunocaptured on protein chip arrays is described. The underglycosylation of glycoproteins in CDG-I produces glycoforms of transferrin with masses lower than that of the normal fully glycosylated transferrin. Immobilisation of antitransferrin antibodies on reactive-surface protein chip arrays (RS100) selectively enriched transferrin by at least 100-fold and allowed the detection of patterns of transferrin glycoforms by SELDI-TOF-MS using approximately 0.3 microL of serum/plasma. Abnormal patterns of immunocaptured transferrin were detected in patients with known defects in glycosylation (CDG-Ia, CDG-Ib, CDG-Ic, CDG-If and CDG-Ih) and in patients in whom the basic defect has not yet been identified (CDG-Ix). The correction of the N-glycosylation defect in a patient with CDG-Ib after mannose therapy was readily detected. A patient who had an abnormal transferrin profile by IEF but a normal profile by SELDI-TOF-MS analysis was shown to have an amino acid polymorphism by sequencing transferrin by quadrupole-TOF MS. Complete agreement was obtained between analysis of immunocaptured transferrin by SELDI-TOF-MS and the IEF profile of transferrin, the clinical severity of the disease and the levels of aspartylglucosaminidase activity (a surrogate marker for the diagnosis of CDG-I). SELDI-TOF-MS of transferrin immunocaptured on protein chip arrays is a highly sensitive diagnostic method for CDG-I, which could be fully automated using microtitre plates and robotics.     

Patients with unsolved congenital disorders of glycosylation type II can be subdivided in six distinct biochemical groups

Defects in the biosynthesis of N- and core 1 O-glycans may be found by isoelectric focusing (IEF) of plasma transferrin and apolipoprotein C-III (apoC-III). We hypothesized that IEF of transferrin and apoC-III in combination with sodium dodecyl sulphate-polyacrylamide gel electrophoresis (SDS-PAGE) of apoC-III may provide a classification for congenital disorders of glycosylation (CDG) patients. We analyzed plasma from 22 patients with eight different and well-characterized CDG subtypes and 19 cases with unsolved CDG. Transferrin IEF (TIEF) has been used to distinguish between N-glycan assembly (type 1 profile) and processing (type 2 profile) defects. We differentiated two different CDG type 2 TIEF profiles: The "asialo profile" characterized by elevated levels of asialo- and monosialotransferrin and the "disialo profile" characterized by increased levels of disialo- and trisialotransferrin. ApoC-III IEF gave two abnormal profiles ("apoC-III(0)" and "apoC-III(1)" profiles). The results for the eight established CDG forms exactly matched the theoretical expectations, providing a validation for the study approach. The combination of the three electrophoretic techniques was not additionally informative for the CDG-Ix patients as they had normal apoC-III IEF patterns. However, the CDG-IIx patients could be further subdivided into six biochemical subgroups. The robustness of the methodology was supported by the fact that three patients with similar clinical features ended in the same subgroup and that another patient, classified in the "CDG-IIe subgroup," turned out to have a similar defect. Dividing the CDG-IIx patients in six subgroups narrows down drastically the options of the primary defect in each of the subgroups and will be helpful to define new CDG type II defects.     

Glycoproteomics of N-glycosylation by in-gel deglycosylation and matrix-assisted laser desorption/ionisation-time of flight mass spectrometry mapping: application to congenital disorders of glycosylation

A general strategy for the structural evaluation of N-glycosylation, a common post-translational protein modification, is presented. The methods for the release of N-linked glycans from the gel-separated proteins, their isolation, purification and matrix-assisted laser desorption/ionisation-mass spectrometry (MALDI-MS) analysis of their mixtures were optimised. Since many glycoproteins are available only at low quantities from sodium dodecyl sulphate-polyacrylamide gel electrophoresis or two-dimensional gels, high attention was paid to obtain N-glycan mixtures representing their actual composition in human plasma by in-gel deglycosylation. The relative sensitivity of solid MALDI matrices for MS analysis of acidic N-glycans was compared. The most favourable results for native acidic N-glycans were obtained with 2,4,6-trihydroxyacetophenone monohydrate/diammoniumcitrate as a matrix. This matrix provided good results for both neutral and acidic mixtures as well as for methylated N-glycans. In the second part of this paper the potential of such an optimised MS strategy alone or in combination with high pH anion-exchange chromatography profiling for the clinical diagnosis of congenital disorders of glycosylation is presented.     

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.     

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.     

Multi-allelic origin of congenital disorder of glycosylation (CDG)-Ic

Congenital disorders of glycosylation (CDG), formerly known as carbohydrate-deficient glycoprotein syndrome, represent a family of genetic diseases with variable clinical presentations. Common to all types of CDG characterized to date is a defective Asn-linked glycosylation caused by enzymatic defects of N-glycan synthesis. Previously, we have identified a mutation in the ALG6 alpha1,3 glucosyltransferase gene as the cause of CDG-Ic in four related patients. Here, we present the identification of seven additional cases of CDG-Ic among a group of 35 untyped CDG patients. Analysis of lipid-linked oligosaccharides in fibroblasts confirmed the accumulation of dolichyl pyrophosphate-Man9GlcNAc2 in the CDG-Ic patients. The genomic organization of the human ALG6 gene was determined, revealing 14 exons spread over 55 kb. By polymerase chain reaction amplification and sequencing of ALG6 exons, three mutations, in addition to the previously described A333 V substitution, were detected in CDG-Ic patients. The detrimental effect of these mutations on ALG6 activity was confirmed by complementation of alg6 yeast mutants. Haplotype analysis of CDG-Ic patients revealed a founder effect for the ALG6 allele bearing the A333 V mutation. Although more than 80% of CDG are type Ia, CDG-Ic may be the second most common form of the disease.     

Characterization of human transferrin glycoforms by capillary electrophoresis and electrospray ionization mass spectrometry

Carbohydrate Deficient Glycoprotein Syndrome (CDGS) is an inherited metabolic disease affecting all parts of the body. The biochemical diagnosis of this syndrome is based on the presence of a special marker in blood, Carbohydrate Deficient Transferrin (CDT), which is also a marker of chronic alcohol abuse. CDT is characterized by abnormal glycoforms of serum transferrin (Tf). In the present study, electrophoretic separation of human serum transferrin glycoforms was carried out using a bare fused-silica capillary and the glycoforms present in commercial Tf were baseline separated. The limit of detection (LOD) of human Tf was around the nmol concentration range. The LOD of the trisialo- and disialo-Tf, expressed as percentages of the tetrasialo-Tf peak area, were 0.5% for trisialo-Tf and 0.4% for disialo-Tf, and these values were appropriate for CDGS diagnosis. Moreover, Tf glycoforms were characterized using mass spectrometry (MS). The method was applied to the analysis of normal and pathological serum samples, after dilution. The results obtained suggest a way of making a rapid and simple CDGS diagnosis.     

An unbiased approach for analysis of protein glycosylation and application to influenza vaccine hemagglutinin

Here a mass spectrometry-based platform for the analysis of glycoproteins is presented. Glycopeptides and released glycans are analyzed, the former by quadrupole orthogonal time-of-flight liquid chromatography/mass spectrometry (QoTOF LC/MS) and the latter by permethylation analysis using matrix-assisted laser desorption/ionization (MALDI)–TOF MS. QoTOF LC/MS analysis reveals the stochastic distribution of glycoforms at occupied sequons, and the latter provides a semiquantitative assessment of overall protein glycosylation. Hydrophilic interaction chromatography (HILIC) was used for unbiased enrichment of glycopeptides and was validated using five model N-glycoproteins bearing a wide array of glycans, including high-mannose, complex, and hybrid subtypes such as sulfo and sialyl forms. Sialyl and especially sulfated glycans are difficult to analyze because these substitutions are labile. The conditions used here allow detection of these compounds quantitatively, intact, and in the context of overall glycosylation. As a test case, we analyzed influenza B/Malaysia/2506/2004 hemagglutinin, a component of the 2006–2007 influenza vaccine. It bears 11 glycosylation sites. Approximately 90% of its glycans are high mannose, and 10% are present as complex and hybrid types, including those with sulfate. The stochastic distribution of glycoforms at glycosylation sites is revealed. This platform should have wide applications to glycoproteins in basic sciences and industry because no apparent bias for any glycoforms is observed.     

Characterization of the glycosylation sites in cyclooxygenase-2 using mass spectrometry

Cyclooxygenase is involved in the biosynthesis and function of prostaglandins. It is a glycoprotein located in the endoplasmic reticulum and in the nuclear envelope, and it has been found to have two isoforms termed COX-1 and COX-2. This paper reports on the glycosylation site analysis of recombinant COX-2 using matrix-assisted laser desorption/ionization (MALDI) time-of-flight (TOF) mass spectrometry (MS) and nanoelectrospray (nanoESI) quadrupole-TOF (Q-TOF) MS. The nanoESI MS analysis of COX-2 revealed the presence of three glycoforms at average molecular masses of 71.4, 72.7, and 73.9 kDa. Each glycoform contained a number of peaks differing by 162 Da indicating heterogeneity and suggesting the presence of high-mannose sugars. The masses of the glycoforms indicate that oligosaccharides occupy two to four sites and a single N-acetylglucosamine (GlcNAc) residue occupied up to two sites. The MALDI MS analysis of a tryptic digest of the protein showed a number of potential glycopeptides. The peptides differed by 162 Da which further suggested high-mannose sugars. Nanoelectrospray MS/MS experiments confirmed glycosylation at the Asn 53 and Asn 130 sites and confirmed the presence of the peptides Asn 396-Arg 414 + GlcNAc and Thr 576-Arg 587 + GlcNAc containing Asn 580. It was not possible to conclusively determine whether the Asn 396 site was glycosylated via an MS/MS experiment, so the tryptic digest was deglycosylated to confirm the presence of the glycopeptides. Finally, a non-glycosylated tryptic peptide was observed containing the Asn 592.     

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.     

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.     

Heterogeneous glycosylation of immunoglobulin E constructs characterized by top-down high-resolution 2-D mass spectrometry

Posttranslational glycosylation is critical for biological function of many proteins, but its structural characterization is complicated by natural heterogeneity, multiple glycosylation sites, and different forms. Here, a top-down mass spectrometry (MS) characterization is applied to three constructs of the Fc segment of IgE: Fcepsilon(3-4) (52 kDa) and Fcepsilon(2-3-4)(2) (76 kDa) disulfide-bonded homodimers. Fourier transform MS of a reduced sample of Fcepsilon(2-3-4) gave molecular masses of 37 527, 37 689, 37 851, and 38 014 Da, directly characterizing multiple glycoforms (hexose = 162 Da) without chromatographic separation. Limited proteolysis of the nonreduced Fcepsilon(2-3-4)(2) protein yielded a peptide mixture with molecular weight values that agreed with those expected from the DNA sequence. The single glycosylation site in these constructs was identified, and quantities were determined of five glycoforms that agreed within +/-2% of the molecular ion values. The 2-D mass spectrum of two glycosylated peptides showed these to have high-mannose structures, -GlcNAc-(hex)(n)(), demonstrating that Fcepsilon(2-3-4) has a single such structure of n = 5-9. For a mutated sample of Fcepsilon(3-4), in addition to five glycoforms, MS showed a molecular discrepancy that could be assigned with proteolysis and 2-D mass spectra to the oxidation of two methionines and an additional residue difference.     

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.     

Sequence and glycosylation site identity of two distinct glycoforms of nonspecific cross-reacting antigen as demonstrated by sequence analysis and fast atom bombardment mass spectrometry

Nonspecific cross-reacting antigen (NCA) is a highly glycosylated membrane protein which is immunologically and structurally related to carcinoembryonic antigen, an important tumor-associated antigen. Two glycoforms of NCA were purified from a single liver metastasis of a colonic carcinoma and characterized with respect to their primary sequence and position of glycosylation sites. The two glycoforms (designated TEX (tumor-extracted antigen), Mr 75,000, and NCA, Mr 45,000) each showed a deglycosylated Mr of 35,000 and yielded identical peptide maps. The structural characterization of TEX and NCA and the assignment of glycosylation sites was performed by fast atom bombardment mass spectrometry and microsequence analysis of the resulting peptides. This approach showed that TEX and NCA were identical with respect to primary sequence and provided direct evidence that 11 of the 12 predicted asparagine-linked glycosylation sites were glycosylated in both TEX and NCA. Indirect evidence was obtained for glycosylation at the other site. Both glycoforms also contain ethanolamine linked to Gly-286, a finding consistent with the conclusion that these proteins are anchored to the plasma membrane through a glycosyl-phosphatidylinositol tail. The large difference in the molecular weights of glycosylated TEX and NCA suggests significant variations in their oligosaccharide structures.     

Modeling human congenital disorder of glycosylation type IIa in the mouse: conservation of asparagine-linked glycan-dependent functions in mammalian physiology and insights into disease pathogenesis.

The congenital disorders of glycosylation (CDGs) are recent additions to the repertoire of inherited human genetic diseases. Frequency of CDGs is unknown since most cases are believed to be misdiagnosed or unrecognized. With few patients identified and heterogeneity in disease signs noted, studies of animal models may provide increased understanding of pathogenic mechanisms. However, features of mammalian glycan biosynthesis and species-specific variations in glycan repertoires have cast doubt on whether animal models of human genetic defects in protein glycosylation will reproduce pathogenic events and disease signs. We have introduced a mutation into the mouse germline that recapitulates the glycan biosynthetic defect responsible for human CDG type IIa (CDG-IIa). Mice lacking the Mgat2 gene were deficient in GlcNAcT-II glycosyltransferase activity and complex N-glycans, resulting in severe gastrointestinal, hematologic, and osteogenic abnormalities. With use of a lectin-based diagnostic screen for CDG-IIa, we found that all Mgat2-null mice died in early postnatal development. However, crossing the Mgat2 mutation into a distinct genetic background resulted in a low frequency of survivors. Mice deficient in complex N-glycans exhibited most CDG-IIa disease signs; however, some signs were unique to the aged mouse or are prognostic in human CDG-IIa. Unexpectedly, analyses of N-glycan structures in Mgat2-null mice revealed a novel oligosaccharide branch on the "bisecting" N-acetylglucosamine. These genetic, biochemical, and physiologic studies indicate conserved functions for N-glycan branches produced in the Golgi apparatus among two mammalian species and suggest possible therapeutic approaches to GlcNAcT-II deficiency. Our findings indicate that human genetic disease due to aberrant protein glycosylation can be modeled in the mouse to gain insights into N-glycan-dependent physiology and the pathogenesis of CDG-IIa.     

Significant impact of single N-glycan residues on the biological activity of Fc-based antibody-like fragments

Recent studies have demonstrated that IgG-Fc fragments (Fcabs) can be engineered to form antigen-binding sites with antibody properties. Thus they may serve as an attractive alternative to conventional antibodies in therapeutic applications. The critical influence of Fc glycosylation on effector functions of IgGs is well documented; however, whether this applies to Fcabs is not known. Here we used human cells, wild type, and glycoengineered plants to generate four different glycoforms of H10-03-6, an Fcab with engineered HER2/neu-binding sites. Plant-derived H10-03-6 differed in the presence/absence of single oligosaccharide residues, i.e., core fucose and xylose, and terminal galactose. All of the glycoforms had similar binding to HER2/neu expressed on human tumor cells. By contrast, glycoforms that lacked core oligosaccharide modifications (i.e., core α1,3-fucose and β1,2-xylose) showed significantly enhanced binding to the Fcγ receptor IIIa, irrespective of whether plant or human expression systems were used. Consistent with this finding, plant-derived H10-03-6 glycoforms lacking core N-glycan residues mediated higher antibody-dependent cellular cytotoxicity against human tumor cells. No alteration in γ-receptor binding and antibody-dependent cellular cytotoxicity activity was observed upon decoration of N-glycans by terminal galactose. The results point to a significant impact of distinct N-glycan residues on effector functions of Fcabs. Moreover, the outcomes imply that the effector functions mediated by H10-03-6 can be optimized by altering the N-glycosylation profile. Biasing vaccine-induced immune responses toward optimal Fc glycosylation patterns could result in improved vaccine efficacy.