A mutation in SLC37A4 causes a dominantly inherited congenital disorder of glycosylation characterized by liver dysfunction.

SLC37A4 encodes an endoplasmic reticulum (ER)-localized multitransmembrane protein required for transporting glucose-6-phosphate (Glc-6P) into the ER. Once transported into the ER, Glc-6P is subsequently hydrolyzed by tissue-specific phosphatases to glucose and inorganic phosphate during times of glucose depletion. Pathogenic variants in SLC37A4 cause an established recessive disorder known as glycogen storage disorder 1b characterized by liver and kidney dysfunction with neutropenia. We report seven individuals who presented with liver dysfunction multifactorial coagulation deficiency and cardiac issues and were heterozygous for the same variant, c.1267C>T (p.Arg423∗), in SLC37A4; the affected individuals were from four unrelated families. Serum samples from affected individuals showed profound accumulation of both high mannose and hybrid type N-glycans, while N-glycans in fibroblasts and undifferentiated iPSC were normal. Due to the liver-specific nature of this disorder, we generated a CRISPR base-edited hepatoma cell line harboring the c.1267C>T (p.Arg423∗) variant. These cells replicated the secreted abnormalities seen in serum N-glycosylation, and a portion of the mutant protein appears to relocate to a distinct, non-Golgi compartment, possibly ER exit sites. These cells also show a gene dosage-dependent alteration in the Golgi morphology and reduced intraluminal pH that may account for the altered glycosylation. In summary, we identify a recurrent mutation in SLC37A4 that causes a dominantly inherited congenital disorder of glycosylation characterized by coagulopathy and liver dysfunction with abnormal serum N-glycans.

Glycan Profiles of gp120 Protein Vaccines from Four Major HIV-1 Subtypes Produced from Different Host Cell Lines under Non-GMP or GMP Conditions.

Envelope (Env) glycoprotein of human immunodeficiency virus type 1 (HIV-1) is an important target for the development of an HIV vaccine. Extensive glycosylation of Env is an important feature that both protects the virus from antibody responses and serves as a target for some highly potent broadly neutralizing antibodies. Therefore, analysis of glycans on recombinant Env proteins is highly significant. Here, we present glycosylation profiles of recombinant gp120 proteins from four major clades of HIV-1 (A, B, C, and AE), produced either as research-grade material in 293 and CHO cells or as two independent lots of clinical material under good manufacturing practice (GMP) conditions. Almost all potential N-linked glycosylation sites were at least partially occupied in all proteins. The occupancy rates were largely consistent among proteins produced under different conditions, although a few sites showed substantial variability even between the two GMP lots. Our data confirmed previous studies in the field, showing an abundance of oligomannose on Env protein, with 40 to 50% of glycans being Man5 to Man9 on all four proteins under all production conditions. Overall, the differences in occupancy and glycan forms among different Env subtypes produced under different conditions were less dramatic than anticipated, and antigenicity analysis with a panel of six monoclonal antibodies, including antibodies that recognize glycan forms, showed that all four gp120s maintained their antibody-binding profiles. Such findings have major implications for the final production of a clinical HIV vaccine with Env glycoprotein components.IMPORTANCE HIV-1 Env protein is a major target for the development of an HIV-1 vaccine. Env is covered with a large number of sugar-based glycan forms; about 50% of the Env molecular weight is composed of glycans. Glycan analysis of recombinant Env is important for understanding its roles in viral pathogenesis and immune responses. The current report presents the first extensive comparison of glycosylation patterns of recombinant gp120 proteins from four major clades of HIV-1 produced in two different cell lines, grown either under laboratory conditions or at 50-liter GMP scale in different lots. Information learned in this study is valuable for the further design and production of HIV-1 Env proteins as the critical components of HIV-1 vaccine formulations.

Mass Spectrometric Quantification of N-Linked Glycans by Reference to Exogenous Standards.

Environmental and metabolic processes shape the profile of glycoprotein glycans expressed by cells, whether in culture, developing tissues, or mature organisms. Quantitative characterization of glycomic changes associated with these conditions has been achieved historically by reductive coupling of oligosaccharides to various fluorophores following release from glycoprotein and subsequent HPLC or capillary electrophoretic separation. Such labeling-based approaches provide a robust means of quantifying glycan amount based on fluorescence yield. Mass spectrometry, on the other hand, has generally been limited to relative quantification in which the contribution of the signal intensity for an individual glycan is expressed as a percent of the signal intensity summed over the total profile. Relative quantification has been valuable for highlighting changes in glycan expression between samples; sensitivity is high, and structural information can be derived by fragmentation. We have investigated whether MS-based glycomics is amenable to absolute quantification by referencing signal intensities to well-characterized oligosaccharide standards. We report the qualification of a set of N-linked oligosaccharide standards by NMR, HPLC, and MS. We also demonstrate the dynamic range, sensitivity, and recovery from complex biological matrices for these standards in their permethylated form. Our results indicate that absolute quantification for MS-based glycomic analysis is reproducible and robust utilizing currently available glycan standards.

Discrimination between adenocarcinoma and normal pancreatic ductal fluid by proteomic and glycomic analysis.

Sensitive and specific biomarkers for pancreatic cancer are currently unavailable. The high mortality associated with adenocarcinoma of the pancreatic epithelium justifies the broadest possible search for new biomarkers that can facilitate early detection or monitor treatment efficacy. Protein glycosylation is altered in many cancers, leading many to propose that glycoproteomic changes may provide suitable biomarkers. In order to assess this possibility for pancreatic cancer, we have performed an in-depth LC-MS/MS analysis of the proteome and MS(n)-based characterization of the N-linked glycome of a small set of pancreatic ductal fluid obtained from normal, pancreatitis, intraductal papillary mucinous neoplasm (IPMN), and pancreatic adenocarcinoma patients. Our results identify a set of seven proteins that were consistently increased in cancer ductal fluid compared to normal (AMYP, PRSS1, GP2-1, CCDC132, REG1A, REG1B, and REG3A) and one protein that was consistently decreased (LIPR2). These proteins are all directly or indirectly associated with the secretory pathway in normal pancreatic cells. Validation of these changes in abundance by Western blotting revealed increased REG protein glycoform diversity in cancer. Characterization of the total N-linked glycome of normal, IPMN, and adenocarcinoma ductal fluid clustered samples into three discrete groups based on the prevalence of six dominant glycans. Within each group, the profiles of less prevalent glycans were able to distinguish normal from cancer on this small set of samples. Our results emphasize that individual variation in protein glycosylation must be considered when assessing the value of a glycoproteomic marker, but also indicate that glycosylation diversity across human subjects can be reduced to simpler clusters of individuals whose N-linked glycans share structural features.

Regulation of glycan structures in murine embryonic stem cells: combined transcript profiling of glycan-related genes and glycan structural analysis.

The abundance and structural diversity of glycans on glycoproteins and glycolipids are highly regulated and play important roles during vertebrate development. Because of the challenges associated with studying glycan regulation in vertebrate embryos, we have chosen to study mouse embryonic stem (ES) cells as they differentiate into embryoid bodies (EBs) or into extraembryonic endodermal (ExE) cells as a model for cellular differentiation. We profiled N- and O-glycan structures isolated from these cell populations and examined transcripts encoding the corresponding enzymatic machinery for glycan biosynthesis in an effort to probe the mechanisms that drive the regulation of glycan diversity. During differentiation from mouse ES cells to either EBs or ExE cells, general trends were detected. The predominance of high mannose N-glycans in ES cells shifted to an equal abundance of complex and high mannose structures, increased sialylation, and increased α-Gal termination in the differentiated cell populations. Whereas core 1 O-glycan structures predominated in all three cell populations, increased sialylation and increased core diversity characterized the O-glycans of both differentiated cell types. Increased polysialylation was also found in both differentiated cell types. Differences between the two differentiated cell types included greater sialylation of N-glycans in EBs, whereas α-Gal-capped structures were more prevalent in ExE cells. Changes in glycan structures generally, but not uniformly, correlated with alterations in transcript abundance for the corresponding biosynthetic enzymes, suggesting that transcriptional regulation contributes significantly to the regulation of glycan expression. Knowledge of glycan structural diversity and transcript regulation should provide greater understanding of the roles of protein glycosylation in vertebrate development.

Glycomic analyses of mouse models of congenital muscular dystrophy.

Dystroglycanopathies are a subset of congenital muscular dystrophies wherein α-dystroglycan (α-DG) is hypoglycosylated. α-DG is an extensively O-glycosylated extracellular matrix-binding protein and a key component of the dystrophin-glycoprotein complex. Previous studies have shown α-DG to be post-translationally modified by both O-GalNAc- and O-mannose-initiated glycan structures. Mutations in defined or putative glycosyltransferase genes involved in O-mannosylation are associated with a loss of ligand-binding activity of α-DG and are causal for various forms of congenital muscular dystrophy. In this study, we sought to perform glycomic analysis on brain O-linked glycan structures released from proteins of three different knock-out mouse models associated with O-mannosylation (POMGnT1, LARGE (Myd), and DAG1(-/-)). Using mass spectrometry approaches, we were able to identify nine O-mannose-initiated and 25 O-GalNAc-initiated glycan structures in wild-type littermate control mouse brains. Through our analysis, we were able to confirm that POMGnT1 is essential for the extension of all observed O-mannose glycan structures with ß1,2-linked GlcNAc. Loss of LARGE expression in the Myd mouse had no observable effect on the O-mannose-initiated glycan structures characterized here. Interestingly, we also determined that similar amounts of O-mannose-initiated glycan structures are present on brain proteins from α-DG-lacking mice (DAG1) compared with wild-type mice, indicating that there must be additional proteins that are O-mannosylated in the mammalian brain. Our findings illustrate that classical ß1,2-elongation and ß1,6-GlcNAc branching of O-mannose glycan structures are dependent upon the POMGnT1 enzyme and that O-mannosylation is not limited solely to α-DG in the brain.

Site mapping and characterization of O-glycan structures on alpha-dystroglycan isolated from rabbit skeletal muscle.

The main extracellular matrix binding component of the dystrophin-glycoprotein complex, alpha-dystroglycan (alpha-DG), which was originally isolated from rabbit skeletal muscle, is an extensively O-glycosylated protein. Previous studies have shown alpha-DG to be modified by both O-GalNAc- and O-mannose-initiated glycan structures. O-Mannosylation, which accounts for up to 30% of the reported O-linked structures in certain tissues, has been rarely observed on mammalian proteins. Mutations in multiple genes encoding defined or putative glycosyltransferases involved in O-mannosylation are causal for various forms of congenital muscular dystrophy. Here, we explore the glycosylation of purified rabbit skeletal muscle alpha-DG in detail. Using tandem mass spectrometry approaches, we identify 4 O-mannose-initiated and 17 O-GalNAc-initiated structures on alpha-DG isolated from rabbit skeletal muscle. Additionally, we demonstrate the use of tandem mass spectrometry-based workflows to directly analyze glycopeptides generated from the purified protein. By combining glycomics and tandem mass spectrometry analysis of 91 glycopeptides from alpha-DG, we were able to assign 21 different residues as being modified by O-glycosylation with differing degrees of microheterogeneity; 9 sites of O-mannosylation and 14 sites of O-GalNAcylation were observed with only two sites definitively exhibiting occupancy by either type of glycan. The distribution of identified sites of O-mannosylation suggests a limited role for local primary sequence in dictating sites of attachment.

Phosphatidic acid phospholipase A1 mediates ER-Golgi transit of a family of G protein-coupled receptors.

The coat protein II (COPII)-coated vesicular system transports newly synthesized secretory and membrane proteins from the endoplasmic reticulum (ER) to the Golgi complex. Recruitment of cargo into COPII vesicles requires an interaction of COPII proteins either with the cargo molecules directly or with cargo receptors for anterograde trafficking. We show that cytosolic phosphatidic acid phospholipase A1 (PAPLA1) interacts with COPII protein family members and is required for the transport of Rh1 (rhodopsin 1), an N-glycosylated G protein-coupled receptor (GPCR), from the ER to the Golgi complex. In papla1 mutants, in the absence of transport to the Golgi, Rh1 is aberrantly glycosylated and is mislocalized. These defects lead to decreased levels of the protein and decreased sensitivity of the photoreceptors to light. Several GPCRs, including other rhodopsins and Bride of sevenless, are similarly affected. Our findings show that a cytosolic protein is necessary for transit of selective transmembrane receptor cargo by the COPII coat for anterograde trafficking.

The diversity of O-linked glycans expressed during Drosophila melanogaster development reflects stage- and tissue-specific requirements for cell signaling.

Appropriate glycoprotein O-glycosylation is essential for normal development and tissue function in multicellular organisms. To comprehensively assess the developmental and functional impact of altered O-glycosylation, we have extensively analyzed the non-glycosaminoglycan, O-linked glycans expressed in Drosophila embryos. Through multidimensional mass spectrometric analysis of glycans released from glycoproteins by beta-elimination, we detected novel as well as previously reported O-glycans that exhibit developmentally modulated expression. The core 1 mucin-type disaccharide (Galbeta1-3GalNAc) is the predominant glycan in the total profile. HexNAcitol, hexitol, xylosylated hexitol, and branching extension of core 1 with HexNAc (to generate core 2 glycans) were also evident following release and reduction. After Galbeta1-3GalNAc, the next most prevalent glycans were a mixture of novel, isobaric, linear, and branched forms of a glucuronyl core 1 disaccharide. Other less prevalent structures were also extended with HexA, including an O-fucose glycan. Although the expected disaccharide product of the Fringe glycosyltransferase, (GlcNAcbeta1-3)fucitol, was not detectable in whole embryos, mass spectrometry fragmentation and exoglycosidase sensitivity defined a novel glucuronyl trisaccharide as GlcNAcbeta1-3(GlcAbeta1-4)fucitol. Consistent with the spatial distribution of the Fringe function, the GlcA-extended form of the Fringe product was enriched in the dorsal portion of the wing imaginal disc. Furthermore, loss of Fringe activity reduced the prevalence of the O-Fuc trisaccharide. Therefore, O-Fuc glycans necessary for the modulation of important signaling events in Drosophila are, as in vertebrates, substrates for extension beyond the addition of a single HexNAc.

Targeted glycoproteomic identification of biomarkers for human breast carcinoma.

Glycosylation is a dynamic post-translational modification that changes during the development and progression of various malignancies. During the oncogenesis of breast carcinoma, the glycosyltransferase known as N-acetylglucosaminyltransferase Va (GnT-Va) transcript levels and activity are increased due to activated oncogenic signaling pathways. Elevated GnT-V levels leads to increased beta(1,6)-branched N-linked glycan structures on glycoproteins that can be measured using a specific carbohydrate binding protein or lectin known as L-PHA. L-PHA does not bind to nondiseased breast epithelial cells, but during the progression to invasive carcinoma, cells show a progressive increase in L-PHA binding. We have developed a procedure for intact protein L-PHA-affinity enrichment, followed by nanospray ionization mass spectrometry (NSI-MS/MS), to identify potential biomarkers for breast carcinoma. We identified L-PHA reactive glycoproteins from matched normal (nondiseased) and malignant tissue isolated from patients with invasive ductal breast carcinoma. Comparison analysis of the data identified 34 proteins that were enriched by L-PHA fractionation in tumor relative to normal tissue for at least 2 cases of ductal invasive breast carcinoma. Of these 34 L-PHA tumor enriched proteins, 12 are common to all 4 matched cases analyzed. These results indicate that lectin enrichment strategies targeting a particular glycan change associated with malignancy can be an effective method of identifying potential biomarkers for breast carcinoma.

Identification of N-glycosylated proteins from the central nervous system of Drosophila melanogaster.

Although the function of many glycoproteins in the nervous system of fruit flies is well understood, information about the glycosylation profile and glycan attachment sites for such proteins is scarce. In order to fill this gap and to facilitate the analysis of N-linked glycosylation in the nervous system, we have performed an extensive survey of membrane-associated glycoproteins and their N-glycosylation sites isolated from the adult Drosophila brain. Following subcellular fractionation and trypsin digestion, we used different lectin affinity chromatography steps to isolate N-glycosylated glycopeptides. We identified a total of 205 glycoproteins carrying N-linked glycans and revealed their 307 N-glycan attachment sites. The size of the resulting dataset furthermore allowed the statistical characterization of amino acid distribution around the N-linked glycosylation sites. Glycan profiles were analyzed separately for glycopeptides that were strongly and weakly bound to Concanavalin A (Con A), or that failed to bind Concanavalin A, but did bind to wheat germ agglutinin (WGA). High- or paucimannosidic glycans dominated each of the profiles, although the wheat germ agglutinin-bound glycan population was enriched in more extensively processed structures. A sialylated glycan structure was unambiguously detected in the wheat germ agglutinin-bound fraction. Despite the large amount of starting material, insufficient amount of glycopeptides was retained by the Wisteria floribunda (WFA) and Sambucus nigra columns to allow glycan or glycoprotein identification, providing further evidence that the vast majority of glycoproteins in the adult Drosophila brain carry primarily high-mannose, paucimannose, and hybrid glycans. The obtained results should facilitate future genetic and molecular approaches addressing the role of N-glycosylation in the central nervous system (CNS) of Drosophila.