TY - JOUR
AU - Lefeber, Dirk, J
AB - Protein glycosylation is increasingly recognized as a crucial modulator of protein function, offering a third layer of biological information over genomics and proteomics. Modern tools for analyzing released N-glycans from cells and body fluids, i.e., the glycome, have shown abnormal protein glycosylation in numerous human diseases. These include both genetic and acquired diseases, ranging from diabetes, cancer, and inflammatory disease to neurodegenerative and neuromuscular disease. Insights from this novel field in human medicine provide exciting perspectives toward understanding disease processes, identifying therapeutic targets, and designing individualized diagnostics based on protein concentrations and glycosylation status. However, the main question is how we can translate this information into concrete biomarkers in a clinical diagnostic setting, with high demands on technical robustness and the ability to interpret results within specific patient groups. Unlike the genetic template for protein synthesis, the process of protein glycosylation is not directly encoded by the genome. Protein N-glycosylation, the best-studied ubiquitous type of glycosylation, follows a sequential pathway in the organelles of the secretory pathway. First, a dolichol-linked glycan composed of 14 monosaccharides is assembled in the endoplasmic reticulum (ER)2 and transferred to nascent proteins that enter the ER via the translocon complex. In the final stages in the ER, and further in the Golgi apparatus, the protein-bound glycan is remodeled to a mature glycan that is present on cellular receptors, ion channels, and secreted proteins in plasma. It is becoming increasingly apparent that this process is influenced by many factors, genetic, metabolic, and environmental (such as medication and alcohol abuse). The congenital disorders of glycosylation (CDG) form an ideal group of well-defined genetic defects to derive important lessons on novel biochemical mechanisms and clinical diagnostic approaches with relevance to more common diseases. More than 100 different genetic defects in various glycosylation pathways are already known, approximately 50 of which affect the N-glycosylation pathway (1). Diagnostic screening for CDG with abnormal N-glycosylation is commonly carried out by isofocusing, capillary electrophoresis, or HPLC of plasma transferrin, an abundant liver-derived protein with 2 N-glycan structures. In patients with CDG-I subtypes, the genetic defect is located in the cytosol or ER. Transferrin lacks the addition of complete glycans, resulting in unoccupied glycosylation sites. In patients with CDG-II subtypes caused by a defect in the cytosol or Golgi apparatus, transferrin is observed with abnormal truncated glycans. Profiling of total plasma N-glycans, i.e., the release and analysis of N-glycans bound to all plasma proteins, provides information on glycan structures and therefore usually reflects Golgi glycosylation status (2, 3). Lessons from ALG1-CDG In this issue of Clinical Chemistry, Zhang et al. (4) report on the identification of a novel N-tetrasaccharide that is highly characteristic for a few very specific CDG-I subtypes: asparagine-linked glycosylation protein 1 (ALG1)-CDG, phosphomannomutase 2 (PMM2)-CDG, and phosphomannose isomerase (MPI)-CDG (Fig. 1). They clearly illustrate the biochemical and diagnostic lessons that can be learned by studying CDG. The authors applied total plasma and fibroblast N-glycan profiling to several CDG-I subtypes. Surprisingly, zooming in on the low molecular mass range highlighted the accumulation of several small, truncated glycan structures. These structures were visible only after PNGaseF digestion, indicating their protein-bound nature. In patients with ALG1-CDG, they identified a novel protein-linked N-tetrasaccharide (Neu5Acα2,6Galβ1,4GlcNAcβ1,4GlcNAc), a trisaccharide (Galβ1,4GlcNAcβ1,4GlcNAc), and the chitobiose GlcNAcβ1,4GlcNAc. These glycans were also present in lower amounts in the PMM2-CDG and MPI-CDG patients tested. In PMM2-CDG and MPI-CDG, the Man3GlcNAc2 and Man4GlcNAc2 glycans were also increased. Mass spectrometry analysis of plasma transferrin indicated the presence of the N-tetrasaccharide in ALG1-CDG patients, but not in patients with PMM2- or MPI-CDG. In addition, the accumulation of Man3GlcNAc2 and Man4GlcNAc2 glycans was not visible on the transferrin protein. Mutations in ALG1 (ALG1, chitobiosyldiphosphodolichol beta-mannosyltransferase), PMM2 (phosphomannomutase 2), and MPI (mannose phosphate isomerase), 3 defects in the early steps of N-glycosylation in the endoplasmic reticulum, give rise to the presence of a unique N-tetrasaccharide on plasma proteins. Fig. 1. Open in new tabDownload slide For ALG1, PMM2, and MPI, the N-tetrasaccharide can be seen on total plasma proteins, but only for ALG1 on transferrin. Total plasma glycan profiling shows additional accumulation of Man4- and Man3GlcNAc2 glycans for PMM2 and MPI; however, they are not detectable on transferrin. These data from Zhang et al. (4) indicate that proteins other than transferrin contain diagnostic glycan markers, showing the potential for protein-specific glycan profiling in patient diagnostics. Fig. 1. Open in new tabDownload slide For ALG1, PMM2, and MPI, the N-tetrasaccharide can be seen on total plasma proteins, but only for ALG1 on transferrin. Total plasma glycan profiling shows additional accumulation of Man4- and Man3GlcNAc2 glycans for PMM2 and MPI; however, they are not detectable on transferrin. These data from Zhang et al. (4) indicate that proteins other than transferrin contain diagnostic glycan markers, showing the potential for protein-specific glycan profiling in patient diagnostics. Previously, the presence of chitobiose has been reported on proteins in ALG1 mutant yeast. Interestingly, the protein-linked chitobiose was also observed in the T24 bladder carcinoma cell line grown under glucose deprivation, indicating a more general disease mechanism (5). ALG1 encodes the first mannosyltransferase, responsible for the elongation of dolichol-PP-GlcNAc2. Apparently, deficiency of ALG1 results in accumulating GlcNAc2 (chitobiose) structures that can be transferred onto nascent proteins. Subsequently, the GlcNAc2 structure can be elongated in the Golgi apparatus by galactosyl and sialyl transferases to yield the N-tetrasaccharide. The low efficiency of this process accounts for the low abundance of these diagnostic markers. In addition to the biochemical lessons, the study by Zhang et al. clearly shows the added value of highly sensitive and specific mass spectrometry techniques to identify unanticipated glycan-based biomarkers for disease. CDG-I subtyping has previously been based on the laborious analysis of lipid-linked oligosaccharides in fibroblasts, requiring a skin biopsy and radioactive labeling. More recently, next-generation sequencing has replaced several biochemical methods. The results from Zhang's study, however, reveal that the ALG1-CDG subtype can already be identified in the initial screening for CDG-I if mass spectrometry of transferrin is performed. In addition, as suggested by the authors, PMM2-CDG might be diagnosed by total plasma N-glycan profiling by mass spectrometry. Protein-Specific Glycoprofiling Glycoprofiling of transferrin by mass spectrometry was first introduced in a diagnostic setting in the Mayo medical laboratory by use of electrospray LC-MS for detection of CDG-I defects and alcohol abuse (6), being more specific than commonly used methods such as isofocusing, HPLC, or capillary electrophoresis. More recently, introduction of high-resolution quadrupole time-of-flight MS instruments (7) has allowed accurate annotation of glycan structures on the intact transferrin protein and thereby direct diagnosis of several CDG-II subtypes, as shown for mannosyl-oligosaccharide 1,2-α-mannosidase (MAN1B1)-CDG (8) and phosphoglucomutase 1 (PGM1)-CDG (9). Although transferrin is a very useful diagnostic biomarker for most CDG types with deficient N-glycosylation, several defects in the N-glycosylation pathway with a normal transferrin profile are known. These include CDG-I subtypes [tumor suppressor candidate 3 (TUSC3)-CDG, ALG14-CDG], CDG-II subtypes with abnormal Golgi glycosylation [mannosyl-oligosaccharide glucosidase (GCS1)-CDG, solute carrier 35A3 (SLC35A3)-CDG, and SLC35C1-CDG], as well as defects in sugar metabolism [glucosamine (UDP-N-acetyl)-2-epimerase/N-acetylmannosamine kinase (GNE)-CDG, PGM3-CDG, and glucosamine-fructose-6-phosphate transaminase 1 (GFPT1)-CDG]. Additionally, novel Golgi homeostasis defects are being identified with abnormal plasma N-glycans but normal transferrin N-glycosylation, as shown for Cohen syndrome caused by mutations in VPS13B [vacuolar protein sorting 13 homolog B (yeast)]3 (10). As shown by Zhang et al. (4), PMM2- and MPI-CDG can be added to this growing list, since they identified abnormal Man3GlcNAc2 and Man4GlcNAc2 glycans in total plasma profiles, while analysis of transferrin glycosylation showed only the expected lack of glycans. Total plasma N-glycan profiling is influenced by variations in protein concentration; for example, increased immunoglobulins in case of infections. Protein-specific glycoprofiling eliminates this variation. The vast majority of plasma proteins are glycosylated, holding enormous potential to identify specific glycoprotein biomarkers for a wide range of diseases, based on protein concentration and glycosylation signature. It could be envisioned that combination of several plasma proteins with a more heterogeneous glycan composition and increased fucosylation compared to transferrin could be used in a panel to even further increase the diagnostic efficiency for CDG-I and CDG-II. As an example, abnormal glycosylation of IgG was observed in GCS1-CDG patients with normal transferrin glycosylation (11), although GCS1 is ubiquitously expressed. New technologies for analysis of complex glycopeptide mixtures are emerging and show tissue-specific glycosylation profiles of individual proteins (12). Such technologies will pave the way to identify additional glycoproteins that can be used as biomarkers for CDG diagnostics and beyond. Quantification of such glycoproteins at the level of the intact protein, or by use of multiple reaction monitoring of glycopeptides (13), as commonly used in clinical chemistry laboratories, will allow the translation of glycomics information into useful clinical biomarkers by mass spectrometry. 2 Nonstandard abbreviations ER endoplasmic reticulum CDG congenital disorders of glycosylation ALG1 asparagine-linked glycosylation protein 1 PMM2 phosphomannomutase 2 MPI phosphomannose isomerase MAN1B1 mannosyl-oligosaccharide 1,2-α-mannosidase PGM1 phosphoglucomutase 1 TUSC3 tumor suppressor candidate 3 SLC35A3 solute carrier 35A3 GNE glucosamine (UDP-N-acetyl)-2-epimerase/N-acetylmannosamine kinase GFPT1 glucosamine—fructose-6-phosphate transaminase 1. 3 VPS13B vacuolar protein sorting 13 homolog B (yeast) ALG1 ALG1, chitobiosyldiphosphodolichol beta-mannosyltransferase PMM2 phosphomannomutase 2 MPI mannose phosphate isomerase. " (see article on page 208) " Author Contributions:All authors confirmed they have contributed to the intellectual content of this paper and have met the following 3 requirements: (a) significant contributions to the conception and design, acquisition of data, or analysis and interpretation of data; (b) drafting or revising the article for intellectual content; and (c) final approval of the published article. " Authors' Disclosures or Potential Conflicts of Interest:No authors declared any potential conflicts of interest. References 1. Freeze HH , Chong JX, Bamshad MJ, Ng BG . Solving glycosylation disorders: fundamental approaches reveal complicated pathways . Am J Hum Genet 2014 ; 94 : 161 – 75 . Google Scholar Crossref Search ADS PubMed WorldCat 2. Morelle W , Michalski JC . Analysis of protein glycosylation by mass spectrometry . Nat Protoc 2007 ; 2 : 1585 – 602 . Google Scholar Crossref Search ADS PubMed WorldCat 3. 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Google Scholar Crossref Search ADS PubMed WorldCat © 2016 The American Association for Clinical Chemistry This article is published and distributed under the terms of the Oxford University Press, Standard Journals Publication Model (https://academic.oup.com/journals/pages/open_access/funder_policies/chorus/standard_publication_model)
TI - Protein-Specific Glycoprofiling for Patient Diagnostics
JF - Clinical Chemistry
DO - 10.1373/clinchem.2015.248518
DA - 2016-01-01
UR - https://www.deepdyve.com/lp/oxford-university-press/protein-specific-glycoprofiling-for-patient-diagnostics-rtGpranwD9
SP - 9
VL - 62
IS - 1
DP - DeepDyve
ER -