Quantitation of Saccharide Compositions of O-glycans by Mass Spectrometry of Glycopeptides and Its Application to Rheumatoid Arthritis Yoshinao Wada,*,† Michiko Tajiri,†,‡ and Shiro Ohshima§ Department of Molecular Medicine, Osaka Medical Center and Research Institute for Maternal and Child Health, 840 Murodo-cho Izumi, Osaka 594-1101, Japan, CREST, Japan Science and Technology Agency, 4-1-8 Honcho Kawaguchi, Saitama 332-0012, Japan, and Department of Clinical Research, NHO Osaka Minami Medical Center, 2-1 Kidohigashimachi, Kawachinagano, Osaka 586-8521, Japan Received October 10, 2009
Profiling of oligosaccharide structures is widely utilized for both identification and evaluation of glycobiomarkers, and site-specific profiling of N-linked glycans of glycoproteins is conducted by mass spectrometry of glycopeptides. However, our knowledge of mucin-type O-glycans including site occupancy and profile variance, as well as attachment sites, is quite limited. Saccharide compositions and site-occupancy of O-glycans were calculated from the signal intensity of glycopeptide ions in the mass spectra and tandem mass spectra from electron transfer dissociation. The results for two major plasma glycoproteins, IgA1 and hemopexin, representing clustered and scattered O-glycan attachments, respectively, indicated that the variability in modifications among individuals is so small as to justify rigorous standards enabling reliable detection of disease-related alterations. Indeed, this method revealed a novel abnormality associated with rheumatoid arthritis: a significant decrease in the N-acetylgalactosamine content of IgA1 O-glycans, indicating that the glycosylation abnormality is not limited to hypogalactosylation of IgG N-glycans in chronic inflammatory conditions. Keywords: mucin-type O-glycan • glycopeptide • mass spectrometry • quantitation • rheumatoid arthritis • galactose
Introduction Glycosylation alters the physicochemical properties and biological activities of proteins.1,2 Even a subtle change such as addition or depletion of a single sugar unit in the glycan moiety potentially affects the functions of proteins and thereby cellular phenotypes, as has been observed in a wide range of diseases such as cancer and infection.3-5 These effects are typically recognized as diverse and multisystemic abnormalities found in a group of diseases, congenital disorders of glycosylation (CDG), most of which are due to enzymatic defects in the N-glycan synthesis pathway.6 N-glycosylation occurs at Asn in the sequence Asn-Xaa-Ser/Thr, and approximately 16 000 N-glycosylation sites have been recorded in the database. On the other hand, O-glycosylation occurring at the Ser/Thr residues includes more diverse classes of structures, among which the mucin-type O-glycans are the most typical structure in mammals.7 Our knowledge of the disorders related to O-glycosylation is small compared with that of N-glycosylation, but the recent identification of the UDP-N-acetyl-R-D-galactosamine: polypeptide N-acetylgalactosaminyltransferase 3 * To whom correspondence should be addressed. Tel: +81-725-57-4105. E-mail:
[email protected]. † Osaka Medical Center and Research Institute for Maternal and Child Health. ‡ CREST, Japan Science and Technology Agency. § NHO Osaka Minami Medical Center. 10.1021/pr900913k
2010 American Chemical Society
gene (GALNT3) as being responsible for familial tumoral calcinosis (FTC) has clearly illustrated the biological significance of mucin-type O-glycosylation.8 In a proposed scheme of FTC pathogenesis, the defective O-glycosylation in the vicinity of the proteolytic cleavage site Arg-179 of FGF23 enhances intracellular processing of this phosphaturic molecule, resulting in the hyperphosphatemic phenotype.9 These studies indicated that the mapping of glycans at specific sites and related information such as occupancy are essential for understanding and specifying the molecular pathology of glycosylation-related disorders.10 The mucin-type O-glycosylation is initiated by linking Nacetylgalactosamine (GalNAc) to the Ser or Thr of the protein backbone and this reaction is catalyzed by GalNAc transferases (GalNAc-Ts).7,11 The linkage is found in characteristic peptide regions: clusters of Ser/Thr residues with a β-turn near Pro and at a distance from charged amino acids, but no specific consensus sequence has been identified. This is due to several factors including the overlapping specificity of the GalNAc-Ts, which constitute a family with up to 20 members in humans, and it is noteworthy that some of these enzymes work in concert in a hierarchical manner to form clustered O-glycans.11 From the technical point of view, one major obstacle is the relatively low frequency of residues such as Lys or Arg in the vicinities of O-glycosylation sites. The rare occurrence of these proteolytic sites makes the glycopeptides large and results in Journal of Proteome Research 2010, 9, 1367–1373 1367 Published on Web 01/27/2010
research articles low abundance of product ions derived from peptide backbone cleavage. In addition, the high occurrence of Pro residues near O-glycosylation sites yields problems collectively termed the “proline effect”.12,13 These factors compromise the capacity for tandem MS utilizing collision-induced dissociation. Furthermore, the O-glycosylation occupancy is usually partial, resulting in oversight by Edman-based amino acid sequencing methods which rely on the depression or absence of an expected signal at particular cycles as suggested by Renfrow et al..14 Thus, the information on mucin-type O-glycosylation is still quite limited as only 235 sites in 66 human proteins have been experimentally verified and recorded in a protein database (”O-linked GalNAc” in UniProt 15.6, 28-Jul-2009), and this lack of information critically hampers the usage of O-glycosylation as a biomarker. Mass spectrometry (MS) of N-glycopeptides has been established as a tool for profiling glycans15-17 as well as for relative quantitation,18 and the strategy for the determination of site occupancy on a proteomic scale has been reported.19 MS of O-glycopeptides has been used for attachment site determination in the last two decades.20-23 However, to our knowledge, the quantitation of O-glycans and measurements of siteoccupancy with this method have not been adequately studied. In the present study, two abundant plasma proteins, immunoglobulin A1 (IgA1) and hemopexin, both bearing mucin-type core-1 O-glycans, with Galβ1-3GlcNAc-(Ser/Thr) as the core structure but different patterns of glycan distribution, were analyzed by MS incorporating electron transfer dissociation (ETD).24,25 This is an effective tool, with a technology similar to electron capture dissociation (ECD), for site-determination of mucin-type O-glycans and O-GlcNAc.26-30 The mass spectra from various MS modes indicated that quantitation of the saccharide composition as well as site occupancy by glycans can be determined by simple calculations, based on the peak intensity. Our calculation results revealed that variances with respect to attachment site, occupancy, and saccharide composition are minimal among healthy individuals. The subtlety of these variances allows evaluation of altered O-glycosylation associated with diseases, as demonstrated by the discovery of a novel abnormality related to rheumatoid arthritis (RA).
Experimental Section Materials and Patients. IgA and hemopexin from 20 µL of serum were purified by affinity chromatography using polyclonal antibody-coupled agarose beads. IgG from 10 µL of serum was purified using a Hi-Trap Protein G column (GE Healthcare). Proteins (5-100 µg) were dissolved in a 0.5 mL solution of 6 M guanidine and 0.25 M Tris-HCl, pH 8.0, reduced with 0.13 M dithiothreitol at 50 °C for 1 h and then Scarbamidomethylated with 0.22 M iodoacetamide for 30 min at room temperature. After reaction, these chemicals were removed by gel filtration using a NAP5 column (GE Healthcare) equilibrated with 0.05 N HCl, and a 1/10 solution of 1.5 M TrisHCl was added to raise the pH to 8.2. A total of 0.5 µg each of lysylendopeptidase (Achromobacter protease I, Wako Pure Chemical, Osaka, Japan) and trypsin (Sequence grade Modified Trypsin, from porcine pancreas, Promega) was added to the solution which was then incubated at 37 °C for 6 h for digestion. Glycopeptides in the digests were extracted according to a method described previously.31 Briefly, a 50 µg digest was mixed with a 15 µL packed volume of Sepharose CL4B (GE Healthcare) in 1 mL of an organic solvent of 1-butanol/ethanol/ H2O (4:1:1, v/v). After gentle shaking for 45 min, the gel was 1368
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Wada et al. washed twice with the same organic solvent. To recover the glycopeptides, the gel was incubated with an aqueous solvent of ethanol/H2O (1:1, v/v) for 30 min, and the solution phase was dried using a vacuum concentrator. In an experiment including the nonglycosylated peptide, the fraction containing glycosylated and nonglycosylated N-terminal peptides corresponding to positions 1-26 of hemopexin was collected by reversed phase chromatography, which was carried out on a C18 column (150 × 1.0 mm) with a linear gradient elution of acetonitrile (1-30%, v/v) in 0.1% (v/v) trifluoroacetic acid (TFA). Desialylation of glycopeptides was carried out by incubation in 2 M acetic acid at 80 °C for 2 h. Twenty-six RA, two SLE, and one Castleman disease patients and nine healthy volunteers were enrolled in this study. All RA patients fulfilled the revised criteria of the American College of Rheumatology (formerly, the American Rheumatism Association).32 This study was approved by the ethics committees of Osaka Medical Center and the Research Institute for Maternal and Child Health and of NHO Osaka Minami Medical Center. Mass Spectrometry. MALDI-TOF-MS was carried out on a Voyager DE Pro MALDI-TOF mass spectrometer with a nitrogen pulsed laser (337 nm) (Applied Biosystems, Foster City, CA). The peptide sample was mixed with 10 mg/mL of DHB dissolved in a 0.1% (v/v) TFA and 50% (v/v) acetonitrile solution for measurement. The measurements were carried out in positive ion and linear TOF mode. The mass spectra acquired by at least 200 laser shots were accumulated, and the measurement was repeated at least three times.17 ESI MS was carried out using an LTQ XL ion trap mass spectrometer (Thermo Fisher Scientific). Glycopeptide samples were dissolved in a 0.1% acetic acid and 50% (v/v) methanol solution and directly infused into the mass spectrometer using a nanospray tip. The mass spectra of 20 scans were accumulated. For ETD, 106 anions of fluoranthene were used for reaction, and the ion/ion reaction time was set to 100 ms. The ETD MS/MS mass spectra was acquired by 200 scans.
Results Nonclustered O-glycosylation of Hemopexin. The N-terminal Thr of hemopexin is the only attachment site of O-linked glycan recorded in the database. The peptide fraction containing the N-terminal tryptic peptide was collected by HPLC and subjected to matrix-assisted laser desorption/ionization (MALDI) linear time-of-flight (TOF) MS (Figure 1a). In the MALDI linear-TOF and electrospray ionization (ESI) mass spectra acquired after desialylation, multiple O-glycosylations with different numbers, from 0 to 3, of GalNAc-Gal disaccharide (Tantigen) were evident in the N-terminal region (residues 1-26) (Figure 1b,c). No substantial amounts of the GalNAc monosaccharide (Tn antigen) were attached, and the glycopeptide ions containing a single O-glycan were most abundant. The percent content of each glycopeptide species was calculated using eq 1 as reported by Rebecchi et al.18 In the case of the ESI mass spectrum, the relative abundances of the peaks for the same glycopeptide composition but differing in the charge state are combined for calculation.18 (Glyco)peptide Peak % ) [(Glyco)peptide Peak Intensity]/ [Total(Glyco)peptide Intensity) × 102
(1)
The (glycol)peptide peak % for each peptide bearing 0, 1, 2, 3, or 4 glycans was 2.5, 68.0, 25.4, 4.1, or 0.0%, respectively,
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Figure 1. Mass spectra of glycopeptides from hemopexin. (a and b) MALDI mass spectra of an HPLC fraction containing the N-terminal tryptic peptide. Before (a) and after (b) desialylation. Linear TOF mode operation. (c) ESI (nanospray) mass spectrum of desialylated glycopeptides. The number in parentheses is the charge state of the ions. The peaks are protonated molecules. N, GalNAc; H, Gal; NA, N-acetylneuraminic acid (Neu5Ac). The peak intensities after desialylation (b and c) were used for calculation. The peaks with asterisks were derived from other origins.
from the MALDI mass spectrum (Figure 1b), and 3.7, 66.7, 23.7, 5.9, or 0.0%, respectively, from the ESI mass spectrum (Figure 1c), indicating the profiles given by MALDI and ESI MS to be consistent with each other. The molar content of O-glycans attached to this region was calculated using eq 2. Glycan Content (mol/peptide) )
∑ {[(Glyco)peptide Peak %] × [Number of Glycans attached to(Glyco)peptide]} × 10-2
(2)
For example, the values of 1.31 and 1.32 mol for the glycans attached to this region were obtained from the MALDI and ESI mass spectra, respectively. Although eqs 1 and 2 are primarily applied to glycosylated peptides, nonglycosylated species may be included in the calculation, when the glycan moiety of the glycosylated counterparts is small and neutral, like the GalNAcGal oligosaccharides in this case. This assumption was justified by the following ETD MS/MS.
Figure 2. ETD MS/MS spectrum of the (peptide + GalNAc2Gal2 + 4H)4+ ion at m/z 861.2. Most peaks were assigned as N- or C-terminal fragment ions, but only those of interest are indicated. N, GalNAc; H, Gal. Note the absence of the nonglycosylated c5 ion and the c7 ion containing GalNAc-Gal from the spectrum (broken arrows).
Glycopeptides were isolated from other peptides by the hydrophilic affinity method31 and subjected to site-specific analysis by ETD. In the ETD mass spectrum of the precursor ions containing two glycans (Figure 2), the absence of the peak at m/z 523.3 for N-terminal c5 fragment ions and the distinct peak at m/z 888.1 indicated full occupancy at the N-terminal Thr. The ratio of 7.9 for the intensity of the peak at m/z 1354.5 to that at m/z 989.3, which corresponded to the c6 ions containing two or one glycan, respectively, suggested approximately 90% occupancy at Thr-6. This calculation was verified by the C-terminal (z-type) fragment ions, in which the ratio of the peak at m/z 2093.9 to that at m/z 2458.9 corresponding to the z20 ions without or with a single glycan, respectively, was 7.8 consistent with the ratio of the c6 ions described above. It is noteworthy here that the ions at m/z 2093.9 were not glycosylated, indicating that the quantitation by eq 1 can be applied to the partially occupied case of core-1 O-glycosylation. The peak for c7 ions was observed at m/z 1441.5 but not at m/z 1089.3, indicating attachment of the second glycan to be at either Thr-6 or Ser-7 exclusively, such that the occupancy at Ser-7 in this diglycosylated peptide was 10%. In a similar manner, the occupancy at each glycosylation site can be calculated from individual glycopeptide peaks, and the overall sitespecific occupancy is subsequently computed using eq 3. Occupancy % at Specific Site )
∑ {(Glycopeptide Peak %) ×
(Occupancy % at Specific Site in the Precursor Glycopeptide Ion)} × 10-2
(3)
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Figure 4. ETD MS/MS spectrum of the (peptide + GalNAc5Gal4 + 4H)4+ ion at m/z 1451.6.The saccharide compositions of the fragment ions are indicated in parentheses.
GalNAc-Gal disaccharides attached to this region (Figure 3b). The molar content of the component saccharides, GalNAc and Gal, is calculated by eq 1 followed by eq 4. GalNAc, Gal (mol/glycopeptide) ) Figure 3. MALDI TOF mass spectra of glycopeptides from IgA1. (a and b) IgA1 from a healthy individual. Before (a) and after (b) desialylation. (c) IgA1 from an IgA nephropathy patient. (d) IgA1 from an RA patient. Linear TOF mode operation. The peak intensities after desialylation (b-d) are used for calculation. N, GalNAc; H, Gal; NA, Neu5Ac.
Thr-6, Ser-7 and Thr-17 are novel O-glycosylation sites, and the occupancy at these sites decreases stepwise as 22.5 ( 4.3, 6.1 ( 2.0, and 0.5) and 3.24 (P < 0.01) (mol/peptide) for GalNAc and Gal, respectively. Renfrow et al. localized the O-glycosylation sites of the precursor glycopeptide ions containing four GalNAc-Gal disaccharides and one GalNAc monosaccharide from IgA1 myeloma proteins using ECD.14 The same glycopeptide species from healthy individuals were analyzed by ETD in the present study, and the masses of c17, z13 and z22 ions indicated a consistent result with respect to the disaccharide attachment sites at Thr-225, Thr-228, Ser-230, and Ser-232 (Figure 4). In addition, the z10 and z13 ions indicated that a monosaccharide, GalNAc, attached to Thr-233 and Thr-236 at approximate ratios of 30% and 70%, respectively, while the previous study detected no product ions suitable for discrimination of these sites. Abnormal O-glycosylation in RA. Human serum IgG molecules contain a single conserved N-linked glycosylation site at Asn-297 in each constant domain of the Fc region. The major glycan is the biantennary type with the arms terminating with GlcNAc or GlcNAc-Gal, giving three different species: ungalactosylated (G0), monogalactosylated (G1), and digalactosylated
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and GalNAc on IgA1 O-glycans (Figure 5), but there was no significant correlation between the galactosylation levels of IgG N-glycans and IgA1 O-glycans nor between Gal and GalNAc on IgA1 O-glycans (Figure 5 and Supplementary Figure 2), suggesting the glycosylation abnormality associated with RA and other inflammatory diseases to involve different classes of immunoglobulins and to manifest as different molecular phenotypes.
Discussion
Figure 5. Correlations between the contents of galactose on IgG N-glycan and of N-acetylgalactosamine on IgA1 O-glycan (a) and between the contents of N-acetylgalactosamine on IgA O-glycan and galactose on IgA O-glycan (b). Healthy control (O), RA (2), SLE (9), Castleman disease (1).
(G2) structures with core fucose.4,17 Reduction of the galactosylation of IgG N-glycans has classically been described in RA, but has also been found in other chronic inflammatory conditions such as Crohn’s disease, juvenile onset chronic arthritis, systemic lupus erythematosus and tuberculosis.36-38 In a previous study comparing various analytical methods, MALDI linear-TOF MS of glycopeptides was found to be reliable in measuring the galactosylation levels of IgG N-glycans.17 The molar content of Gal in the glycan was calculated using eq 4, and the average value of nine healthy individuals was 1.19 ( 0.16 (mol/glycan). Furthermore, decreased galactosylation levels in RA patients were clearly demonstrated by this calculation; for example, 0.78 (mol/glycan) for an RA patient (see Supplementary Figure 1), representing a significant reduction versus healthy individuals (P < 0.05). Galactosylation of protein N-linked oligosaccharides is catalyzed by UDP-N-acetyl-R-D-gal:polypeptide N-acetylglucosamine β1-4 galactosyltransferase I (β4Gal-TI).39,40 The reduction of galactosylation of IgG-Fc glycans is associated with reduced galactosyltransferase activity in B cells.41 In contrast to IgG, the N-glycan structures on the IgA1 Fc region are more completely processed, with only a trace level (1.3%) of the agalactosylated oligosaccharide (G0) being detectable,33,42 and the increase in the G0 level, only 2.3% for RA patients, is too small to be significant.42 On the other hand, the IgA1 O-glycans in RA patients have not yet been studied in sufficient detail. Figure 3d shows the MALDI linear-TOF mass spectrum of IgA1 O-glycopeptides from the same RA patient. The GalNAc content (4.35 mol/peptide; P < 0.01) was significantly decreased in this patient, while the Gal content was not reduced (3.71 mol/ peptide). This reduction in GalNAc of IgA1 O-glycans was found in many patients with RA and other inflammatory diseases, as shown in Figure 5. Interestingly, regression analysis revealed a positive linear relationship between Gal on IgG N-glycans
There are two classes of mucin-type O-glycosylation with respect to the density on a protein sequence: that is, clustered and nonclustered attachment. The latter is subdivided into solitary and scattered attachments, represented by apolipoprotein C-III (apoC-III) and hemopexin, respectively. To date, the occupancy variance in healthy individuals has not been addressed, especially for the clustered and scattered subclasses, because conventional analysis of glycoprotein glycans is conducted after release from proteins and thus does not address the occupancy issue. On the other hand, MS of glycopeptides has recently been used as an emerging method for site-specific analysis of glycoprotein glycans.17 However, it is mostly directed to N-glycosylation, and quantitation based on the mass spectra of glycopeptides is performed on an oligosaccharide basis but does not refer to saccharide compositions. In the present study, quantitation clearly characterized alterations of O-glycosylation associated with IgA nephropathy and RA. Galactosylation to form core-1 O-glycan is catalyzed by a core-1 β1-3 galactosyltransferase (C1 Gal-T1).43 Recently, the reduced galactosylation of IgA glycans in IgA nephropathy has been ascribed to concerted reductions in the expressions of C1 Gal-T1 and its molecular chaperone Cosmc.44 The hypogalactosylation of IgA1 is not shared by IgD in IgA nephropathy, suggesting that Ig O-glycosylation is differentially controlled during B cell maturation.45 On the other hand, the reduced galactosylation of IgG N-glycans in RA and other chronic inflammatory diseases results from a defect in the galactosyltransferase enzyme, namely, β4Gal-TI, in lymphocytes.46 However, the present study revealed that the molecular phenotype of IgA1 in RA involves a reduction of GalNAc, most likely indicating reduced O-glycosylation, distinct from the hypogalactosylation found in IgA nephropathy. The mechanism underlying the reduced galactosylation of IgG N-glycans may impact other glycosylation enzymes in IgA-producing B cells either primarily or secondarily, as suggested by the cytokinemediated alterations in the activity of enzymes that synthesize branching mucin carbohydrate structures in airway epithelial cells.47 Our observations on the O-glycosylation site of IgA1 have biological and pathological implications. O-glycosylation is initiated by GalNAc-Ts. In a report by Iwasaki et al., among six GalNAc transferases expressed in B cells, GalNAc-T2 had the highest catalytic activity in transferring GalNAc to a 20 residuelong synthetic peptide corresponding to positions 222-241.48 The same authors concluded that Thr-225 was not the liable attachment site and that Thr-233 next to the constantly glycosylated Ser-232 was never glycosylated based on their assay using recombinant GalNAc-T2 and a synthetic peptide substrate. On the contrary, in the present study, species modified at both Ser-232 and Thr-233 were demonstrated. In addition, Thr-225 is fully occupied, which is consistent with the previous mass spectrometric analysis of glycopeptides from IgA myeloma proteins.14 These results indicate that the synJournal of Proteome Research • Vol. 9, No. 3, 2010 1371
research articles thetic peptide used for the in vitro-assay by Iwasaki et al. was presumably too small to give or even mimic the real conformation of IgA1 protein. Alternatively, other GalNAc-Ts might be responsible for the glycosylation of Thr-225 and Ser-232 in vivo. Characterization of the O-glycans of hemopexin (Figure 1) is anticipated to provide a new diagnostic tool for CDG, applicable to O-glycosylation defects, while isoelectric focusing of apoC-III is currently used for subdividing unclassified CDGIIx disorders.49,50 Considering the high abundance of hemopexin (0.5-1.0 mg/mL plasma) as compared with apoCIII (80 µg/mL plasma) and the presence of a major Oglycosylation site with constant occupancy, hemopexin would be a potential alternative allowing detailed analysis of abnormalities in mucin-type O-glycan structures. In conclusion, the label-free quantitation of O-glycans described herein is anticipated to broaden the scope of glycopeptide analysis. Although this method is not applicable to sialylated species,17 the results for the saccharide compositions of GalNAc and Gal as well as site-occupancy determinations for IgA1 and hemopexin have demonstrated the usefulness of this method. Furthermore, this approach will provide the rigorous evaluation standards necessary for evaluating glycosylation status in various diseases, as demonstrated by our novel findings of IgA1 O-glycans from RA patients.
Acknowledgment. We are indebted to Prof. Yoshiyuki Hiki (Fujita Health University) for the generous gift of serum from an IgA nephropathy patient and Machiko Kadoya for technical assistance. This study was supported in part by a grant-in-aid for Scientific Research (B) #19390093 from the Ministry of Education, Culture, Sports, Science and Technology, Japan, and by the Takeda Science Foundation. Supporting Information Available: MALDI TOF mass spectra of glycopeptides from IgG Fc region and correlations between galactose contents on IgG N-glycan and IgA O-glycan. This material is available free of charge via the Internet at http://pubs.acs.org. References (1) Ohtsubo, K.; Marth, J. D. Glycosylation in cellular mechanisms of health and disease. Cell 2006, 126, 855–867. (2) Shental-Bechor, D.; Levy, Y. Folding of glycoproteins: toward understanding the biophysics of the glycosylation code. Curr. Opin. Struct. Biol. 2009, 19, 524–533. (3) Dube, D. H.; Bertozzi, C. R. Glycans in cancer and inflammation-potential for therapeutics and diagnostics. Nat. Rev. Drug Discovery 2005, 4, 477–488. (4) Arnold, J. N.; Saldova, R.; Hamid, U. M.; Rudd, P. M. Evaluation of the serum N-linked glycome for the diagnosis of cancer and chronic inflammation. Proteomics 2008, 8, 3284–3293. (5) Takahashi, M.; Kuroki, Y.; Ohtsubo, K.; Taniguchi, N. Core fucose and bisecting GlcNAc, the direct modifiers of the N-glycan core: their functions and target proteins. Carbohydr. Res. 2009, 344, 1387–1390. (6) Jaeken, J.; Matthijs, G. Congenital disorders of glycosylation: a rapidly expanding disease family. Annu. Rev. Genomics Hum. Genet. 2007, 8, 261–278. (7) Tian, E.; Ten Hagen, K. G. Recent insights into the biological roles of mucin-type O-glycosylation. Glycoconjugate J. 2009, 26, 325– 334. (8) Topaz, O.; Shurman, D. L.; Bergman, R.; Indelman, M.; Ratajczak, P.; Mizrachi, M.; Khamaysi, Z.; Behar, D.; Petronius, D.; Friedman, V.; Zelikovic, I.; Raimer, S.; Metzker, A.; Richard, G.; Sprecher, E. Mutations in GALNT3, encoding a protein involved in O-linked glycosylation, cause familial tumoral calcinosis. Nat. Genet. 2004, 36, 579–581. (9) Kato, K.; Jeanneau, C.; Tarp, M. A.; Benet-Pages, A.; LorenzDepiereux, B.; Bennett, E. P.; Mandel, U.; Strom, T. M.; Clausen, H. Polypeptide GalNAc-transferase T3 and familial tumoral cal-
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