POGLUT1 biallelic mutations cause myopathy with reduced satellite cells, α-dystroglycan hypoglycosylation and a distinctive radiological pattern.

Protein O-glucosyltransferase 1 (POGLUT1) activity is critical for the Notch signaling pathway, being one of the main enzymes responsible for the glycosylation of the extracellular domain of Notch receptors. A biallelic mutation in the POGLUT1 gene has been reported in one family as the cause of an adult-onset limb-girdle muscular dystrophy (LGMD R21; OMIM# 617232). As the result of a collaborative international effort, we have identified the first cohort of 15 patients with LGMD R21, from nine unrelated families coming from different countries, providing a reliable phenotype-genotype and mechanistic insight. Patients carrying novel mutations in POGLUT1 all displayed a clinical picture of limb-girdle muscle weakness. However, the age at onset was broadened from adult to congenital and infantile onset. Moreover, we now report that the unique muscle imaging pattern of "inside-to-outside" fatty degeneration observed in the original cases is indeed a defining feature of POGLUT1 muscular dystrophy. Experiments on muscle biopsies from patients revealed a remarkable and consistent decrease in the level of the NOTCH1 intracellular domain, reduction of the pool of satellite cells (SC), and evidence of α-dystroglycan hypoglycosylation. In vitro biochemical and cell-based assays suggested a pathogenic role of the novel POGLUT1 mutations, leading to reduced enzymatic activity and/or protein stability. The association between the POGLUT1 variants and the muscular phenotype was established by in vivo experiments analyzing the indirect flight muscle development in transgenic Drosophila, showing that the human POGLUT1 mutations reduced its myogenic activity. In line with the well-known role of the Notch pathway in the homeostasis of SC and muscle regeneration, SC-derived myoblasts from patients' muscle samples showed decreased proliferation and facilitated differentiation. Together, these observations suggest that alterations in SC biology caused by reduced Notch1 signaling result in muscular dystrophy in LGMD R21 patients, likely with additional contribution from α-dystroglycan hypoglycosylation. This study settles the muscular clinical phenotype linked to POGLUT1 mutations and establishes the pathogenic mechanism underlying this muscle disorder. The description of a specific imaging pattern of fatty degeneration and muscle pathology with a decrease of α-dystroglycan glycosylation provides excellent tools which will help diagnose and follow up LGMD R21 patients.

Molecular evolution of protein O-fucosyltransferase genes and splice variants.

O-Fucose has been described on both epidermal growth factor-like (EGF-like) repeats and Thrombospondin type 1 repeats (TSRs). The enzyme adding fucose to EGF-like repeats, protein O-fucosyltransferase 1 (Pofut1), is a soluble protein located in the lumen of endoplasmic reticulum (ER). A second protein O-fucosyltransferase, Pofut2, quite divergent from its homolog Pofut1, has recently been shown to O-fucosylate TSRs but not EGF-like repeats. To date, Pofut1 genes have only been characterized in human, mouse, and fly, and Pofut2 in mouse, fly, and partially in the nematode Caenorhabditis elegans. Here, we report cDNA sequences and genomic structures of bovine Pofut1 and Pofut2 genes and describe for the first time five alternative spliced transcripts for each gene. Only one transcript for both Pofut1 and Pofut2 encodes an active bovine O-fucosyltransferase. Variant transcript distribution was examined in 13 bovine tissues. Transcripts encoding active forms are ubiquitous, whereas other forms possess a more restricted tissue-expression profile. Sequence comparison and phylogenetic analyses revealed that both Pofut genes are present as a single copy in animal genomes, and their exon-intron organizations are conserved among vertebrates. The last common ancestor of all analyzed bilaterian species would be predicted to possess polyexonic Pofut genes in their genome.

O-fucose modifications of epidermal growth factor-like repeats and thrombospondin type 1 repeats: unusual modifications in unusual places.

Recent discoveries revealing that carbohydrate modifications play critical roles in a wide variety of biological processes have brought wide recognition to the field of glycobiology. Growing attention has focused on the function of unusual O-linked carbohydrate modifications such as O-fucose. O-fucose modifications have been described in several different protein contexts, including epidermal growth factor-like repeats and thrombospondin type 1 repeats. The O-fucose modifications on thrombospondin type 1 repeats have only recently been described, but the site of modification occurs in a region proposed to play a role in cell adhesion. O-fucose modifications on epidermal growth factor-like repeats have been described as important players in several signal transduction systems. For instance, Notch, a cell-surface signaling receptor required for many developmental events, bears multiple O-fucose saccharides on the epidermal growth factor-like repeat of its extracellular domain. The O-fucose moieties serve as a substrate for the beta1,3 N-acetylglucosaminyltransferase activity of Fringe, a known modifier of Notch function. The alteration of O-fucose structures by Fringe influences the ability of Notch ligands to activate the receptor and provides a means to regulate Notch signaling. Thus, O-fucose and Fringe provide a clear example of how carbohydrate modifications can have direct functional consequences on the proteins they modify.

Modification of epidermal growth factor-like repeats with O-fucose. Molecular cloning and expression of a novel GDP-fucose protein O-fucosyltransferase.

The O-fucose modification is found on epidermal growth factor-like repeats of a number of cell surface and secreted proteins. O-Fucose glycans play important roles in ligand-induced receptor signaling. For example, elongation of O-fucose on Notch by the beta1,3-N-acetylglucosaminyltransferase Fringe modulates the ability of Notch to respond to its ligands. The enzyme that adds O-fucose to epidermal growth factor-like repeats, GDP-fucose protein O-fucosyltransferase (O-FucT-1), was purified previously from Chinese hamster ovary (CHO) cells. Here we report the isolation of a cDNA that encodes human O-FucT-1. A probe deduced from N-terminal sequence analysis of purified CHO O-FucT-1 was used to screen a human heart cDNA library and expressed sequence tag and genomic data bases. The cDNA contains an open reading frame encoding a protein of 388 amino acids with a predicted N-terminal transmembrane sequence typical of a type II membrane orientation. Likewise, the mouse homolog obtained from an expressed sequence tag and 5'-rapid amplification of cDNA ends of a mouse liver cDNA library encodes a type II transmembrane protein of 393 amino acids with 90.4% identity to human O-FucT-1. Homologs were also found in Drosophila and Caenorhabditis elegans with 41.2 and 29.4% identity to human O-FucT-1, respectively. The human gene (POFUT1) is on chromosome 20 between PLAGL2 and KIF3B, near the centromere at 20p11. The mouse gene (Pofut1) maps near Plagl2 on a homologous region of mouse chromosome 2. POFUT1 gene transcripts were expressed in all tissues examined, consistent with the widespread localization of the modification. Expression of a soluble form of human O-FucT-1 in insect cells yielded a protein of the predicted molecular weight with O-FucT-1 kinetic and enzymatic properties similar to those of O-FucT-1 purified from CHO cells. The identification of the gene encoding protein O-fucosyltransferase I now makes possible mutational strategies to examine the functions of the unusual O-fucose post-translational modification.

Fringe is a glycosyltransferase that modifies Notch.

Notch receptors function in highly conserved intercellular signalling pathways that direct cell-fate decisions, proliferation and apoptosis in metazoans. Fringe proteins can positively and negatively modulate the ability of Notch ligands to activate the Notch receptor. Here we establish the biochemical mechanism of Fringe action. Drosophila and mammalian Fringe proteins possess a fucose-specific beta1,3 N-acetylglucosaminyltransferase activity that initiates elongation of O-linked fucose residues attached to epidermal growth factor-like sequence repeats of Notch. We obtained biological evidence that Fringe-dependent elongation of O-linked fucose on Notch modulates Notch signalling by using co-culture assays in mammalian cells and by expression of an enzymatically inactive Fringe mutant in Drosophila. The post-translational modification of Notch by Fringe represents a striking example of modulation of a signalling event by differential receptor glycosylation and identifies a mechanism that is likely to be relevant to other signalling pathways.

Mammalian Notch1 is modified with two unusual forms of O-linked glycosylation found on epidermal growth factor-like modules.

Notch is a large cell-surface receptor known to be an essential player in a wide variety of developmental cascades. Here we show that Notch1 endogenously expressed in Chinese hamster ovary cells is modified with O-linked fucose and O-linked glucose saccharides, two unusual forms of O-linked glycosylation found on epidermal growth factor-like (EGF) modules. Interestingly, both modifications occur as monosaccharide and oligosaccharide species. Through exoglycosidase digestions we determined that the O-linked fucose oligosaccharide is a tetrasaccharide with a structure identical to that found on human clotting factor IX: Sia-alpha2,3-Gal-beta1, 4-GlcNAc-beta1,3-Fuc-alpha1-O-Ser/Thr. The elongated form of O-linked glucose appears to be a trisaccharide. Notch1 is the first membrane-associated protein identified with either O-linked fucose or O-linked glucose modifications. It also represents the second protein discovered with an elongated form of O-linked fucose. The sites of glycosylation, which fall within the multiple EGF modules of Notch, are highly conserved across species and within Notch homologs. Since Notch is known to interact with its ligands through subsets of EGF modules, these results suggest that the O-linked carbohydrate modifications of these modules may influence receptor-ligand interactions.

The O-linked fucose glycosylation pathway: identification and characterization of a uridine diphosphoglucose: fucose-beta1,3-glucosyltransferase activity from Chinese hamster ovary cells.

O-Linked fucose is an unusual carbohydrate modification in which fucose is linked directly to the hydroxyl groups of serines or threonines. It has been found on the epidermal growth factor-like modules of several secreted proteins involved in blood coagulation and fibrinolysis. We have recently reported the existence of an elongated form of O-linked fucose in Chinese hamster ovary cells consisting of a glucose linked to the 3'-hydroxyl of fucose (Glcbeta1,3Fuc- O-Ser/Thr). This structure is highly unusual for two reasons. First, in mammalian systems fucose is usually a terminal modification of N - and O-linked oligosaccharides. Here the fucose is internal. Secondly, terminal beta-linked glucose is extremely rare on mammalian glycoconjugates. Thus, the Glcbeta1,3Fuc structure is a very unique mammalian carbohydrate structure. Here we report the identification and initial characterization of a novel enzyme activity capable of forming this unique linkage: UDP-glucose: O-linked fucose beta1,3 glucosyltransferase. The enzyme utilizes UDP-glucose as the high energy donor and transfers glucose to alpha-linked fucose residues. The activity is linearly dependent on time, enzyme, and substrate concentrations and is enhanced in the presence of manganese ions. Activity is present in extracts of cultured cells from a variety of species (hamster, human, mouse, rat, chicken) and is enriched in brain and spleen of a normal adult rat. Thus, while this glycosyltransferase appears to be widespread in biology, it forms a very unique linkage, and it represents the first mammalian enzyme identified capable of elongating fucose.

SV40 large T antigen is modified with O-linked N-acetylglucosamine but not with other forms of glycosylation.

SV40 large T antigen has been reported to be modified with several different sugars including N-acetylglucosamine, galactose, and mannose. In this report we have reexamined the glycosylation of T antigen and found that while we could detect modification with N-acetylglucosamine, we could not detect any other sugars on the protein. Surprisingly, even though [3H]galactose could be metabolically incorporated into the protein, analysis showed that all of the radioactivity in T antigen had been converted to other species. The N-acetylglucosamine was demonstrated to be linked to the protein in the form of O-linked N-acetylglucosamine, the best characterized form of nuclear and cytoplasmic glycosylation in mammalian systems. We have localized the major site of glycosylation to the amino terminal portion of the molecule. Analysis of mutated T antigen where serines 111/112 were substituted with alanine suggest that these residues constitute a glycosylation site on the protein. These two serines fall within a typical O-linked N-acetylglucosamine glycosylation site (PSS) and are also known to be phosphorylated. Thus, it is likely that competition between phosphorylation and glycosylation occurs at this site.

Modulation of O-linked N-acetylglucosamine levels on nuclear and cytoplasmic proteins in vivo using the peptide O-GlcNAc-beta-N-acetylglucosaminidase inhibitor O-(2-acetamido-2-deoxy-D-glucopyranosylidene)amino-N-phenylcarbamate.

O-Linked N-acetylglucosamine (O-GlcNAc) is a ubiquitous and abundant post-translational modification found on nuclear and cytoplasmic proteins and is thought to be a dynamically regulated modification much like phosphorylation. In this study we have demonstrated that O-(2-acetamido-2-deoxy-D-glucopyranosylidene)amino-N-phenylcarbama te (PUGNAc), a potent in vitro inhibitor of the enzyme responsible for the removal of O-GlcNAc from proteins (peptide O-GlcNAc-beta-N-acetylglucosaminidase), can be used to increase O-GlcNAc levels on nuclear and cytoplasmic proteins in vivo. Overall, PUGNAc caused approximately a 2-fold increase in O-GlcNAc levels in the human colon cancer cells, HT29, although the effects on individual proteins varied. The increase appeared to be the result of the direct inhibition of the peptide O-GlcNAc-beta-N-acetylglucosaminidase since neither the O-GlcNAc transferase nor UDP-GlcNAc levels were affected by the treatment. O-GlcNAc levels in other cell lines tested (NIH 3T3, CV-1, and HeLa) were also affected by PUGNAc, although the effects on HeLa cells were minimal. At the concentrations tested, PUGNAc was non-toxic and had no affect on the growth rate of any of the cell lines examined. Interestingly, we demonstrated that an increase in O-GlcNAc levels on the transcription factor Sp1 resulted in a reciprocal decrease in its level of phosphorylation, supporting the hypothesis that O-GlcNAc competes with phosphate on some proteins. These studies demonstrate that PUGNAc is an effective inhibitor of O-GlcNAc turnover within cells and can be used to selectively alter the extent of O-GlcNAc on cellular proteins.

Calf thymus high mobility group proteins are nonenzymatically glycated but not significantly glycosylated.

Over the past decade, there have been many reports suggesting the presence of complex carbohydrates on nuclear and cytoplasmic proteins in mammalian cells. Some of the most often cited of these reports deal with the glycosylation of the high mobility group (HMG) proteins. These are relatively abundant chromosomal proteins that are known to be associated with nucleosomes and actively transcribed regions of chromatin. The original report describing HMG protein glycosylation presented several lines of evidence suggesting that these proteins are glycosylated, including carbohydrate compositional analysis and periodic-acid Schiff staining. We have attempted to repeat these observations with more highly purified protein than was utilized in the original study. Using carbohydrate compositional analysis performed by high pH anion exchange chromatography coupled to pulsed-amperometric detection, we saw no evidence for significant glycosylation of these proteins. In addition, we found no evidence for the presence of O-GlcNAc, a well known form of nuclear glycosylation. The HMG proteins did react with periodate, suggesting the presence of a modification containing cis-diols on the protein. Several tryptic peptides isolated from HMG 14 and 17 which retained the periodate reactivity had in common lysine residues, suggesting a potential modification of the straightepsilon-amino groups of lysines such as nonenzymatic glycation. Western blot analysis of the HMG proteins using anti-advanced glycation endproducts (AGE) antibodies confirmed the presence of glycation products on the HMG proteins.

The O-linked fucose glycosylation pathway. Evidence for protein-specific elongation of o-linked fucose in Chinese hamster ovary cells.

O-Linked fucose is an unusual form of glycosylation recently shown to modify the hydroxyls of serine or threonine residues at a strict consensus site within epidermal growth factor-like domains of several serum proteins. Here we demonstrate that Chinese hamster ovary cells modify numerous proteins with O-linked fucose and that the fucose is elongated on specific proteins. We have identified at least two forms of O-linked fucose elongation in Chinese hamster ovary cells: a disaccharide (Glcbeta1,3Fuc) and a larger oligosaccharide of indeterminate structure. Interestingly, it appears that the level of monosaccharide accumulates in the cells over time whereas the disaccharide does not. Analysis of the O-linked fucose-containing saccharides on individual proteins revealed that some proteins are modified with the monosaccharide only, whereas others are modified with monosaccharide and disaccharide, or monosaccharide and oligosaccharide. These results suggest that elongation of the O-linked fucose monosaccharide is a protein-specific phenomena. The presence of elongated O-linked fucose moieties suggests that a novel glycosylation pathway exists in mammalian cells with O-linked fucose as the core.

Mitotic arrest with nocodazole induces selective changes in the level of O-linked N-acetylglucosamine and accumulation of incompletely processed N-glycans on proteins from HT29 cells.

O-Linked N-acetylglucosamine (O-GlcNAc) is a ubiquitous and abundant protein modification found on nuclear and cytoplasmic proteins. Several lines of evidence suggest that it is a highly dynamic modification and that the levels of this sugar on proteins may be regulated. Previous workers (Chou, C. F., and Omary, M. B. (1993) J. Biol. Chem. 268, 4465-4472) have shown that mitotic arrest with microtubule-destabilizing agents such as nocodazole causes an increase in the O-GlcNAc levels on keratins in the human colon cancer cell line HT29. We have sought to determine whether this increase in glycosylation is a general (i.e. occurring on many proteins) or a limited (i.e. occurring only on the keratins) process. A general increase would suggest that the microtubule-destabilizing agents were somehow affecting the enzymes responsible for addition and/or removal of O-GlcNAc. Our results suggest that the changes in O-GlcNAc induced by nocodazole are selective for the keratins. The levels of O-GlcNAc on other proteins, including the nuclear pore protein p62 and the transcription factor Sp1, are not significantly affected by this treatment. In agreement with these findings, nocodazole treatment caused no change in the activity of the enzymes responsible for addition or removal of O-GlcNAc as determined by direct in vitro assay. Interestingly, nocodazole treatment did cause a dramatic increase in modification of N-glycans with terminal GlcNAc residues on numerous proteins. Potential mechanisms for this and the change in glycosylation of the keratins are discussed.

Synergy between trehalose and Hsp104 for thermotolerance in Saccharomyces cerevisiae.

We isolated a mutant strain unable to acquire heat shock resistance in stationary phase. Two mutations contributed to this phenotype. One mutation was at the TPS2 locus, which encodes trehalose-6-phosphate phosphatase. The mutant fails to make trehalose and accumulates trehalose-6-phosphate. The other mutation was at the HSP104 locus. Gene disruptions showed that tps2 and hsp104 null mutants each produced moderate heat shock sensitivity in stationary phase cells. The two mutations were synergistic and the double mutant had little or no stationary phase-induced heat shock resistance. The same effect was seen in the tps1 (trehalose-6-phosphate synthase) hsp104 double mutant, suggesting that the extreme heat shock sensitivity was due mainly to a lack of trehalose rather than to the presence of trehalose-6-phosphate. However, accumulation of trehalose-6-phosphate did cause some phenotypes in the tps2 mutant, such as temperature sensitivity for growth. Finally, we isolated a high copy number suppressor of the temperature sensitivity of tps2, which we call PMU1, which reduced the levels of trehalose-6-phosphate in tps2 mutants. The encoded protein has a region homologous to the active site of phosphomutases.

Core fucosylation of high-mannose-type oligosaccharides in GlcNAc transferase I-deficient (Lec1) CHO cells.

During studies on the fucosylation of endogenous proteins in parental (Pro5) and N-acetyl-D-glucosamine (GlcNAc) transferase I-deficient (Lec1) Chinese hamster ovary (CHO) cells, we observed that Lec1 cells incorporate approximately 10-fold less [3H]fucose into macromolecules than Pro5 cells. Interestingly, most of the labelled oligosaccharides from both cell types could be released from the macromolecules by digestion with peptide N-glycosidase F (PNGase F). This was unexpected for Lec1 cells because they do not synthesize complex- or hybrid-type N-glycans. Structural analyses of the fucosylated oligosaccharides from Lec1 cells showed the fucose to be in an alpha 1,6 linkage to the core GlcNAc of relatively small oligomannose N-glycans (Man4GlcNAc2 and Man5GlcNAc2, where Man is D-mannose). Comparing the sizes of oligomannose N-glycans from Pro5 and Lec1 cells demonstrated a much higher proportion of the small (Man4GlcNAc2 and Man5GlcNAc2) oligomannose species in Lec1 cells. These results suggest that the core alpha 1,6 fucosyltransferase will fucosylate small (Man4-Man5GlcNAc2), but not large (Man8-Man9GlcNAc2) oligomannose N-glycans.

Localization of O-GlcNAc modification on the serum response transcription factor.

A unique form of nucleoplasmic and cytoplasmic protein glycosylation, O-linked GlcNAc, has previously been detected, using Gal transferase labeling techniques, on a myriad of proteins (for review see Hart, G. W., Haltiwanger, R. S., Holt, G. D., and Kelly, W. G. (1989a) Annu. Rev. Biochem. 58, 841-874), including many RNA polymerase II transcription factors (Jackson, S. P., and Tjian, R. (1988) Cell 55, 125-133). However, virtually nothing is known about the degree of glycosylation at individual sites, or, indeed, the actual sites of attachment of O-GlcNAc on transcription factors. In this paper we provide rigorous evidence for the occurrence and locations of O-GlcNAc on the c-fos transcription factor, serum response factor (SRF), expressed in an insect cell line. Fast atom bombardment mass spectrometry (FAB-MS) of proteolytic digests of SRF provides evidence for the presence of a single substoichiometric O-GlcNAc residue on each of four peptides isolated after sequential cyanogen bromide, tryptic, and proline specific enzyme digestion: these peptides are 306VSASVSP312, 274GTTSTIQTAP283, 313SAVSSADGTVLK324, and 374DSSTDLTQTSSSGTVTLP391. Using an array of techniques, including manual Edman degradation, aminopeptidase, and elastase digestion, together with FAB-MS, the major sites of O-GlcNAc attachment were shown to be serine residues within short tandem repeat regions. The highest level of glycosylation was found on the SSS tandem repeat of peptide (374-391) which is situated within the transcriptional activation domain of SRF. The other glycosylation sites observed in SRF are located in the region of the protein between the DNA binding domain and the transcriptional activation domain. Glycosylation of peptides (274-283) and (313-324) was found to occur on the serine in the TTST tandem repeat and on serine 316 in the SS repeat, respectively. The lowest level of glycosylation was recovered in peptide (306-312) which lacks tandem repeats. All the glycosylation sites identified in SRF are situated in a relatively short region of the primary sequence close to or within the transcriptional activation domain which is distant from the major sites of phosphorylation catalyzed by casein kinase II.

Glycosylation of nuclear and cytoplasmic proteins. Purification and characterization of a uridine diphospho-N-acetylglucosamine:polypeptide beta-N-acetylglucosaminyltransferase.

Using a combination of conventional and affinity chromatographic techniques, we have purified a uridine diphospho-N-acetylglucosamine:polypeptide beta-N-acetylglucosaminyltransferase (O-GlcNAc transferase) over 30,000-fold from rat liver cytosol. The transferase is soluble and very large, migrating with an apparent molecular weight of 340,000 on molecular sieve chromatography. Analysis of the purified enzyme on sodium dodecyl sulfate-polyacrylamide gel electrophoresis reveals two protein species migrating at 110 (alpha subunit) and 78 (beta subunit) kDa in approximately a two-to-one ratio. Thus, the enzyme likely exists as a heterotrimer complex with two subunits of 110 kDa and one of 78 kDa (alpha 2 beta). The alpha subunit appears to contain the enzyme's active site since it is selectively radiolabeled by a specific photoaffinity probe (4-[beta-32P]thiouridine diphosphate). Photoinactivation and photolabeling of the enzyme are dependent on time and long wavelength ultraviolet light. Photolabeling of the alpha subunit is specifically blocked by UDP. The enzyme has an extremely high affinity for UDP-GlcNAc (Km = 545 nM). This unusually high affinity for the sugar nucleotide donor probably provides the enzyme an advantage over the nucleotide transporters in the endoplasmic reticulum and Golgi apparatus which compete for available cytoplasmic UDP-GlcNAc. The multimeric state and large size of the O-GlcNAc transferase imply that its activity may be highly regulated within the cell.

High-sensitivity FAB-MS strategies for O-GlcNAc characterization.

In this paper we report the first application of fast atom bombardment mass spectrometry (FAB-MS) to O-linked N-acetylglucosamine (O-GlcNAc)-bearing glycopeptides. Using N-acetylgalactosamine (GalNAc)- and Gal-GalNAc-containing glycopeptides (isolated from Tn glycophorin and desialylated normal glycophorin, respectively) as readily available model compounds, rapid and sensitive derivatization/FAB-MS strategies applicable to serine/threonine-rich glycopeptides have been devised. Peptides and glycopeptides were propionylated in a 1 min reaction using a mixture of trifluoroacetic anhydride and propionic acid, and the product mixtures were analysed directly by FAB-MS. Glycopeptides and peptides rich in hydroxylated residues afforded characteristic clusters of molecular ions at high sensitivity. Additional sensitivity enhancement was achieved by prior esterification of carboxyl groups. These methods were used in a study of O-GlcNAc glycopeptides produced by purified O-GlcNAc transferase addition of GlcNAc to the synthetic peptides YSDSPSTST and YSGSPSTST in which Y is tyrosine, S is serine, D is aspartic acid, P is proline, T is threonine and G is glycine. The propionyl derivatives afforded high-quality spectra which unequivocally showed that the majority of the glycopeptides were substituted with a single GlcNAc residue. Low pmol quantities of material gave detectable signals. The propionylation/FAB-MS procedure has been combined with gas-phase sequencing strategies and shows promise for defining the sites of glycosylation of O-GlcNAc glycopeptides that are available in limited quantities.