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.     

Schistosoma mansoni synthesizes glycoproteins containing terminal O-linked N-acetylglucosamine residues

In this report, we describe our studies on the structures of the O-linked oligosaccharides in glycoproteins synthesized by the human blood fluke Schistosoma mansoni. Adult male schistosomes were incubated with either [2-3H]mannose, [6-3H]glucosamine, or [6-3H]galactose to metabolically radiolabel newly synthesized glycoproteins. Sodium dodecyl sulfate/polyacrylamide gel electrophoresis and fluorographic analyses indicated that many glycoproteins were labeled by each of the radioactive precursors. Glycopeptides were prepared from radiolabeled glycoproteins by pronase treatment and fractionated on columns of concanavalin A-Sepharose and pea lectin-agarose. The O-linked oligosaccharides were released from glycopeptides by treatment with mild base/borohydride. All O-linked material was found in glycopeptides not bound by either of the immobilized lectins. The structures of the released chains were then analyzed by a variety of techniques. Our results demonstrate that the schistosomes synthesize glycoproteins containing two major types of simple O-linked sugar chains. One type, which represents a minor fraction of the O-linked oligosaccharides, contains N-acetylgalactosamine linked to peptide. These O-linked chains occur as terminal O-linked N-acetylgalactosamine and the O-linked disaccharide, galactose----N-acetylgalactosamine. Sialic acid was not present in either of these O-linked chains or in any other glycopeptides derived from adult male schistosomes. However, the major type of O-linked chain in glycoproteins synthesized by adult schistosomes is an unusual terminal O-linked N-acetylglucosamine linked to peptide. This latter structure represents approximately 10% of the total radioactive N-acetylglucosamine recovered in all glycopeptides. Our results also suggest the possibility that the O-linked oligosaccharides are highly clustered on the glycopeptides.     

Carbohydrate components of lipovitellin of the sand crab Emerita asiatica

Lipovitellin II (Lv II), the major yolk protein of the anomuran crab Emerita asiatica, was purified using heparin-sepharose affinity column chromatography. The purified Lv II was a glycoprotein as it was stainable with periodic acid-Schiff's reagent. Quantitative analysis of sugars showed the presence of fucose, mannose, galactosamine, N-linked oligosaccharides, as well as O-linked oligosaccharides containing N-acetyl hexosamine as the terminal residue. The amount of N-linked oligosaccharides is higher than that of the O-linked oligosaccharides. Biogel P-4 column chromatographic separation of the radiolabeled oligosaccharides of Lv II showed the presence of five different O-linked oligosaccharides and four different N-linked oligosaccharide species. HPTLC separation of the neoglycolipids prepared from the O-linked oligosaccharides also showed the presence of five different O-linked oligosaccharide species. N-linked oligosaccharides contain significant quantities of mannose. Unisil column chromatographic purification in conjunction with HPTLC separation revealed three neutral glycolipid species such as monoglycosylceramide, diglycosylceramide, and triglycosylceramide in the Lv II. The functional significance of these carbohydrate components of the major yolk protein during embryogenesis of the sand crab is discussed.     

Deletion of clustered O-linked carbohydrates does not impair function of low density lipoprotein receptor in transfected fibroblasts

A single exon in the gene for the receptor for plasma low density lipoprotein (LDL) encodes a region of clustered serine and threonine residues that is immediately external to the membrane-spanning sequence. This region has been proposed as the site of clustered O-linked carbohydrate chains. In the current studies we have deleted the 144 base pairs (48 amino acids) that encode this serine- and threonine-rich region from the cDNA for the human LDL receptor. Upon transfection into receptor-deficient hamster fibroblasts, this mutated cDNA encoded a shortened receptor that no longer showed an anomalously high molecular weight on sodium dodecyl sulfate-polyacrylamide gel electrophoresis. Labeling with [3H]glucosamine confirmed the lack of clustered O-linked sugars and further revealed that the shortened receptor and the normal receptor both contained isolated O-linked carbohydrate chains attached to the NH2-terminal portion of the protein. The ratio of clustered to isolated O-linked sugar chains in the normal receptor was estimated to be approximately 4-6 to 1. Despite the loss of clustered O-linked carbohydrate, the LDL receptor encoded by the deletion-bearing cDNA bound and internalized LDL normally. It also recycled normally and exhibited a normal half-life. We conclude that: 1) the serine- and threonine-rich region of the LDL receptor is the site for addition of clustered O-linked carbohydrates; 2) the receptor contains a small number of isolated chains of O-linked carbohydrates in addition to the clustered chains; and 3) the clustered O-linked carbohydrates are not essential for LDL receptor function in cultured hamster fibroblasts.     

O-Linked glycome and proteome of high-molecular-mass proteins in human ovarian cancer ascites: Identification of sulfation, disialic acid and O-linked fucose

The O-linked glycosylation of the main acidic high-molecular-weight glycoprotein from ascites fluid from patients with ovarian cancer were analyzed. The O-linked oligosaccharides were shown to consist of mainly highly sialylated core 1 and 2 structures with a smaller amount of sulfated core 2 structures. These structures were shown to be able to be further extended into small keratan sulfate (KS)-type oligosaccharides with up to four N-acetyllactosamine units. Proteomic studies of the acidic fraction of ascites fluid from patients with ovarian cancer showed that this fraction was enriched in proteoglycans. Among them, lumican, agrin, versican and dystroglycans were potential candidates, with threonine- and serine-rich domains that could carry a significant amount of O-linked glycosylation, including also the O-linked KS. Glycomic analysis using liquid chromatography (LC)-tandem mass spectrometry (MS/MS) also showed that the disialic acid NeuAc-NeuAc- was frequently found as the terminating structure on the O-linked core 1 and 2 oligosaccharides from one ascites sample. Also, a small amount of the epidermal growth factor (EGF)-associated O-linked fucose structure Gal-GlcNAc-Fucitol was detected with and without sialic acid in the LC-MS/MS analysis. Candidate proteins containing O-linked fucose were suggested to be proteoglycan-type molecules containing the O-linked fucose EGF consensus domain.     

The chemistry and biology of mucin-type O-linked glycosylation

Mucin-type O-linked glycosylation is a fundamental post-translational modification that is involved in a variety of important biological processes. However, the lack of chemical tools to study mucin-type O-linked glycosylation has hindered our molecular understanding of O-linked glycans in many biological contexts. The review discusses the significance of mucin-type O-linked glycosylation initiated by the polypeptide N-acetylgalactosaminyltransferases in biology and development of chemical tools to study these enzymes and their substrates.     

Ataxin-10 interacts with O-GlcNAc transferase OGT in pancreatic beta cells

Several nuclear and cytoplasmic proteins in metazoans are modified by O-linked N-acetylglucosamine (O-GlcNAc). This modification is dynamic and reversible similar to phosphorylation and is catalyzed by the O-linked GlcNAc transferase (OGT). Hyperglycemia has been shown to increase O-GlcNAc levels in pancreatic beta cells, which appears to interfere with beta-cell function. To obtain a better understanding of the role of O-linked GlcNAc modification in beta cells, we have isolated OGT interacting proteins from a cDNA library made from the mouse insulinoma MIN6 cell line. We describe here the identification of Ataxin-10, encoded by the SCA10 (spinocerebellar ataxia type 10) gene as an OGT interacting protein. Mutations in the SCA10 gene cause progressive cerebellar ataxias and seizures. We demonstrate that SCA10 interacts with OGT in vivo and is modified by O-linked glycosylation in MIN6 cells, suggesting a novel role for the Ataxin-10 protein in pancreatic beta cells.     

Glycoproteomics of milk: differences in sugar epitopes on human and bovine milk fat globule membranes

Oligosaccharides from human and bovine milk fat globule membranes were analyzed by LC-MS and LC-MS/MS. Global release of N-linked and O-linked oligosaccharides showed both to be highly sialylated, with bovine peak-lactating milk O-linked oligosaccharides presenting as mono- and disialylated core 1 oligosaccharides (Galbeta1-3GalNAcol), while human milk had core type 2 oligosaccharides (Galbeta1-3(GlcNAcbeta1-6)GalNAcol) with sialylation on the C-3 branch. The C-6 branch of these structures was extended with branched and unbranched N-acetyllactosamine units terminating in blood group H and Lewis type epitopes. These epitopes were also presented on the reducing terminus of the human, but not the bovine, N-linked oligosaccharides. The O-linked structures were found to be attached to the high molecular mass mucins isolated by agarose-polyacrylamide composite gel electrophoresis, where MUC1 and MUC4 were present. Analysis of bovine colostrum showed that O-linked core 2 oligosaccharides are present at the early stage (3 days after birth) but are down-regulated as lactation develops. This data indicates that human milk may provide different innate immune protection against pathogens compared to bovine milk, as evidenced by the presence of Lewis b epitope, a target for the Helicobacter pylori bacteria, on human, but not bovine, milk fat globule membrane mucins. In addition, non-mucin-type O-linked fucosylated oligosaccharides were found (NeuAc-Gal-GlcNAc1-3Fuc-ol in bovine milk and Gal-GlcNAc1-3Fuc-ol in human milk). The O-linked fucose structure in human milk is the first to our knowledge to be found on high molecular mass mucin-type molecules.     

O-linked mannose composition of secreted invertase of Saccharomyces cerevisiae

The secreted invertase (EC 3.2.1.26) of Saccharomyces cerevisiae is a glycoenzyme that contains N- and O-linked mannoses in 40/1 proportion. The small amount of mannose chains O-linked to invertase is distributed as follows: mannose (20%), mannobiose (50%), mannotriose (6%), mannotetraose (7%) and mannopentaose (17%).     

O-linked oligosaccharides are acquired by herpes simplex virus glycoproteins in the Golgi apparatus

The O-linked oligosaccharides on mature forms of herpes simplex virus type 1 (HSV1) glycoproteins were characterized, and were found to account largely for the lower electrophoretic mobilities of these forms relative to the mobilities of immature forms. Other posttranslational modifications of HSV1 glycoproteins (designated gB, gC, gD and gE) were related temporally to the discrete shifts in electrophoretic mobilities that signal acquisition of the O-linked oligosaccharides. Fatty acid acylation (principally of gE) could be detected just prior to the shifts, whereas conversion of high-mannosetype N-linked oligosaccharides to the complex type occurred coincident with the shifts. The addition of O-linked oligosaccharides did not occur in cells treated with the ionophore monensin or in a ricinresistant cell line defective in the processing of N-linked oligosaccharides. We conclude that extension of O-linked oligosaccharide chains on HSV1 glycoproteins, and probably also attachment of the first O-linked sugars, occurs as a late posttranslational modification in the Golgi apparatus.     

Studying O-linked protein glycosylations in human plasma

Recent investigations have implicated aberrant glycosylations in various malignancies, including epithelial ovarian cancer (EOC). The protocol here identifies O-linked carbohydrate patterns in EOC plasma glycoproteins through chemical cleavage and purification of these glycans. Dialyzed plasma is subjected to reductive beta-elimination with alkaline borohydride to release O-linked oligosaccharides from glycoproteins. Enrichment of released glycans, as well as removal of peptide and other contaminants, is followed by carbohydrate pattern analysis with MALDI-FT-ICR-MS.     

O-linked glycan expression during Drosophila development

Mucin-type O-linked glycosylation is an evolutionarily conserved protein modification that is essential for viability in Drosophila melanogaster. However, the exact role of O-glycans and the identity of the crucial apoproteins modified with O-linked N-acetylgalactosamine (O-GalNAc) remain unknown. In an effort to elucidate the O-linked glycans expressed during Drosophila development, we have employed fluorescent confocal microscopy using a battery of lectins and an antibody specific for the GalNAcalpha-Ser/Thr structure (Tn antigen). Confocal microscopy provides high-resolution images of the diversity of glycans expressed in many developing organ systems. In particular, O-glycans are highly expressed on a number of ectodermally derived tissues such as the salivary glands, developing gut, and the tracheal system, suggesting a role for O-glycans in cell polarity and tube formation common to these organs. Additionally, O-glycans are found in the developing nervous system and within subregions of developing tissues known to be active in cell signaling events. This study provides us with temporal and spatial information regarding O-glycan expression as well as a set of reagents for the isolation of glycoproteins from specific developmental stages and organ systems. This information will aid us in identifying the in vivo substrates of the UDP-GalNAc: polypeptide N-acetylgalactosaminyltranferases, in a continuing effort to define the biological role of O-linked glycoproteins during development.     

The role of O-linked sugars in determining the very low density lipoprotein receptor stability or release from the cell.

The very low density lipoprotein receptor is a member of the low density lipoprotein receptor supergene family for which two isoforms have been reported, one lacking and the other containing an O-linked sugar domain. In order to gain insight into their functionality, transient and stable transformants separately overexpressing previously cloned bovine variants were analyzed. We report evidence that the variant lacking the O-linked sugar domain presented a rapid cleavage from the cell and that a large amino-terminal very low density lipoprotein receptor fragment was released into the culture medium. As only minor proteolysis was involved in the other very low density lipoprotein receptor variant, the clustered O-linked sugar domain may be responsible for blocking the access to the protease-sensitive site(s). To test this hypothesis, a mutant Chinese hamster ovary cell line, ldlD, with a reversible defect in the protein O-glycosylation, was used. The instability of the O-linked sugar-deficient very low density lipoprotein receptor on the cell surface was comparable to that induced by the proteolysis of the variant lacking the O-linked sugar domain. Moreover, our data suggest that the O-linked sugar domain may also protect the very low density lipoprotein receptor against unspecific proteolysis. Taken together, these results indicate that the presence of the O-linked sugar domain may be required for the stable expression of the very low density lipoprotein receptor on the cell surface and its absence may be required for release of the receptor to the extracellular space. The exclusive expression of the variant lacking the O-linked sugar domain in the bovine aortic endothelium opens new perspectives in the physiological significance of the very low density lipoprotein receptor.     

Isolation of glycopeptides containing O-linked oligosaccharides by lectin affinity chromatography on jacalin-agarose

Jacalin is a lectin which has high specificity and affinity for the core disaccharide, 1-beta-galactopyranosyl-3-(alpha-2-acetamido-2-deoxygalactopyranoside ), in O-linked oligosaccharides. Here, it is shown that this lectin can be used for isolation of glycopeptides bearing O-linked oligosaccharides. Peptides produced by digestion of reduced and carboxamidomethylated human plasminogen or of bovine protein Z were chromatographed on a column of jacalin-agarose. Reverse-phase high-performance liquid chromatography revealed that two peptides from plasminogen and one from protein Z were eluted from the jacalin-agarose column by alpha-methylgalactopyranoside. Amino acid sequence and compositional analysis showed that both of the peptides from plasminogen consisted of residues 330-357 and that the single peptide from protein Z represented residues 385-396. These sequences contain the single known site of attachment of O-linked oligosaccharides to these proteins. The present analysis suggested that there may be a fraction of plasminogen with two sites of O-linked glycosylation. The two tryptic peptides isolated from plasminogen represented the same segment of the protein but sequence analysis showed that one peptide was modified only at Thr346, the known site of glycosylation, and the other peptide contained a modification of Ser339 as well. Results of the present study indicate that lectin affinity chromatography using jacalin-agarose can be a useful technique for isolating glycopeptides containing O-linked oligosaccharides and thereby localizing sites of attachment of these oligosaccharides.     

Protein glycosylation in bacteria: sweeter than ever

Investigations into bacterial protein glycosylation continue to progress rapidly. It is now established that bacteria possess both N-linked and O-linked glycosylation pathways that display many commonalities with their eukaryotic and archaeal counterparts as well as some unexpected variations. In bacteria, protein glycosylation is not restricted to pathogens but also exists in commensal organisms such as certain Bacteroides species, and both the N-linked and O-linked glycosylation pathways can modify multiple proteins. Improving our understanding of the intricacies of bacterial protein glycosylation systems should lead to new opportunities to manipulate these pathways in order to engineer glycoproteins with potential value as novel vaccines.     

An inhibitor of O-glycosylation induces apoptosis in NIH3T3 cells and developing mouse embryonic mandibular tissues

The family of UDP-GalNAc:polypeptide N-acetylgalactosaminyltransferases (ppGaNTases) is responsible for initiating mucin-type O-linked glycosylation in higher eukaryotes. To begin to examine the biological role of O-linked glycosylation, mammalian cells were treated with a small molecule inhibitor (designated 1-68A, Ref. 15) of ppGaNTase activity. NIH3T3 cells exposed to the inhibitor were shown to undergo a significant reduction in cell surface O-glycosylation as detected by staining with jacalin and peanut agglutinin lectins after 30 min of treatment; no reduction in staining using antibodies to O-linked N-acetylglucosamine or the lectin concanavalin A was detected. Apoptosis was also observed in treated cells after 45 min of exposure, ostensibly following the O-glycosylation reduction. Overexpression of several different ppGaNTase isoforms restored cell surface O-glycosylation and rescued inhibitor-induced apoptosis. Additionally, mouse embryonic mandibular organ cultures exposed to 1-68A developed abnormally, presumably because of epithelial and mesenchymal apoptosis that followed a reduction in jacalin and peanut agglutinin staining. Our studies suggest that mucin-type O-linked glycosylation may be required for normal development and that ppGaNTases may play a role in the regulation of apoptosis.     

Histochemical analysis of glycoconjugates in the domestic cat testis

The localization and characterization of oligosaccharide sequences in the cat testis was investigated using 12 lectins in combination with the beta-elimination reaction, N-Glycosidase F and sialidase digestion. Leydig cells expressed O-linked glycans with terminal alphaGalNAc (HPA reactivity) and N-glycans with terminal/internal alphaMan (Con A affinity). The basement membrane showed terminal Neu5Acalpha2,6Gal/GalNAc, Galbeta1,3GalNAc, alpha/betaGalNAc, and GlcNAc (SNA, PNA, HPA, SBA, GSA II reactivity) in O-linked oligosaccharides, terminal Galbeta1,4GlcNAc (RCA120 staining) and alphaMan in N-linked oligosaccharides; in addition, terminal Neu5acalpha2,3Galbeta1,4GlcNac, Forssman pentasaccharide, alphaGal, alphaL-Fuc and internal GlcNAc (MAL II, DBA, GSA I-B4, UEA I, KOH-sialidase-WGA affinity) formed both O- and N-linked oligosaccharides. The Sertoli cells cytoplasm contained terminal Neu5Ac-Galbeta1,4GlcNAc, Neu5Ac-betaGalNAc as well as internal GlcNAc in O-linked glycans, alphaMan in N-linked glycoproteins and terminal Neu5Acalpha2,6Gal/ GalNAc in both O- and N-linked oligosaccharides. Spermatogonia exhibited cytoplasmic N-linked glycoproteins with alphaMan residues. The spermatocytes cytoplasm expressed terminal Neu5Acalpha2,3Galbeta1,4 GlcNAc and Galbeta1,3GalNAc in O-linked oligosaccharides, terminal Galbeta1,4GlcNAc and alpha/betaGalNAc in N-linked glycoconjugates. The Golgi region showed terminal Neu5Acalpha2,3Galbeta1,4GlcNac, Galbeta1,4GlcNAc, Forssman pentasaccharide, and alphaGalNAc in O-linked oligosaccharides, alphaMan and terminal betaGal in N-linked oligosaccharides. The acrosomes of Golgi-phase spermatids expressed terminal Galbeta1,3GalNAc, Galbeta1,4GlcNAc, Forssmann pentasaccharide, alpha/betaGalNAc, alphaGal and internal GlcNAc in O-linked oligosaccharides, terminal alpha/betaGalNAc, alphaGal and terminal/internal alphaMan in N-linked glycoproteins. The acrosomes of cap-phase spermatids lacked internal Forssman pentasaccharide and alphaGal, while having increased alpha/betaGalNAc. The acrosomes of elongated spermatids did not show terminal Galbeta1,3GalNAc, displayed terminal Galbeta1,4GlcNAc and alpha/betaGalNAc in N-glycans and Neu5Ac-Galbeta1,3GalNAc in O-linked oligosaccharides.     

The subcellular distribution of terminal N-acetylglucosamine moieties. Localization of a novel protein-saccharide linkage, O-linked GlcNAc

Previously our laboratory reported the discovery of a novel protein-saccharide linkage in which single N-acetylglucosamine (GlcNAc) residues are attached in O-linkages to protein (Torres, C. R., and Hart, G. W. (1984) J. Biol. Chem. 259, 3308-3317). This linkage was first found on plasma membrane proteins of living cells by galactosylation with bovine milk galactosyltransferase. Here we report the distribution of O-linked GlcNAc in highly enriched rat liver subcellular organelles. Nonidet P-40 solubilized organelles were labeled by galactosyltransferase with UDP-[3H]galactose, and the amount of radiolabel occurring on GlcNAc residues in O-linkages was assessed by its sensitivity to beta-elimination and by its resistance to deglycosylation with endo-beta-N-acetylglucosaminidase F. The presence of galactose-labeled O-linked GlcNAc residues was confirmed by high voltage paper electrophoresis. There is a 17-fold range per mg of protein in the amount of galactosylatable terminal GlcNAc residues found in the various organelles, as well as a wide range in the organelles' apparent content of O-linked GlcNAc residues. Nuclei and the soluble fraction of rat liver cells are particularly enriched with proteins bearing O-linked GlcNAc residues, although these residues are demonstrable in virtually all organelles tested. Furthermore, examination by sodium dodecyl sulfate-polyacrylamide gel electrophoresis reveals that many different organelle-specific proteins are glycosylated with O-linked GlcNAc residues. Because of the wide occurrence of this unique linkage, these data suggest that glycosylation with O-linked GlcNAc residues is not an exclusive marker for a particular organelle. In addition, we have surveyed the organelles for their content of glycoproteins bearing GlcNAc-terminated N-linked oligosaccharides. Our data demonstrate that there are significant amounts of these oligosaccharides in rough and stripped microsomes, nuclei, and nuclear envelopes. In light of evidence that terminal GlcNAc transferases are localized to the Golgi complex, these data suggest that there are glycoproteins which enter into the Golgi for processing and then are transported back into the rough endoplasmic reticulum, and possibly the nucleus.     

Structure and cell surface maturation of the attachment glycoprotein of human respiratory syncytial virus in a cell line deficient in O glycosylation.

The synthesis of the extensively O-glycosylated attachment protein, G, of human respiratory syncytial virus and its expression on the cell surface were examined in a mutant Chinese hamster ovary (CHO) cell line, ldlD, which has a defect in protein O glycosylation. These cells, used in conjunction with an inhibitor of N-linked oligosaccharide synthesis, can be used to establish conditions in which no carbohydrate addition occurs or in which either N-linked or O-linked carbohydrate addition occurs exclusively. A recombinant vaccinia virus expression vector for the G protein was constructed which, as well as containing the human respiratory syncytial virus G gene, contained a portion of the cowpox virus genome that circumvents the normal host range restriction of vaccinia virus in CHO cells. The recombinant vector expressed high levels of G protein in both mutant ldlD and wild-type CHO cells. Several immature forms of the G protein were identified that contained exclusively N-linked or O-linked oligosaccharide side chains. Metabolic pulse-chase studies indicated that the pathway of maturation for the G protein proceeds from synthesis of the 32-kilodalton (kDa) polypeptide accompanied by cotranslational attachment of high-mannose N-linked sugars to form an intermediate with an apparent mass of 45 kDa. This step is followed by the Golgi-associated conversion of the N-linked sugars to the complex type and the completion of the O-linked oligosaccharides to achieve the mature 90-kDa form of G. Maturation from the 45-kDa N-linked form to the mature 90-kDa form occurred only in the presence of O-linked sugar addition, confirming that O-linked oligosaccharides constitute a significant proportion of the mass of the mature G protein. In the absence of O glycosylation, forms of G bearing galactose-deficient truncated N-linked and fully mature N-linked oligosaccharides were observed. The effects of N- and O-linked sugar addition on the transport of G to the cell surface were measured. Indirect immunofluorescence and flow cytometry showed that G protein could be expressed on the cell surface in the absence of either O glycosylation or N glycosylation. However, cell surface expression of G lacking both N- and O-linked oligosaccharides was severely depressed.     

Sequential analysis of N- and O-linked glycosylation of 2D-PAGE separated glycoproteins

A robust method has been developed that allows analysis of both N- and O-linked oligosaccharides released from glycoproteins separated using 2D-PAGE and then electroblotted to PVDF membrane. This analysis provides efficient oligosaccharide profiling applicable to glycoproteomic analysis. The method involves the enzymatic release of N-linked oligosaccharides using PNGase F followed by the chemical release of O-linked oligosaccharides using reductive beta-elimination and analysis using LC-ESI-MS. Oligosaccharides from the major plasma glycoproteins with a pI between 4 and 7 were characterized from the glycoforms of haptoglobin, alpha2-HS-glycoprotein, serotransferrin, alpha1-antitrypsin, and alpha1-antichymotrypsin. It was shown that the separation of protein glycoforms evident in 2D-PAGE is partially due to the combined sialylation of the O-linked and N-linked oligosaccharides. Bi-, tri- and tetra-antennary N-linked structures, which had differing levels of sialylation and fucosylation, were found to be present on the glycoproteins analyzed, together with O-linked oligosaccharides such as mono-, and disialylated T-antigen and a disialylated core type 2 hexasaccharide. In addition, N-linked site-specific information was obtained by MALDI-MS analysis using tryptic digestion after PNGase F release of the oligosaccharides.