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O-Linked Chains of Mucin 27327323O-Linked Chain GlycosyltransferasesInka Brockhausen1. IntroductionThe complex O-linked oligosaccharide chains (O-glycans) attached to thepolypeptide backbone of mucins are assembled by glycosyltransferases. Theseenzymes act in the Golgi apparatus in a controlled sequence that is determined bytheir substrate specificities, their localization in Golgi compartments, and theirrelative catalytic activities (1). Activities are controlled by many factors, includ-ing the membrane environment, metal ions, concentrations of donor and acceptorsubstrates, cofactors, and, in some cases, posttranslational modifications ofenzymes. Cloning of glycosyltransferases has revealed the existence of families ofhomologous glycosyltransferases with similar actions but encoded by differentgenes. Thus, many steps in the pathways of O-glycosylation appear to be cata-lyzed by several related glycosyltransferases that may show slight differences inproperties and substrate specificities. The relative expression levels of theseenzymes is cell typespecific and appears to be regulated during the growth anddifferentiation of cells and, during tissue development, and is altered in manydisease states (2,3).Figure 1 shows the biosynthetic pathways of O-glycans with the commonmucin O-glycans core structures 1–4. The biosynthesis of other less common corestructures (1) has not been studied in detail. Core structures can be elongatedby repeating GlcNAcβ1-3Galβ1-4 or GlcNAc1-3Galβ1-3 structures (poly-N-acetyllactosamine chains, i antigens). Poly-N-acetyllactosamine chains may containbranches of GlcNAcβ1-6 residues linked to Gal- (I antigen), and may be terminatedby blood group or tissue antigens (blood group ABO and Lewis antigens) or bysialic acid and sulfate. Many of the enzymes involved in these elongation andtermination reactions also act on N-linked oligosaccharides of glycoproteins andon glycolipids.From: Methods in Molecular Biology, Vol. 125: Glycoprotein Methods and Protocols: The MucinsEdited by: A. Corfield © Humana Press Inc., Totowa, NJ 274 BrockhausenFig. 1. Pathways of O-glycan biosynthesis. The first step of O-glycosylation is cata-lyzed by polypeptide α-GalNAc-transferase (path a), which acts preferably on Thr invitro. The occurrence of various O-glycan structures is cell type specific and varies withcellular activation and differentiation, and in disease states. Core 1 and 2 structures arethe most common core structures in mucins, and are synthesized by core 1 β3-Gal-trans-ferase (path b) and core 2 β6-GlcNAc-transferase (path d). Core 3 is synthesized by core 3β3-GlcNAc-transferase (path c) and core 4 by core 4 β6-GlcNAc-transferase (path e).GalNAc- may be sialylated by α6-sialyltransferases (path j) to form sialylα2-6GalNAc-,which cannot be converted to any of the core structures. After the synthesis of cores,chains may be elongated, sulfated, fucosylated, or sialylated, and blood group and otherantigenic determinants may be added. Core 1 is sialylated by α3-sialyltransferase (path l),and this reaction blocks core 1 branching and elongation with the exception of α6-sialylation by α6-sialyltransferase (path m), which may differ from those catalyzingpath j. The α3-sialyltransferase can also act on the Gal residue of core 2. The Gal residueof core 1 may be sulfated by Gal 3-sulfotransferase (path k). Sulfation will also blockcore 1 elongation and branching. Cores 1 and 2 are elongated by elongation β3-GlcNAc-transferase (path h). On galactosylation of the GlcNAc residue of core 2 by β4-Gal-trans-ferase (path f), the poly-N-acetyllactosamine chains can be assembled by the repeatedactions of β4-Gal-transferase and i β3-GlcNAc-transferase (paths f and g, respectively).GlcNAcβ1-6 Gal branches (I antigen) may be introduced into these chains by I β6-GlcNAc-transferase (path i). O-Linked Chains of Mucin 275Golgi glycosyltransferases have a domain structure characteristic of type IImembrane proteins; the amino terminus extends into the cytoplasm, followed by amembrane anchor domain, and a catalytic domain at the carboxy terminus, whichextends into the lumen of the Golgi. Mainly the membrane anchor and adjacentamino acid sequences, but also other protein determinants of these enzymes, aswell as the membrane structure, determine the localization of transferases in vari-ous Golgi compartments (4). The donor substrates for mucin glycosyltransferasesare nucleotide sugars:nucleotide-sugar + acceptor → product + nucleotide (1)Mucin sulfotransferases transfer sulfate from 3'-phosphoadenosine 5'-phosphosulfate(PAPS) to the hydroxyls of sugars:PAPS + acceptor → product + PAP (2)These donor substrates are synthesized in the cytosol, with the exception ofCMP-sialic acid, which is made in the nuclear compartment, and are transported intothe Golgi by specific transporter systems (5).In the sequences of glycosylation reactions, glycosyltransferases often competefor a common substrate. For example, the enzymes that synthesize cores 1 and 3(Fig. 1, paths b and c) compete for GalNAc-R substrates. Conversely, certain productsformed may block further reactions; for example, no glycosyl transferase acts onsialylα2-6GalNAc (Fig. 1, path j), which therefore blocks extension of chains. Alter-natively, certain reactions may be required prior to further conversions. For example,core 1 has to be formed before a GlcNAcβ1-6 residue can be added to GalNAc in thesynthesis of core 2 (Fig. 1, path d). The distinct specificities of glycosyltransferasestherefore regulate the pathways, and thus the relative amounts of final O-glycan struc-tures found in mucins. The peptide backbones as well as existing glycosylation ofsubstrates near the O-glycosylation sites also have an important function in regulatingO-glycosylation (6). Thus, primary O-glycosylation as well as the synthesis of variousO-glycan core structures appear to be sitedirected by peptide sequences and theirglycosylation patterns.Based on many different studies, O-glycosylation appears to be initiated mainlyin early Golgi compartments. The first enzyme in the O-glycosylation pathways,polypeptide α-GalNAc-transferase (Fig. 1, path a), has been localized to the cisGolgi compartment in porcine submaxillary gland (7) but can be more broadly dis-tributed throughout the Golgi in other cell types (8). The various members of thisglycosyltransferase family have slightly different specificities toward their peptidesubstrates, have different cell type–specific expression patterns, and may be local-ized to different subcellular compartments (9,10). Polypeptide α-GalNAc-trans-ferase does not require a specific peptide sequence in the substrate; however,particular charged amino acids (11) as well as existing glycosylation (12) influencethe activity.Most mucins and other glycoproteins contain O-glycans with the core 1 structure,and the enzyme synthesizing core 1, core 1 β3-Gal-transferase (Fig. 1, path b), is a 276 Brockhausenubiquitous enzyme (1,13). The activity is a prerequisite for the synthesis of T-antigensand sialylated core 1 structures, as well as core 2 structures (14). The peptide sequencesof glycopeptide substrates and the existing glycosylation determine the activity of core 1β3-Gal-transferase (15). Erythrocytes from patients with permanent mixed-fieldpolyagglutinability (16), human T-lymphoblastoid Jurkat cells (17), and human coloncancer cells LSC (18) lack the enzyme and therefore cannot make cores 1 and 2 struc-tures. A similar effect can be introduced by the use of GalNAcα-benzyl, which is analternative substrate for enzymes extending O-glycan chains, and which can penetratecell membranes to compete with endogenous substrates of mucin. Thus, cells treatedwith this O-glycosylation inhibitor exhibit truncated O-glycans, terminating mainly inGalNAc (19). These truncated chains no longer carry ligands for cell-cell interactions,and the binding of colon cancer cells to the endothelium via E-selectin is significantlyreduced (20).The synthesis of core 2 (Fig. 1, path d) is catalyzed by core 2 β6-GlcNAc-trans-ferase (21). Several apparently related β6-GlcNAc-transferases exist that synthesizeGlcNAcβ1-6 branches on Gal or GalNAc (1,22,23). The L-type core 2 β6-GlcNAc-transferase occurs in leukocytes and other cells and only synthesizes core 2. TheM-type enzyme is found in most mucin-secreting cell types and can synthesize theGlcNAcβ1-6 branch of core 2, core 4 (Fig. 1, path e), and the I antigen (Fig. 1,path i) (24). The L-enzyme activity increases during cellular activation and differ-entiation (25,26). The M enzyme may be affected in cancer cells (3,27). Core 2β6-GlcNAc-transferase appears to be localized to cis and medial Golgi com-partments (27a,28), which is in agreement with its role in synthesizing a centralO-glycan core structure.The enzymes synthesizing O-glycan cores 3 and 4 (Fig. 1, paths c and e, respec-tively) appear to occur exclusively in mucin-secreting tissues since these coreshave not been found in nonmucin molecules (1). Core 3 is synthesized by core 3β3-GlcNAc-transferase (29). The enzyme is enriched in colonic tissues but reduced incolon cancer tissue (30,31) and is lacking in many other tissues. The activity appar-ently is lacking in colon cancer cell lines (27). The enzyme activity synthesizingcore 4, core 4 β6-GlcNAc-transferase, resides in the M-type core 2 β6-GlcNAc-transferase (24,29).Poly-N-acetyllactosamine chains of mucins are assembled by the repeating actionsof β4-Gal-transferase (Fig. 1, path f) (32) and i β3-GlcNAc-transferase (Fig. 1, path g)(33). These enzymes are ubiquitous, and may be considered as housekeeping enzymes.However, their expression is often up- or downregulated in healthy tissues as well asin a number of disease states (2,3,34). The reaction catalyzed by β4-Gal-transferaseoccurs mainly in the trans-Golgi (35). Yet another elongation β3-GlcNAc-trans-ferase elongates core 1 and 2 structures, also by a GlcNAcβ1-3Gal linkage Fig. 1,path h) (36).Poly-N-acetyllactosamine chains may acquire GlcNAcβ1-6 (GlcNAcβ1-3) Galbranches in a developmentally regulated fashion, which leads to a change from the ito the I antigenicity. Some of the I β6-GlcNAc-transferases (Fig. 1, path i) synthe-sizing the I branch act on terminal Gal residues whereas others recognize internal O-Linked Chains of Mucin 277Gal residues (1,37,38). Most of the enzymes synthesizing blood group ABO, Lewis,and other antigenic determinants act on O- and N-glycans as well as glycolipids. Bycontrast, sialyltransferases often prefer one type of glycoconjugate (39). Twosialyltransferase families, α3- and α6-sialyltransferase, act preferably on mucin-type O-glycans.α3-Sialyltransferase acts on Gal residues of cores 1 and 2 (Fig. 1, path l) (24,40,41).The enzyme is developmentally regulated in thymocytes (42) and increased in leuke-mia cells (43) and several cancer models (3,30). The α3-sialyltransferase has beenlocalized to medial and trans-Golgi compartments (44). The sialylation reaction cata-lyzed by this enzyme has an important role in keeping O-glycan chains short andsialylated. Since the enzyme acts relatively early in the O-glycan extension pathways(Fig. 1, path l), it has the ability to compete with branching and elongation reactions.Once core 1 is α3-sialylated, it is no longer a substrate for extension reactionsalthough it can still be converted to the disialylated core 1 by α6-sialyltransferase(Fig. 1, path m).The α6-sialyltransferase (Fig. 1, path j) that acts on GalNAc-R to form thesialyl-Tn antigen, sialylα2-6GalNAc-Th/Ser (45), requires glycoproteins as substrateand cannot act on GalNAc-benzyl or nitrophenyl substrates (46,47). However,another type of α6-sialyltransferase (α6-sialyltransferase III) does not have a pep-tide requirement, but is specific for the α3-sialylated core 1 structure (48). Thedisialylated core structure can probably be synthesized by α6-sialyltransferase IIIand other α6-sialyltransferases (Fig. 1, path m). Modifications of the sialic acidresidues of mucins include O-acetylation, catalyzed by specific O-acetyltransferasesacting in the Golgi (49).The common sulfate ester linkages in mucins are SO4-6-GlcNAc and SO4-3-Gal.Several types of sulfotransferases have been described that act on the 6-position ofGlcNAc (50) or the 3-position of Gal of core 1 (Fig. 1, path k) and N-acetyllactosaminestructures (51,52). Sulfated oligosaccharides appear to play an important role in celladhesion through binding to selectins and in the control of bacterial binding (53,54).Sulfation also functions in directing the biosynthetic pathways of complex O-glycansby blocking certain reactions. For example, sulfation of core 1 prevents the branchingreaction to form core 2 (51).The enzymes catalyzing the reactions depicted in Fig. 1 assemble mucin-typeO-linked carbohydrate chains and are listed in Table 1, together with their substrates,enzyme products, and the high performance liquid chromatography (HPLC) condi-tions of product separation. Probably none of these enzymes are specific for mucins,but also act on other glycoproteins that carry O-glycans, and can act on various glyco-peptides with O-linkages. In vitro, many of these enzymes utilize synthetic compoundsas substrates in which the peptide chain is replaced by a hydrophobic group. Thesubstrate should be clean, specific, and easy to isolate in order to determine theenzyme activity and specificity accurately. For a few enzymes, purified mucins withdefined glycosylation are available as substrates. However, mucins are usually tooheterogeneous in their carbohydrate structures, and therefore the use of synthetic com-pounds with defined structure is preferred. In addition, it is much easier to determine 278 Brockhausenthe product structure of synthetic substrates as a proof of the assayed activity. When acompound is a potential substrate for several glycosyltransferases present in theenzyme preparation, or several reactions occur in sequence, the various productshave to be separated and identified. This can usually be achieved by HPLC. With theexception of β1,6-GlcNAc-transferases, UDP-sugar binding enzymes require the pres-ence of divalent metal ion for optimal activity. Thus, measuring a β6-GlcNAc-trans-ferase activity in the presence of EDTA will eliminate the activity of otherGlcNAc-transferases potentially acting on the same substrate. Enzymes utilizingCMP-sialic acid may be stimulated by metal ions but usually can act in their absence.Unless they are released and secreted, or are produced as soluble recombinantenzymes, glycosyltransferases are membrane-bound enzymes, and their activities arestimulated by detergents.A convenient way of identifying and quantifying glycosyltransferase products is bythe use of nucleotide-sugar donors that contain 14C or 3H-labeled radioactive sugar.Similarly, the sulfate moiety of PAPS can be labeled with 35S. Calculations ofsulfotransferase activities must take into account the relatively short half-life of35S (about 87 d).2. Materials2.1. Preparation of Enzymes1. 0.25 M Sucrose.2. 0.9% NaCl.3. Potter-Elvehjem hand homogenizer.4. Low-speed centrifuge (10,000g).5. Ultracentrifuge (100,000g).6. Small pieces of tissue, or cells.2.2. Preparation of Substrates and Standard Compounds1. Commercially available oligosaccharides: GlcNAc, GalNAcα-benzyl, Galβ1-3GalNAcα-benzyl, GlcNAcβ1-3 GalNAcα-p-nitrophenyl [pnp], Galβ1-4 GlcNAc,GlcNAcβ1-3 Galβ-methyl (Sigma, St. Louis MO); Galβ1-3 GalNAcα-pnp (TorontoResearch Chemicals, Toronto, Canada).2. Thr-peptides, synthesized by Hans Paulsen, University of Hamburg, Germany(15,55).3. Frozen sheep submaxillary glands (Pel-Freez, Rogers, AR) to isolate ovine submaxillarymucin (OSM), 0.1 N H2SO4, bovine testicular β-galactosidase (Boehringer, Laval,Canada), 0.1 M Na-citrate buffer, Sephadex G25 column.4. Components of enzyme assays to prepare product standards enzymatically GlcNAcβ1-3GalNAcα-benzyl, GlcNAcβ1-6 (GlcNAcβ1-3) GalNAcα-pnp, GlcNAcβ1-3 Galβ1-4GlcNAc, GlcNAcβ1-6 (GlcNAcβ1-3 Galβ1-3) GalNAcα-benzyl, GlcNAcβ1-6(GlcNAcβ1-3) Galβ-methyl, sialylα2-3 Galβ1-3 GalNAcα-pnp, SO4-3 Galβ1-3GalNAcα-benzyl, SO4-3 Galβ1-4 GlcNAc.5. Enzymatically prepared substrates: GlcNAcβ1-6 (Galβ1-3) GalNAcα-benzyl, sialylα2-3Galβ1-3 GalNAcα-pnp O-Linked Chains of Mucin 2796. Nuclear magnetic resonance (NMR) and mass spectrometers, reagents for methylationanalysis.2.3. Separation and Identificationof Glycosyltransferase and Sulfotransferase Products1. HPLC apparatus.2. HPLC columns C18, NH2 (amine), PAC (cyano-amine).3. Acetonitrile/water mixtures.4. Dionex system for high-performance anion-exchange chromatography (HPAEC).5. Bio-Gel P4 or P2 column (80 × 1.6 cm) (Bio-Rad, Hercules, CA).6. Ion-exchange columns (AG1 × 8, 100–200 mesh, Bio-Rad).7. High-voltage electrophoresis apparatus, 1% Na-tetraborate, Whatman No. 1 paper.8. C18 Sep-Pak columns, methanol.9. 0.05 M KOH/1 M NaBH4 for β-elimination.10. Scintillation fluid, scintillation counter.2.4. Polypeptide α-GalNAc-Transferase Assays1. 5% Triton X-100.2. 0.5 M N-morpholino ethanesufonate (MES) buffer, pH 7.3. 0.05 M Adenosine 5'-monophosphate (AMP) to inhibit pyrophosphatases.4. 0.5 M MnCl2.5. 10 mM UDP-GalNAc (2000 dpm/nmol) donor substrate.6. 5 mM Acceptor substrate solution: Thr-containing peptide.7. Enzyme homogenate or solution.2.5.β3- and β6-GlcNAc-Transferase Assays1. 5% Triton X-100.2. 0.5 M MnCl2 (for β3-GlcNAc-transferases only).3. 0.5 M MES buffer, pH 7.4. 0.05 M AMP.5. 0.5 M GlcNAc to inhibit N-acetylglucosaminidases.6. 50 mM γ-galactonolactone (if substrate with terminal Gal is used) to inhibitgalactosidases.7. 10 mM UDP-GlcNAc (2000 dpm/nmol).8. 5 mM Acceptor substrate solution: GalNAcα-benzyl, Galβ1-3 GalNAcα-benzyl, Galβ1-4GlcNAc, GlcNAcβ1-3Galβ-methyl, or GlcNAcβ1-6 (Galβ1-3) GalNAcα-benzyl.9. Enzyme homogenate or solution.2.6. Core 1 β3-Gal- and β4-Gal-Transferase Assays1. 5% Triton X-100.2. 0.5 M MnCl2.3. 0.5 M MES buffer, pH 7.4. 0.05 M AMP.5. 0.05 M γ-galactonolactone.6. 10 mM UDP-Gal (2000 dpm/nmol). 280 Brockhausen7. 5 mM Acceptor substrate solution: GalNAcα-benzyl or GlcNAc.8. Enzyme homogenate or solution.2.7.α3- and α6-Sialyltransferase Assays1. 5% Triton X-100.2. 0.5 M Tri-HCl buffer, pH 7.3. 0.05 M AMP.4. 10 mM CMP-sialic acid (2000 dpm/nmol).5. 5 mM Acceptor substrate solution: DS-OSM. with 3 mM GalNAc concentration, Galβ1-3GalNAcα-pnp, or sialylα2-3 Galβ1-3 GalNAcα-pnp.6. Enzyme homogenate or solution.7. High-voltage electrophoresis apparatus.8. 20 mM EDTA/2 % Na-tetraborate.9. 1% Na-tetraborate.10. Whatman No. 1 paper.11. HPLC apparatus.2.8. Sulfotransferase Assays1. 5% Triton X-100.2. 0.1 M magnesium-acetate.3. 0.1 M NaF to inhibit sulfatases.4. 0.5 M Tris-HCl buffer, pH 75. 0.05 M adenosine triphosphate (ATP).6. 0.1 M 2,3-Mercaptopropanol to inhibit PAPS degradation.7. 0.3 mM PAPS (2000 dpm/nmol).8. 5 mM Acceptor substrate solution: Galβ1-3GalNAcα-benzyl, Galβ1-4 GlcNAc, orGlcNAcβ1-3Galβ-methyl.9. Enzyme-homogenate or solution.10. High-voltage electrophoresis apparatus.11. 20 mM EDTA/2% Na-tetraborate.12. 1% Na-tetraborate.13. Whatman No. 1 paper.14. HPLC apparatus.15. Dionex system for HPAEC.3. Methods3.1. Preparation of EnzymesIdeally, enzymes are present in the highly purified state, and soluble in the assaymixture. A number of enzymes have been purified. However, these proceduresdepend on the specific enzyme and tissue and may take several months or years.Therefore, purification protocols are not described here. Purified enzymes may bestable at 4°C for months but are usually more stable at lower temperatures. Withtissue homogenates or microsomes, however, this is rarely the case. The enzymepreparations inevitably contain interfering substances and degradative enzymes. Forexample pyrophosphatases and phosphatases that degrade nucleotide sugar donors, O-Linked Chains of Mucin 281glycosidases that degrade substrates and products, and proteases that degradethe peptide moiety of substrates and products may be present, and deactivate theenzyme to be assayed. These unwanted reactions can be suppressed with specificinhibitors.1. To prepare crude homogenate, hand homogenize tissue in 10 times the volume of 0.25 Msucrose. For most studies of crude enzymes, this preparation is sufficient. The homoge-nate can be stored at –20°C for a few months, or at –70°C for several years. If sufficientmaterial is available, a more enriched enzyme fraction can be prepared as microsomes.Microsomes may be prepared from homogenates by first removing a low-speed pellet bycentrifugation at 10,000g, followed by the precipitation of microsomes from the superna-tant at 100,000g. The microsomal pellet is hand homogenized in 10 times the volume of0.25 M sucrose.2. Enzymes from cultured cells are prepared similarly. Harvest cells from the culture plate,and wash three times with 0.9% NaCl by gently stirring and centrifuging cells. Afterwashing, hand homogenize cells in 0.25 M sucrose (1 mL/108cells) and store asdescribed in step 1.3.2. Preparation of Substrates and Standard CompoundsSubstrates may be purchased, prepared by chemical synthesis or combined chemi-cal-enzymatic synthesis, or prepared by enzymatic synthesis or degradation fromnatural glycoproteins.1. GalNAc-OSM is prepared from purified sheep submaxillary mucin, ovine submaxil-lary mucin (OSM) (29). Treat OSM with 0.1 N H2SO4for 1 h at 80°C to remove sialicacid and fucose. For high purity, follow by digestion with bovine testicular β-galac-tosidase (56), which removes the small amount of β1-3–linked Gal residues presentin OSM (30).2. Substrate and product compounds that are not commercially available are synthesizedwith a known source of the desired enzyme under the conditions described for the stan-dard transferase assay.3. Low molecular weight compounds are isolated by gel filtration on Bio-Gel P4 or P2 col-umns, followed by HPLC (Table 1). The purity and linkages of all compounds should beverified by mass spectrometry (MS) and 1H-NMR. The concentrations of individual sug-ars can be determined after acid hydrolysis (1 h at 80°C with 33% trifluoroacetic acid forsialic acid–containing compounds, 1 h at 100°C with 6 N HCl for neutral sugars) byHPAEC (Dionex system) as described in Subheading 3.4.5.3.3. Separation and Identificationof Glycosyltransferase and Sulfotransferase ProductsTo demonstrate that an enzyme activity is synthesizing a certain sugar linkage, theproduct has to be isolated and its structure determined. This is especially importantwhen a new enzyme activity is to be assayed or when a novel variant of a knownactivity is expected.1. Produce large amounts of glycosyltransferase product in a standard assay, possibly afterincubation for 8–24 h, and pass through an AG1 × 8 column to remove nucleotide sugar 282 Brockhausenand negatively charged molecules. For sulfotransferase products, run high-voltage elec-trophoresis after the standard assay.2. Purify low molecular weight compounds by HPLC or using the Dionex system, as de-scribed in Subheading 3.4.5. Low molecular weight compounds separated by high-volt-age electrophoresis, can be eluted off the paper with water, by placing the paper intosyringes and centrifuging at low speed. Borate is removed by repeated flash evaporationwith methanol.3. Purify mucin substrates by passing incubation mixtures through AG1 × 8 columns,followed by gel filtration on Bio-Gel P4 or P2. O-glycans are released from mucinsby β-elimination (0.05 N KOH/1 M Na BH4at 45°C for 16 h). After neutralization,purify reduced O-glycan-alditols by gel filtration on Bio-Gel P4 or P2 columns, andby HPLC.4. Carry out structural analysis of all low molecular compounds and oligosaccharide-alditols by NMR, MS (fast atom bombardment, electrospray, or matrix-assisted laserdesorption ionization), and methylation analysis. If small amounts of product areavailable, chromatographic methods, including HPLC and the Dionex system, withthe use of standard compounds, and sequential glycosidase digestion are useful(29,57–60).3.4. Glycosyltransferase Assays (see Notes 1 and 2)3.4.1. Ion-Exchange AssayThe ion exchange assay is simple, quick, and inexpensive, and can be applied to alltransferase assays using neutral acceptor substrates.1. After the incubation, stop the reaction with 100 mL of ice-cold water. Apply mixtureto a column (a Pasteur pipet) of 0.4 mL of AG1 × 8, which removes excess radioac-tive nucleotide sugar. Wash the column three times with 0.6 mL of water and collectthe eluate.2. Add 5 mL of scintillation fluid and estimate radioactivity with a scintillation counter.Since the radioactivity in the eluate includes free radioactive sugar (originating fromnucleotide sugar breakdown) and radioactive products from various endogenous sub-strates, the radioactivity of assays lacking exogenous substrates has to be substractedfrom the disintegrations per minute obtained. The specific enzyme activity is calculatedas nanomoles/(hour.milligrams of protein).3. Regenerate AG1 × 8 columns with 5 M NaCl, followed by thorough washing with water.3.4.2. C18 Column AssayThe C18 column (Sep-Pak) assay can be applied when substrates contain a hydro-phobic group. This method is often not reliable when charged (sialylated or sulfated)products are formed, unless very large hydrophobic groups are present in the enzymeproduct. A methyl aglycone group does not provide sufficient hydrophobicity to bindto Sep-Pak C18 columns.1. After the incubation, apply the mixture onto a Sep-Pak C18 column, previously washedin water. Wash columns with 5 mL of water to elute excess nucleotide sugar and freeradioactive sugar. [...]... 0–20 b Core 1 β3-Gal-T 1. GalNAcα-Bn Galβ 3GalNAcα-Bn C18 10 2. GalNAcα-OSM Galβ3GalNAcα-OSM — — c Core 3 β3-GlcNAc-T 1. GalNAcα-Bn GlcNAcβ3GalNAcα-Bn C18 10 2. GalNAcα-OSM GlcNAcβ3GalNAcα-OSM — — d Core 2 β6-GlcNAc-T Galβ3GalNAcα-pnp GlcNAcβ6(Galβ3)GalNAcα -pnp C18 8 e Core 4 β 6-GlcNAc-T GlcNAcβ3GalNAcα-pnp GlcNAcβ6(GlcNAcβ3)GalNAcα-pnp C18 8 f β 4Gal-T GlcNAc Galβ4GlcNAc NH2 85 gi β3-GlcNAc-T Galβ4GlcNAc... Elongation β3-GlcNAc-T GlcNAcβ6(Galβ3 )- GlcNAcβ6(GlcNAcβ3Galβ3 )- C18 8 GalNAcα-Bn GalNAcα-Bn PAC 82 iI β6-GlcNAc-T GlcNAcβ3Galβ-CH 3 GlcNAcβ6(GlcNAcβ3)Galβ-CH 3 NH2 85 j α6-sialyl-T GalNAcα-OSM Sialylα6GalNAcα-OSM — — 284 O -Linked Chains of Mucin 291 29. Brockhausen, I., Matta, K. L., Orr, J., and Schachter, H. (1985) Mucin synthesis. VI. UDP-GlcNAc:GalNAc-R β3-N-acetylglucosaminyltransferase and UDP-GlcNAc: GlcNAcβ 1-3 GalNAc-R... Barner, M., Granovsky, M., and Brockhausen, I. (1993) Processing O-glycan core 1, Galβ 1-3 GalNAcα-R. Specificities of core 2 UDP-GlcNAc:Galβ 1-3 GalNAc-R β6-N-acetylglucosaminyl- transferase and CMP-SA: Galβ 1-3 GalNAc-R α3-sialyltransferase. Glycoconj. J. 10, 381–394. 25. Piller, F., Piller, V., Fox, R., and Fukuda, M. (1988) Human T-lymphocyte activa- tion is associated with changes in O-glycan biosynthesis.... inhibit N-acetylglucosaminidases. 6. 50 mM γ-galactonolactone (if substrate with terminal Gal is used) to inhibit galactosidases. 7. 10 mM UDP-GlcNAc (2000 dpm/nmol). 8. 5 mM Acceptor substrate solution: GalNAcα-benzyl, Galβ 1-3 GalNAcα-benzyl, Galβ 1-4 GlcNAc, GlcNAcβ 1-3 Galβ-methyl, or GlcNAcβ 1-6 (Galβ 1-3 ) GalNAcα-benzyl. 9. Enzyme homogenate or solution. 2.6. Core 1 β 3-Gal- and β 4-Gal-Transferase... the activity of O-glycan core 1 uridine 5'-diphospho-galactose: N-acetylgalactosamineα-R β3-galactosyl-transferase. Biochemistry 29, 10,206– 10,212. 7. Roth, J., Wang, Y., Eckhardt, A. E., and Hill, R. L. (1994) Subcellular localization of the UDP-N-acetyl-D-galactosamine: polypeptide N-acetylgalactosaminyltransferase-medi- ated O-glycosylation reaction in the submaxillary gland. Proc. Natl. Acad.... with cellular activation and differentiation, and in disease states. Core 1 and 2 structures are the most common core structures in mucins, and are synthesized by core 1 β3-Gal-trans- ferase (path b) and core 2 β6-GlcNAc-transferase (path d). Core 3 is synthesized by core 3 β3-GlcNAc-transferase (path c) and core 4 by core 4 β6-GlcNAc-transferase (path e). GalNAc- may be sialylated by α6-sialyltransferases... J., and Serafini-Cessi, F. (1996) Differ- entiation-dependent expression of human β-galactoside α2,6-sialyltransferase mRNA in colon carcinoma CaCo-2 cells. Glycoconj. J. 13, 115–121. 284Brockhausen Table 1 Mucin Glycosyltransferases, Their Substrates and Products, and HPLC Conditions for Product Isolation HPLC Path Enzyme Substrate Product column % AN a Polypeptide α-GalNAc-T Thr-peptide GalNAc-Thr-peptide... synthesize GlcNAcβ 1-6 branches on Gal or GalNAc (1,22,23). The L-type core 2 β6-GlcNAc- transferase occurs in leukocytes and other cells and only synthesizes core 2. The M-type enzyme is found in most mucin-secreting cell types and can synthesize the GlcNAcβ 1-6 branch of core 2, core 4 (Fig. 1, path e), and the I antigen (Fig. 1, path i) (24). The L-enzyme activity increases during cellular activation and differ- entiation... assembled by the repeated actions of β4-Gal-transferase and i β3-GlcNAc-transferase (paths f and g, respectively). GlcNAcβ 1-6 Gal branches (I antigen) may be introduced into these chains by I β6-GlcNAc- transferase (path i). O -Linked Chains of Mucin 273 273 23 O -Linked Chain Glycosyltransferases Inka Brockhausen 1. Introduction The complex O-linked oligosaccharide chains (O-glycans) attached to the polypeptide... O-glycans with the common mucin O-glycans core structures 1–4. The biosynthesis of other less common core structures (1) has not been studied in detail. Core structures can be elongated by repeating GlcNAcβ 1-3 Galβ 1-4 or GlcNAc 1-3 Galβ 1-3 structures (poly-N- acetyllactosamine chains, i antigens). Poly-N-acetyllactosamine chains may contain branches of GlcNAcβ 1-6 residues linked to Gal- (I antigen), and . synthesis: conversion of R 1- 1-3 Gal-R2to R 1- 1-3 (GlcNAcβ 1-6 )Gal-R 2and of R 1- 1-3 GalNAc-R2to R 1- 1-3 (GlcNAcβ 1-6 )GalNAc-R2by a β6-N-acetyl-glucosaminyltransferase. (GlcNAcβ 1-3 ) GalNAcα-pnp, GlcNAcβ 1-3 Galβ 1-4 GlcNAc, GlcNAcβ 1-6 (GlcNAcβ 1-3 Galβ 1-3 ) GalNAcα-benzyl, GlcNAcβ 1-6 (GlcNAcβ 1-3 ) Galβ-methyl, sialylα 2-3 Galβ 1-3 GalNAcα-pnp,