Glycoprotein methods protocols - biotechnology
Inhibition of Glycosylation of Mucin 26126122Inhibition of Mucin GlycosylationGuillemette Huet, Philippe Delannoy, Thécla Lesuffleur,Sylviane Hennebicq, and Pierre Degand1. IntroductionMucins are secreted or membrane-bound large glycoproteins produced by epithe-lial cells of normal and malignant tissues. The secreted mucins are the major compo-nents of the mucous gel overlaying respiratory, gastrointestinal, or genital epithelia.Mucins constitute a family of extensively O-glycosylated glycoproteins (40–80% byweight) (1,2) encoded by a family of different MUC genes (3). The oligosaccharideside chains substitute threonine or serine residues of tandemly repeated sequences inthe core of the molecule.The biochemical properties and functions of mucins are greatly dependent on theirO-glycosylation state. In particular, mucins can display a role in cellular protection orcellular adhesion (4). On the one hand, filamentous mucins highly substituted by nega-tively charged O-glycans act as a protective barrier for epithelial cells. On the otherhand, the terminal oligosaccharides of mucins can interact with cellular or bacterialreceptors and promote adhesion on the epithelial cells. Mucins display tissue-specificpatterns of O-glycosylation. Alterations in the glycosylation of mucins commonlyoccur in many mucosal diseases. In particular, glycan epitopes of mucins are impor-tant markers in cancer (5,6).Studies on the regulation of the biosynthesis and secretion of mucins have beenimproved by the use of human mucosal cells, which can be grown in long-termculture (7). To address the function of carbohydrates, inhibitors of O-glycosylationhave been used in in vivo experiments on cultured cells (8). For that purpose, aryl-N-acetyl-α-galactosaminides (GalNAcα-O-aryls) have been initially used aspotential competitors of the glycosylation of N-acetylgalactosamine (GalNAc)residues linked to the core protein, since these sugar analogs were suitable substratesFrom: Methods in Molecular Biology, Vol. 125: Glycoprotein Methods and Protocols: The MucinsEdited by: A. Corfield © Humana Press Inc., Totowa, NJ 262 Huet et al.for UDP-GlcNAc:GalNAc-Rβ1, 3-N-acetylglucosaminyltransferase (9). Actually, inin vitro experiments, these compounds inhibit the UDP-Gal:GalNAc-Rβ1,3-galac-tosyltransferase (8). GalNAcα-O-aryls (benzyl-, phenyl-, and p-nitrophenyl deriva-tives of N-acetylgalactosamine), when added in the medium of cultured cells, aremetabolized within the cells and give rise to different internal derivatives. In vivo theresulting effects of GalNAcα-O-aryls on the O-glycosylation of mucins are differentfrom the effects obtained in vitro (8,10–12). In this way, in mucin-secreting coloncancer cells such as the HM7 variant of LSI74T cells (8,10) and the HT-29 MTXsubpopulation (11,12), GalNAcα-O-aryls are highly converted into the disaccharideGalβ1-3GalNAcα-O-aryls, but this conversion does not impair some significant β1,3-galactosylation of O-linked GalNAc to the core protein as well. The disaccharide-formed Galβ1-3GalNAcα-O–aryls have proved, on the contrary, to behave as a strongcompetitive inhibitor of the elongation of the mucin Galβ1-3GalNAcα sequences byN-acetylglucosa-minyltransferases, sialyltransferases, and fucosyltransferases (8,10–12).Hence, in in vivo experiments, GalNAcα-O-aryls mainly act as inhibitors of the elonga-tion of the Galβ1-3GalNAcα sequence (T-antigen) of mucins.The carbohydrate changes induced by GalNAcα-O-aryl treatments can be evaluated bydifferent methods:1. Mucins can be directly analyzed in the cell culture media or in the cell lysates by Westernblotting using lectins and/or antibodies directed against carbohydrate epitopes.2. Mucins can be isolated from cell lysates or culture media by the conventional proceduresusing ultracentrifugation on a cesium bromide gradient (13,14), and analyzed by carbo-hydrate composition or by (enzyme-linked immunosorbent assay (ELISA) with lectinsand/or glycan epitope-specific antibodies.The effects on mucin secretion can be estimated using metabolic labeling with[3H]threonine or by histochemical staining. And, the intracellular metabolization ofGalNAcα-O-aryls can be studied using metabolic labeling with [3H]galactose andreversed-phase, high-performance liquid chromatography (HPLC).2. Materials1. Alcian blue (AB), pH 2.5: 0.1% (w/v) AB in 3% (v/v) acetic acid, pH 2.5.2. Amplify (Amersham, Buchler Gmbh, Braunschweig, Germany).3. Anti-digoxygenin (DIG) Fab fragment conjugated with alkaline phosphatase (BoehringerMannheim, Germany).4. Aryl-N-acetyl-α=D=galactosaminides (Sigma, St. Louis, MD).5. Blocking solutions:a. Blocking solution 1: Tris-buffered saline (TBS), pH 7.5, containing 2% polyvinylpyrrolidone K-30 (Aldrich).b. Blocking solution 2: TBS, pH 7.5, containing 0.5% blocking reagent (BoehringerMannheim). Heat at 60°C for 1 h.c. Blocking solution 3: TBS, pH 7.5, containing 6% bovine serum albumin (BSA).d. Blocking solution 4: 0.01 M phosphate-buffered saline (PBS), pH 6.8, containing 1%(w/v) BSA. Inhibition of Glycosylation of Mucin 2636. Blotting buffers:a. Anode buffer: 0.3 M Tris HCl, pH 10.4, containing 20% methanol.b. Cathode buffer: 0.04 M 6-amino-N-hexanoic acid containing 20% methanol.7. Cell disrupter Sonifer B-30 adapted with an exponential probe (Branson SonicPower).8. Continous flow radiochromatography detector Flo-One/Beta.9. ECL detection kit (Amersham).10. Eukitt (Kindler GMBN, Freeburg, Germany).11. Hibar prepacked column RT 250-4, Lichrosorb RP-18, 5 µm (Merck, Darmstadt,Germany).12. Incubation buffer:a. Incubation buffer 1: TBS, pH 7.5 containing 1% (w/v) BSA, 1% sodium dodecylsulfate (SDS), 1% Triton X-100, 10% fetal calf serum (FCS).b. Incubation buffer 2: TBS, pH 7.5 containing 10% FCS.13. Labeling :a. [3H]-L-threonine (ICN, Costa Mesa, CA) 46 Ci/mmol, 1.7 GBq/mmol (50 mCi/mL).b.D-[6–3H]galactose (Amersham): 20–40 Ci/mmol, 0.7–1.5 TBq/mmol (1 mCi/mL).14. Lectin buffer: TBS, pH 7.5, containing 1 mM MgCl2, 1 mM MnCl2,and 1 mM CaCl2.15. Cell culture media:a. Standard growth medium: Dulbecco’s modified Eagle’s medium containing 2 mMglutamine, 100 UmL of penicillin, 100 mg µL of streptomycin, and 10% fetal bovineserum.b. Growth medium with GalNAcα-O-aryl: The medium in ituma is added with aryl-N-acetyl-α-D-galactosaminide.c. Threonine-free medium (Life Technologies).d. Low-glucose Dulbecco’s minimal essential/H-16 medium (Gibco).16. Molecular weight-markers are the high molecular range weight of Rainbow colored mark-ers (Amersham), maximun 200 kDa (myosin).17. Monoclonal anti-T (Thomsen-Friedenrich) antibody available from commercial suppliers.18. Nuclear Red: 0.1% (w/v) nuclear red in 5% aluminum sulfate solution. Heat to dissolveand filter.19. Paraformaldehyde: 4% (w/v) in 0.1M phosphate buffer, pH 7.4.20. PBS:a. 0.01 M PBS, pH 6.8.b. 0.01 M PBS, pH 6.8, containing 0.1% (v/v) Tween-20.21. Peroxidase-labeled antimouse IgG antibody (Jackson Immunoresearch Laboratories, WestGrove, PA).22. Peroxidase-labeled streptavidin.23. RIPA buffer: 1 mM Tris-HCl, pH 8.0, 0.01 M NaCl, 0.1% SDS, 1% Triton X-100,0.5% sodium deoxycholate, 1% phenylmethylsulfonylfluoride, 1 mM sodium ethylenediaminetetraacetate.24. Stop solution: 1 N HCl.25. Substrates:a. Substrate solution 1: 50 µL of 4-nitroblue tetrazolium chloride (77 mg mL in 70%dimethylformamide) and 37.5 µL of 5-bromo-4-chloro-3-indolyl-phosphate (50 mgmL in dimethylformamide) in 10 mL of 100 mM Tris-HCl, pH 9.5, 50 mMMgCl2, and 100 mM NaCl. 264 Huet et al.b. Substrate solution 2: 1 × 10 mg tablet of O-phenylenediamine dihydrochloride (Sigma)in 10 mL of 0.01 M PBS, pH 5.5. Add 15 µL of 30% hydrogen peroxide immediatelybefore use.26. TBS: 50 mM Tris-HCl, 150 mM sodium chloride, pH 7.5.27. X-O Kodak films (Amersham).28. Whatman 3MM paper.29. SDS-polyacrylamide gels for electrophoresis: stacking gels of 2% and running gels of2–10% polyacrylamide gradient gel (acrylamide/bisacrylamide ratio of 37.5) are preparedat 2-mm thickness.3. Methods3.1. Cell Culture in the Presence of GalNAcα-O-benzyl3.1.1. Conditions of Use of GalNAcα-O-benzylGalNAcα-O-benzyl is directly dissolved in the culture medium for 2 h at roomtemperature with continuous stirring. Then, the medium is sterilized by filtration.GalNAcα-O-benzyl may be used at various concentrations up to 10 mM. A range ofdifferent concentrations should be tested for the viability of the cells and the obtainedeffects. Indeed, the response is expected to be different according to the cell type, and inparticular, its glycosyltransferase pattern.GalNAcα-O-benzyl can be used in the following ways:1. In a short treatment over a 24-h period. The cells are cultured in the standard medium upto their differentiation state into mucin-secreting phenotype, and then for 24 h in themedium enriched in GalNAcα-O-benzyl.2. In a long time period treatment. The cells are cultured in the standard medium up to d 2after seeding, and then in the medium enriched in GalNAcα-O-benzyl. The medium ischanged daily with new medium containing GalNAcα-O-benzyl.3.1.2. Metabolic LabelingTo study the effects of GalNAcα-O-benzyl on the secretion of mucins, cells areseeded on 6-well plates. For metabolic labeling with [3H]Threonine, the currentmedium is substituted by threonine-free medium. [3H]Threonine (50 µCi mL) isadded in the threonine-free medium of control cells and in the threonine-freemedium containing GalNAcα-O-benzyl of treated cells. Then, the culture media arecollected and centrifuged and the cells are lysed in 1 mL of RIPA buffer and centri-fuged. The supernatants are analyzed on 2–10% gels and autoradiography (seeSubheading 3.2.4.).To study the intracellular metabolism of GalNAcα-O-benzyl, cells are seeded into6-well culture plates. For metabolic labeling with [6–3H]galactose, the current mediumis substituted by low glucose medium to facilitate the incorporation of the precursor.[6–3H]Galactose (1 mCi mL) is added in the medium simultaneously with GalNAcα-O-benzyl for up to 72 h. After removing the culture media, cells are lysed by sonica-tion in 1 mL of distilled water and centrifuged at 13,000g. The supernatants arecollected, heat-denaturated, and filtered through 0.22 µm ultrafiltration units. Inhibition of Glycosylation of Mucin 265GalNAcα-O-benzyl derivatives are analyzed by reversed-phase HPLC using aLichrosorb RP-18 column.3.2. Visualization of Mucins after ElectrophoresisSamples of culture media or cell lysates are subjected to electrophoresis on2–10% polyacrylamide gels in the presence of SDS.3.2.1. Transfer to NitrocelluloseProteins are transferred from the gel to nitrocellulose for 1 h using a semi-dryelectroblot apparatus. Six sheets of Whatman 3MM paper are immersed in the anodebuffer and covered with the membrane and then with the gel, both previously rinsed inthe anode buffer. The gel is then covered with six more sheets of Whatman 3MMpaper that have been immersed in the cathode buffer. The transfer is carried out at0.8 mA/cm2.3.2.2. Lectin Staining (see Notes 1–4)All steps should be performed at room temperature with gentle shaking except thecolor development.1. Wash the membrane three times in TBS for 5 min.2. Incubate in blocking solution 1 for 2 h (see Note 1).3. Wash the membrane three times in TBS for 5 min.4. Incubate with DIG-labeled lectin (see Note 2) in lectin buffer for 1 h (see Note 3).5. Wash the membrane in TBS (three times for 5 min).6. Incubate in blocking solution 2 for 1 h.7. Wash the membrane in TBS (three times for 5 min).8. Incubate with anti-DIG Fab fragment conjugated with alkaline-phosphatase diluted 1000-fold in TBS (1 µg mL).9. Wash the membrane in TBS (three times for 5 min).10. Incubate the membrane in substrate solution 1 until color development (see Note 4).11. Stop the reaction by immersion and gentle shaking in distilled water.12. Dry the nitrocellulose membrane at room temperature.3.2.3. Antibody StainingAll steps should be performed with gentle shaking.1. Wash the membrane in TBS for 15 min.2. Incubate the membrane in blocking solution 3 for 1 h at 37°C.3. Wash the membrane twice in TBS containing 0.1% Tween-20 for 15 min.4. Incubate the membrane at room temperature with the first antibody diluted 1000-fold inthe incubation buffer 1 for 2 h.5. Wash the membrane in TBS containing 0.1% Tween-20 (two times for 15 min).6. Incubate the membrane at room temperature with the peroxidase-conjugated second anti-body diluted 4000-fold in incubation buffer 2 for 2 h. 266 Huet et al.7. Wash the membrane in TBS containing 0.1% Tween-20 (two times for 15 min).8. Incubate with the ECL solution for 1 min.9. Expose to hyperfilm.3.2.4. Autoradiography1. Fix the gel overnight in 40% ethanol, 10% glycerol, 10% acetic acid (v/v/v).2. Soak in Amplify for 20 min.3. Dry on Whatman 3MM paper.4. Expose to X-O Kodak film (Amersham).3.3. ELISA of Purified MucinsFigure 1 gives an example of ELISAs of purified mucins.3.3.1. Coating1. Solubilize the mucins in PBS buffer overnight at 4°C.2. For coating, incubate different amounts of mucins (from 10 to 1000 ng) in wellsof a 96-well plate overnight at 4°C, and then empty the plate.3. Incubate the plate with blocking solution 4 at room temperature for 2 h.4. Wash the plate in PBS (three time for 5 min.).3.3.2. Lectin Staining1. Incubate for 1 h with biotinylated lectin diluted in blocking solution 4 (from 1 to 10µg mL).2. Wash in PBS containing 0.1% (v/v) Tween-20.3. Wash three times in PBS.4. Incubate for 90 min with peroxidase-labeled streptavidin diluted in blocking solution 4(2 µg mL).5. Repeat steps 2 and 3.6. Develop color with substrate solution 2.7. Stop the reaction by adding 50 µL of 1 N HCl.8. Read the optical density at 492 nm.3.3.3. Antibody Staining1. Incubate for 1 h with first antibody diluted in blocking solution 4.2. Wash in PBS containing 0.1% (v/v) Tween-20.3. Wash three times in PBS.4. Incubate for 90 min with peroxidase-labeled second antibody at 0.2 µg mL in blockingsolution 4.5. Repeat steps 2 and 3.6. Develop color with substrate solution 2.7. Stop the reaction by adding 50 µL of 1 N HCl.8. Read the optical density at 492 nm. Inhibition of Glycosylation of Mucin 2673.4. AB Staining on Cryostat Sections of Cell Layer Rolls3.4.1. Cryostat Sections of Cell Layer RollsCells grown in 25-cm2 flasks are rinsed twice in PBS, and dry scraped with a rubberpoliceman, and the cell pellet is frozen in liquid nitrogen. Cryostat sections (6 µm) of thecell pellet are performed and fixed using 4% paraformaldehyde (15 min), followed bywashing in PBS (two times for 15 min). After drying, slides can be stored at –20°C (15).3.4.2. AB Staining of Cryostat Sections1. Wash slides in distilled water for 5 min.2. Incubate in AB for 15–30 min.3. Wash in distilled water (three times for 5 min).4. Counterstain using nuclear red for 5 min.5. Wash in distilled water (three times for 5 min).6. Dehydrate in alcohol (70°, 95°, 100°) and toluol before mounting in Eukitt.3.5. Analysis of GalNAcα-O-aryl Derivatives3.5.1. Preparation of SamplesAfter continuous labeling with [6–3H]Galactose, the culture media are collectedfrom the 6-well culture plates and cells are washed three times with sterile PBS. Cellsare then harvested and lysed in 1 mL of distilled water by sonication for 2 min using aFig. 1. Example of ELISAs of purified mucins from control (ᮀ), and fromGalNAcα-O-benzyl–treated cells () with monoclonal antibody (MAb) BM 22.19detecting T-antigen (A), and with MAb Tn-5 detecting Tn-antigen (B) (12). 268 Huet et al.cell disrupter adapted with an exponential probe. Cell lysates are centrifuged for15 min at 13,000g. The supernatants are heat denatured (5 min at 100°C) and filteredthrough onto 0.22 µm ultrafiltration units prior the injection.For the analysis of the GalNAcα-O-benzyl derivatives secreted in the culture media,medium samples are heat-denatured (5 min at 100°C), centrifuged for 15 min at13,000g, and filtered through onto 0.22-µm ultrafiltration units prior to injection on anHPLC column.3.5.2. Reversed-Phase HPLC Fractionationof GalNAcα-O-benzyl DerivativesOne hundred microliters of sample are injected at a flow rate of 1 mL min andeluted isocratically with distilled water for 10 min. An acetonitrile gradient is thenapplied by increasing the percentage of acetonitrile from 0 to 50% in 20 min. Thepercentage is still maintained at 50% for 15 min and the column is reequilibrated inwater for 20 min prior to the next injection. Detection of the radioactive compoundsis performed using a continuous flow radiochromatography detector. Figure 2 givesan example of a separation profile.3.5.3. Identification of GalNAcα-O-benzyl DerivativesIdentification is performed by the coinjection of [14C]labeled radioactive standards(i.e., [14C]Gal, [14C]Galβ1-3GalNAcα-O-benzyl, [14C]NeuAcα2-3Galβ1-3GalNAcα-O-benzyl) and dual-label scintillation counting.4. Notes1. The lectin staining protocol is an evolution of the Glycan Detection protocol fromBoehringer Mannheim, provider of the DIG-labeled lectins. Several modifications havebeen made to optimize the initial procedure. The main change concerns the substitutionof the first blocking solution (step 1) by polyvinyl pyrrolidone K-30 at 2% in TBS (block-ing solution 1) prior to the incubation with DIG-labeled lectins. This change was made todecrease the background owing to the nonspecific binding of lectins to the blocking rea-gent, initially used for the saturation of the membrane after Western blotting. The block-ing reagent (commercially available from Boehringer Mannheim, cat. no. 1 096 176) isused in a second step of blocking (blocking solution 2, step 6) prior to incubation with thenitrocellulose membrane with the anti-DIG Fab fragments.2. The DIG-labeled lectins are used at the following concentrations: Amaranthin fromAmaranthus caudatus (ACA-dig) 2.5 µg mL; Maackia amurensis agglutinin (MAA-dig)5 µg mL; peanut (Arachis hypogaea) agglutinin (PNA-dig) 2 µg mL; Sambucusnigra agglutinin (SNA-dig) 2µg mL.3. PNA recognizes T-antigen (Galβ1-3GalNAc-R) only when the disaccharide is unsub-stituted by sialic acid, either linked on 3 position to Gal, or on 6 position to GalNAc. It ispossible to visualize and estimate the total amount of T-antigen (sialylated or not) by totaldesialylation of mucins after transfer onto the nitrocellulose membrane. Desialylation ofthe blot is performed as follows: After step 2, incubate the membrane in a plastic bag Inhibition of Glycosylation of Mucin 269Fig. 2. Example of separation by reversed-phase HPLC of cell extracts from control andGalNAcα-O-benzyl–treated cells after metabolic labeling with [3H]Gal (12). Incorporation of[3H]Galactose was examined after 5 h (A), 24 h (B), and 48 h (C) of exposure to 5 mMGalNAcα-O-benzyl. The HPLC profile of control cells (D) is shown for 48 h of incubationwith [3H]Gal. The retention times of [14C]labeled standards are indicated: 1, [14C]Gal; 2, [14C]NeuAcα2-3Galβ1-3GalNAcα-O-benzyl; 3, [14C]Galβ1-3GalNAcα-O-benzyl. 270 Huet et al.containing 10 mL of 50 mM citrate buffer, pH 6.0, 0.9% NaCl, 0.1% CaCl2supplementedwith 50 mU mL of sialidase from Clostridium perfringens. After a 16-h of incubation at37°C, go back to the current protocol at step 2.4. Development of the coloration must be performed without shaking. The revelation ofmucins should appear within the first 2 to 3 min. Increasing the times is not advised andmay enhance the nonspecific staining.References1. Roussel, P., Lamblin, G., Lhermitte, M., Houdret, N., Lafitte, J. J., Perini, J. M., Klein, A.,and Scharfman, A. (1988) The carbohydrate diversity of human respiratory mucins : aprotection of the underlying mucosa? Biochimie 70, 1471–1482.2. Strous, G. J. and Dekker, J. (1992) Mucin-type glycoproteins Crit. Rev. Biochem. Mol.Biol. 27, 57–92.3. Verma, M. and Davidson, E. A. (1994) Mucin genes : structure, expression and regulationGlycoconjugate J. 11, 172–179.4. Van Klinken, B. J. W., Dekker, J., Büller, H. A., and Einerhand, A. W. C. (1995) Mucingene structure and expression : protection vs adhesion. Am. J. Physiol. 269, (Gastrointest.Liver Physiol. 32), G613–G627.5. Lesuffleur, T., Zweibaum, A., and Real, F. X. (1994) Mucins in normal and neoplastichuman gastrointestinal tissues Crit. Rev. Oncol. Hematol. 17, 153–180.6. Kim, Y. S., Gum, J. R., and Brockhausen, I. (1996) Mucin glycoproteins in neoplasiaGlycoconjugate J. 13, 693–707.7. Zweibaum, A., Laburthe, M. Grasset, E., and Louvard, D. (1991) Use of cultured cellslines in studies of intestinal cell differentiation and function, in “Intestinal Absorption andSecretion.” Handbook of Physiology. vol. 4 (Field, M. and Frizzel, R. A., eds.), AmericanPhysiological Society, Bethesda, MD, pp. 223–255.8. Kuan, S. F., Byrd, J. C., Basbaum, C., and Kim, Y.S. (1989) Inhibition of mucinglycosylation by aryl-N-acetyl-α-galactosaminides in human colon cancer cells J. Biol.Chem. 264, 19271–19277.9. Brockhausen, I., Rachaman, E. S., Matta, K., and Schachter, H. (1983) The separationby liquid chromatography (under elevated pressure) of phenyl, benzyl, and O-nitro-phenyl glycosides of oligosaccharides. Analysis of substrates and products for fourN-acetyl-D-glucosaminyltransferases involved in mucin synthesis. Carbohydr. Res.120, 3–16.10. Huang, S., Byrd, J. C., Yoor, W. H., and Kim, Y. S. (1992) Effect of benzyl-α-GalNAc, aninhibitor of mucin glycosylation, on cancer-associated antigens, in human colon cancercells Oncol. Res. 4, 507–515.11. Huet, G., Kim, I., de Bolos, C., Lo-Guidice, J. M., Moreau, O., Hemon, B., Richet, C.,Delannoy, P., Real, F. X., and Degand, P. (1995) Characterization of mucins andproteoglycans synthesized by a mucin-secreting HT-29 cell subpopulation. J. Cell Sci.108, 1275–1285.12. Delannoy, P., Kim, I., Emery, N., de Bolos, C., Verbert, A., Degand, P., and Huet, G.(1996) Benzyl-N-acetyl-α-D-galactosaminide inhibits the sialylation and the secretionof mucins by a mucin secreting HT-29 cell subpopulation Glycoconj. J. 13, 717–726.13. Houdret, N., Perini, J. M., Galabert, C., Scharfman, A., Humbert, P., Lamblin, G., andRoussel, P. (1986) The high lipid content of respiratory mucins in cystic fibrosis is relatedto infection. Biochem. Biophys. Acta 880, 54–61. [...]... of GalNAc α -O-benzyl Derivatives Identification is performed by the coinjection of [ 14 C]labeled radioactive standards (i.e., [ 14 C]Gal, [ 14 C]Galβ 1-3 GalNAcα-O-benzyl, [ 14 C]NeuAcα 2-3 Galβ 1-3 GalNAc - O-benzyl) and dual-label scintillation counting. 4. Notes 1. The lectin staining protocol is an evolution of the Glycan Detection protocol from Boehringer Mannheim, provider of the DIG-labeled lectins.... O-glycosylation have been used in in vivo experiments on cultured cells (8). For that purpose, aryl- N-acetyl-α-galactosaminides (GalNAcα-O-aryls) have been initially used as potential competitors of the glycosylation of N-acetylgalactosamine (GalNAc) residues linked to the core protein, since these sugar analogs were suitable substrates From: Methods in Molecular Biology, Vol. 125: Glycoprotein Methods. .. with the anti-DIG Fab fragments. 2. The DIG-labeled lectins are used at the following concentrations: Amaranthin from Amaranthus caudatus (ACA-dig) 2.5 µg mL; Maackia amurensis agglutinin (MAA-dig) 5 µg mL; peanut (Arachis hypogaea) agglutinin (PNA-dig) 2 µg mL; Sambucus nigra agglutinin (SNA-dig) 2µg mL. 3. PNA recognizes T-antigen (Galβ 1-3 GalNAc-R) only when the disaccharide is unsub- stituted by... units prior the injection. For the analysis of the GalNAcα-O-benzyl derivatives secreted in the culture media, medium samples are heat-denatured (5 min at 100°C), centrifuged for 15 min at 13,000g, and filtered through onto 0.2 2- m ultrafiltration units prior to injection on an HPLC column. 3.5.2. Reversed-Phase HPLC Fractionation of GalNAc α -O-benzyl Derivatives One hundred microliters of sample are... and Pierre Degand 1. Introduction Mucins are secreted or membrane-bound large glycoproteins produced by epithe- lial cells of normal and malignant tissues. The secreted mucins are the major compo- nents of the mucous gel overlaying respiratory, gastrointestinal, or genital epithelia. Mucins constitute a family of extensively O-glycosylated glycoproteins (40–80% by weight) (1,2) encoded by a family of... first blocking solution (step 1) by polyvinyl pyrrolidone K-30 at 2% in TBS (block- ing solution 1) prior to the incubation with DIG-labeled lectins. This change was made to decrease the background owing to the nonspecific binding of lectins to the blocking rea- gent, initially used for the saturation of the membrane after Western blotting. The block- ing reagent (commercially available from Boehringer... cells. Mucins display tissue-specific patterns of O-glycosylation. Alterations in the glycosylation of mucins commonly occur in many mucosal diseases. In particular, glycan epitopes of mucins are impor- tant markers in cancer (5,6). Studies on the regulation of the biosynthesis and secretion of mucins have been improved by the use of human mucosal cells, which can be grown in long-term culture (7). To address... core of the molecule. The biochemical properties and functions of mucins are greatly dependent on their O-glycosylation state. In particular, mucins can display a role in cellular protection or cellular adhesion (4). On the one hand, filamentous mucins highly substituted by nega- tively charged O-glycans act as a protective barrier for epithelial cells. On the other hand, the terminal oligosaccharides... (Galβ 1-3 GalNAc-R) only when the disaccharide is unsub- stituted by sialic acid, either linked on 3 position to Gal, or on 6 position to GalNAc. It is possible to visualize and estimate the total amount of T-antigen (sialylated or not) by total desialylation of mucins after transfer onto the nitrocellulose membrane. Desialylation of the blot is performed as follows: After step 2, incubate the membrane in... glycosylation of N-acetylgalactosamine (GalNAc) residues linked to the core protein, since these sugar analogs were suitable substrates From: Methods in Molecular Biology, Vol. 125: Glycoprotein Methods and Protocols: The Mucins Edited by: A. Corfield © Humana Press Inc., Totowa, NJ . with GalNAcα-O-aryl: The medium in ituma is added with aryl-N-acetyl-α-D-galactosaminide.c. Threonine-free medium (Life Technologies).d. Low-glucose Dulbecco’s. UDP-GlcNAc:GalNAc-Rβ1, 3-N-acetylglucosaminyltransferase (9). Actually, inin vitro experiments, these compounds inhibit the UDP-Gal:GalNAc-Rβ1,3-galac-tosyltransferase