Mucin Granule Intraluminal Organization in Living Mucous/Goblet Cells
2005; Elsevier BV; Volume: 281; Issue: 8 Linguagem: Inglês
10.1074/jbc.m510520200
ISSN1083-351X
AutoresJuan Pérez-Vilar, Raean Mabolo, Cheryl T. McVaugh, Carolyn R. Bertozzi, Richard C. Boucher,
Tópico(s)Carbohydrate Chemistry and Synthesis
ResumoRecent studies suggest that the mucin granule lumen consists of a matrix meshwork embedded in a fluid phase. Secretory products can both diffuse, although very slowly, through the meshwork pores and interact noncovalently with the matrix. Using a green fluorescent protein-mucin fusion protein (SHGFP-MUC5AC/CK) as a FRAP (fluorescence recovery after photobleaching) probe, we have assessed in living mucous cells the relative importance of different protein post-translational modifications on the intragranular organization. Long term inhibition of mucin-type O-glycosylation, sialylation, or sulfation altered SHGFP-MUC5AC/CK characteristic diffusion time (t½), whereas all but sulfation diminished its mobile fraction. Reduction of protein disulfide bonds with tris(hydroxypropyl)phosphine resulted in virtually complete immobilization of the SHGFP-MUC5AC/CK intragranular pool. However, when activity of the vacuolar H+-ATPase was also inhibited, disulfide reduction decreased SHGFP-MUC5AC/CK t½ while diminishing its intraluminal concentration. Similar FRAP profiles were observed in granules that remained in the cells after the addition of a mucin secretagogue. Taken together these results suggest that: (a) the relative content of O-glycans and intragranular anionic groups is crucial for protein diffusion through the intragranular meshwork; (b) protein-protein, rather than carbohydrate-mediated, interactions are responsible for binding of SHGFP-MUC5AC/CK to the immobile fraction, although the degree of matrix O-glycosylation and sialylation affects such interactions; (c) intragranular organization does not depend on covalent multimerization of mucins or the presence of native disulfide bonds in the intragranular mucin/proteins, but rather on specific protein-mediated interactions that are important during the early stages of mucin matrix condensation; (d) alterations of the intragranular matrix precede granule discharge, which can be partial and, accordingly, does not necessarily involve the disappearance of the granule. Recent studies suggest that the mucin granule lumen consists of a matrix meshwork embedded in a fluid phase. Secretory products can both diffuse, although very slowly, through the meshwork pores and interact noncovalently with the matrix. Using a green fluorescent protein-mucin fusion protein (SHGFP-MUC5AC/CK) as a FRAP (fluorescence recovery after photobleaching) probe, we have assessed in living mucous cells the relative importance of different protein post-translational modifications on the intragranular organization. Long term inhibition of mucin-type O-glycosylation, sialylation, or sulfation altered SHGFP-MUC5AC/CK characteristic diffusion time (t½), whereas all but sulfation diminished its mobile fraction. Reduction of protein disulfide bonds with tris(hydroxypropyl)phosphine resulted in virtually complete immobilization of the SHGFP-MUC5AC/CK intragranular pool. However, when activity of the vacuolar H+-ATPase was also inhibited, disulfide reduction decreased SHGFP-MUC5AC/CK t½ while diminishing its intraluminal concentration. Similar FRAP profiles were observed in granules that remained in the cells after the addition of a mucin secretagogue. Taken together these results suggest that: (a) the relative content of O-glycans and intragranular anionic groups is crucial for protein diffusion through the intragranular meshwork; (b) protein-protein, rather than carbohydrate-mediated, interactions are responsible for binding of SHGFP-MUC5AC/CK to the immobile fraction, although the degree of matrix O-glycosylation and sialylation affects such interactions; (c) intragranular organization does not depend on covalent multimerization of mucins or the presence of native disulfide bonds in the intragranular mucin/proteins, but rather on specific protein-mediated interactions that are important during the early stages of mucin matrix condensation; (d) alterations of the intragranular matrix precede granule discharge, which can be partial and, accordingly, does not necessarily involve the disappearance of the granule. In response to specific signals, regulated secretion permits the controlled transport of membrane and secretory products (1Kepes F. Rambourg A. Satiat-Jeunemaitre B. Int. Rev. Cytol. 2005; 242: 55-120Crossref PubMed Scopus (47) Google Scholar, 2Burgoyne R.D. Morgan A. Physiol. Rev. 2003; 83: 581-632Crossref PubMed Scopus (555) Google Scholar). This pathway is supported by an efficient mechanism for importing/packing proteins into secretory granules. Indeed, the biogenesis of regulated secretory granules is initiated when secretory proteins aggregate in the trans-Golgi compartments (3Thiele C. Huttner W.B. Cell Dev. Biol. 1998; 9: 511-516Crossref PubMed Scopus (44) Google Scholar). Nevertheless, our understanding of intragranular protein condensation and its regulation still is fragmented and incomplete, especially in exocrine cells. Mucous/goblet cells are specialized in the synthesis and (constitutive/regulated) secretion of a family of glycoproteins known as secreted gel-forming mucins (4Strous G.J. Dekker J. Crit. Rev. Biochem. 1992; 27: 57-92Crossref Scopus (781) Google Scholar, 5Gendler D.J. Spicer A.P. Annu. Rev. Physiol. 1995; 57: 607-634Crossref PubMed Scopus (881) Google Scholar, 6Perez-Vilar J. Hill R.L. J. Biol. Chem. 1999; 274: 31751-31754Abstract Full Text Full Text PDF PubMed Scopus (404) Google Scholar, 7Dekker J. Rossen J.W. Buller H.A. Einerhand A.W. Trends Biochem. Sci. 2002; 27: 126-131Abstract Full Text Full Text PDF PubMed Scopus (333) Google Scholar). Mucins are characterized by their large size, high degree of mucin-type O-glycosylation, and the formation of disulfide-linked oligomers/multimers. Together with water and salts, mucins largely determine the viscoelastic and adhesive properties of the mucus secretions that cover the tracheobronchial, gastrointestinal, urogenital, auditory, and conjunctival epithelia. Although the mucus layer lubricates and protects epithelial cells against pathogenic and noxious agents (8Basbaum C. Lemjabbar H. Longphre M. Li D. Gensch E. McNamara N. Am. J. Respir. Crit. Care Med. 1999; 160: S44-S48Crossref PubMed Scopus (119) Google Scholar, 9Knowles M.R. Boucher R.C. J. Clin. Invest. 2002; 109: 571-577Crossref PubMed Scopus (1009) Google Scholar), overproduction of mucus can be detrimental to health, as is evident in lung diseases characterized by a mucus hypersecretory phenotype such as cystic fibrosis (10Perez-Vilar J. Boucher R.C. Free Rad. Biol. Med. 2004; 37: 1564-1577Crossref PubMed Scopus (83) Google Scholar). The biosynthesis of mucins comprises N-and O-glycosylation, likely C-mannosylation, sulfation, interchain disulfide bond formation, and proteolysis (6Perez-Vilar J. Hill R.L. J. Biol. Chem. 1999; 274: 31751-31754Abstract Full Text Full Text PDF PubMed Scopus (404) Google Scholar, 11Wickstrom C. Carlstedt I. J. Biol. Chem. 2001; 276: 47116-47121Abstract Full Text Full Text PDF PubMed Scopus (28) Google Scholar, 12Lidell M.E. Johansson M.E. Hansson G.C. J. Biol. Chem. 2003; 278: 13944-13951Abstract Full Text Full Text PDF PubMed Scopus (78) Google Scholar, 13Perez-Vilar J. Randell S.H. Boucher R.C. Glycobiology. 2004; 14: 325-337Crossref PubMed Scopus (88) Google Scholar). Processed mucin oligomers/multimers are stored inside mucin granules, where they likely form a highly condensed polyanionic matrix (14Neutra M.R. Phillips T.L. Phillips T.E. CIBA Found. Symp. 1984; 109: 20-39PubMed Google Scholar, 15Rogers D.F. Int. J. Biochem. Cell Biol. 2003; 35: 1-6Crossref PubMed Scopus (171) Google Scholar, 16Verdugo P. Annu. Rev. Physiol. 1990; 52: 157-176Crossref PubMed Scopus (243) Google Scholar, 17Verdugo P. Am. Rev. Respir. Dis. 1991; 144: S33-S37Crossref PubMed Google Scholar). Consistent with this notion, intestinal mucous cells with the characteristic mucin granules are not present in mice lacking expression of mMuc2, the gene encoding the major intestinal gel-forming mucin (18Velcich A. Yang W. Heyer J. Fragale A. Nicholas C. Viani S Kucherlapati R. Lipkin M. Yang K. Augenlicht L. Science. 2002; 295: 1726-1729Crossref PubMed Scopus (747) Google Scholar). Polyanionic matrices and high intraluminal concentrations of Ca2+ and H+ appear to be common features of regulated secretory granules irrespective of species and the cell type (e.g. 1Kepes F. Rambourg A. Satiat-Jeunemaitre B. Int. Rev. Cytol. 2005; 242: 55-120Crossref PubMed Scopus (47) Google Scholar, 19Serafin W.E. Katz H.R. Austen K.F. Stevens R.L. J. Biol. Chem. 1986; 261: 15017-15021Abstract Full Text PDF PubMed Google Scholar, 20Day R. Gorr S.U. Trends Endocrinol. Metab. 2003; 14: 10-13Abstract Full Text Full Text PDF PubMed Scopus (65) Google Scholar, 21Chin W.C. Orellana M.V. Quesada I. Verdugo P. Plant Cell Physiol. 2004; 45: 535-542Crossref PubMed Scopus (62) Google Scholar). Ca2+ and H+ likely shield the negative charges in mucin O-linked oligosaccharide chains, facilitating the emergence of physical interactions among intragranular matrix mucins. It has been proposed that at some point during granule biogenesis, and following the same physical laws that govern phase transitions of synthetic polymers (22Tanaka T. Filmore D.J. J. Chem. Phys. 1979; 70: 1214-1220Crossref Scopus (1515) Google Scholar), polyanionic intragranular proteins in general, and mucin oligomers/multimers in particular, undergo a highly cooperative (likely Ca2+/H+-driven) phase transition (condensation) process, which results in the formation of osmotically inert, condensed aggregates (16Verdugo P. Annu. Rev. Physiol. 1990; 52: 157-176Crossref PubMed Scopus (243) Google Scholar, 17Verdugo P. Am. Rev. Respir. Dis. 1991; 144: S33-S37Crossref PubMed Google Scholar, 23Verdugo P. CIBA Found. Symp. 1984; 109: 212-225PubMed Google Scholar). Moreover, an ion exchange (Ca2+/K+)-triggered phase transition of the matrix is thought to occur prior to and/or during granule exocytosis, resulting in matrix decondensation and hydration and discharge of entrapped secretory proteins (24Marszalek P.E. Farrell B. Verdugo P. Fernandez J.M. Biophys. J. 1997; 73: 1160-1168Abstract Full Text PDF PubMed Scopus (52) Google Scholar, 25Marszalek P.E. Farrell B. Verdugo P. Fernandez J.M. Biophys. J. 1997; 73: 1169-1183Abstract Full Text PDF PubMed Scopus (74) Google Scholar). Recent studies in live mucous/goblet cells have revealed novel aspects on mucous/goblet cells and their granules (26Perez-Vilar J. Olsen J.C. Chua M. Boucher R.C. J. Biol. Chem. 2005; 280: 16868-16881Abstract Full Text Full Text PDF PubMed Scopus (30) Google Scholar, 27Perez-Vilar J. Ribeiro C.M. Salmon W.C. Mabolo R. Boucher R.C. J. Histochem. Cytochem. 2005; 53: 1305-1308Crossref PubMed Scopus (11) Google Scholar). Thus, fluorescence recovery after photobleaching (FRAP) 2The abbreviations used are: FRAP, fluorescence recovery after photobleaching; CHAPS, 3-[(3-cholamidopropyl)dimethylammonio]-1-propanesulfonic acid; DTT, dithiothreitol; GFP, green fluorescent protein; Mf, mobile fraction; PBS, phosphate-buffered saline; SiaNAc, sialic acid; t½, half-recovery time or characteristic diffusion time; THP, tris(hydroxypropyl)phosphine. analyses with a mucin-GFP fusion protein suggested that the mucin granule lumen is compartmentalized into a mobile or fluid phase, in which secretory products diffused very slowly, and an immobile, pH-dependent phase (i.e. a "matrix") to which secretory products were noncovalently bound via pH-dependent interactions (26Perez-Vilar J. Olsen J.C. Chua M. Boucher R.C. J. Biol. Chem. 2005; 280: 16868-16881Abstract Full Text Full Text PDF PubMed Scopus (30) Google Scholar). A two-phase, intraluminal organization was proposed independently on the basis of studies on the Ca2+/K+ intragranular ion exchange mechanism of exocytosis (28Nguyen T. Chin W.C. Verdugo P. Nature. 1998; 395: 908-912Crossref PubMed Scopus (161) Google Scholar, 29Quesada I. Chin W.C. Verdugo P. Biophys. J. 2003; 85: 963-970Abstract Full Text Full Text PDF PubMed Scopus (40) Google Scholar). The facts that the same protein probe diffused ∼100-fold faster in the endoplasmic reticulum lumen than within the mucin granule, whereas its diffusion seemed even faster once it reached the extracellular mucus 3J. Perez-Vilar, unpublished observations. (26Perez-Vilar J. Olsen J.C. Chua M. Boucher R.C. J. Biol. Chem. 2005; 280: 16868-16881Abstract Full Text Full Text PDF PubMed Scopus (30) Google Scholar), supports the notion that the intragranular mucin matrix is in a condensed state. However, the FRAP profiles obtained argued against the existence of an inaccessible, dehydrated condensed mucin matrix surrounded by a dense fluid phase (26Perez-Vilar J. Olsen J.C. Chua M. Boucher R.C. J. Biol. Chem. 2005; 280: 16868-16881Abstract Full Text Full Text PDF PubMed Scopus (30) Google Scholar). Instead, the mucin meshwork is likely embedded in a fluid phase so that proteins diffuse through its pores and have access to, and interact with, the matrix components. Although these studies are not necessarily in conflict with a phase transition-driven mechanism of matrix condensation, they raise the possibility that specific protein-protein interactions have critical roles at some point during the formation of the matrix meshwork. Using time-lapse and semiquantitative FRAP analysis in live mucous cells, we report here studies aimed at identifying the relative roles in mucin granule organization of mucin post-translational modifications, including O-glycosylation, sulfation, sialylation, and protein disulfide bonding. The intraluminal environment of granules remaining after the addition of mucin secretagogue was also investigated. The results suggest that the intragranular environment can be independently regulated by different factors. Maintenance of HT29-SHGFP-MUC5AC/CK Cells and Experimental Treatments—The generation, characterization, and maintenance of HT29-SHGFP-MUC5AC/CK cells were described earlier (26Perez-Vilar J. Olsen J.C. Chua M. Boucher R.C. J. Biol. Chem. 2005; 280: 16868-16881Abstract Full Text Full Text PDF PubMed Scopus (30) Google Scholar). Compound 2-68A (100 μg/ml; Ref. 31Hang H.C. Yu C. Ten Hagen K.G. Tian E. Winans K.A. Tabak L.A. Bertozzi C.R. Chem. Biol. 2004; 11: 337-345Abstract Full Text Full Text PDF PubMed Scopus (56) Google Scholar), sodium chlorate (10 mg/ml; Ref. 32Dekker J. Van Beurden-Lamers W.M. Strous G.J. J. Biol. Chem. 1989; 264: 10431-10437Abstract Full Text PDF PubMed Google Scholar), 4-methylumbelliferyl-β-d-xylopyranoside (10 mm; Ref. 33Lohmander L.S. Hascall V.C. Caplan A.I. J. Biol. Chem. 1979; 254: 10551-10561Abstract Full Text PDF PubMed Google Scholar), the peptides NH2-GNWWWW and NH2-WRGGSG (100 μm; Ref. 34Lee K.Y. Kim H.G. Hwang M.R. Chae J.L. Yang J.M. Lee Y.C. Choo Y.K. Lee Y.I. Lee S.S. Do S.I. J. Biol. Chem. 2002; 277: 49341-49351Abstract Full Text Full Text PDF PubMed Scopus (25) Google Scholar), denoted herein as P6SIAL and P6CON, respectively, tris(hydroxypropyl)phosphine (THP, 5 mm, Calbiochem; Ref. 35Cline D.J. Redding S.E. Brohawn S.G. Psathas J.N. Schneider J.P. Thorpe C. Biochemistry. 2004; 43: 15195-15203Crossref PubMed Scopus (162) Google Scholar), bafilomycin A1 (0.1 μg/ml; Ref. 36Drose S. Altendorf K. J. Exp. Biol. 1997; 200: 1-8Crossref PubMed Google Scholar) or dithiothreitol (DTT, 5 mm; Ref. 37Braakman I. Helenius J. Helenius A. EMBO J. 1992; 11: 1717-1722Crossref PubMed Scopus (336) Google Scholar) were freshly prepared in culture medium, with solubilization in dimethyl sulfoxide when required, and given to the cells for 48 h, in the case of compound 2-68A and sodium chlorate or 72 h in the case of 4-methylumbelliferyl-β-d-xylopyranoside, P6SIAL or P6CON. To induce mucin granule secretion, cells were incubated for at least 30 min in the presence of 3 mm ATP prior to FRAP or biochemical analysis. In all cases, vehicle-incubated control cells were analyzed in parallel under the same conditions. Unless otherwise indicated, all of the reagents and peptides mentioned in this work were obtained from Sigma. Generation of NIH-3T3-SHGFP-MUC5AC/CK Cells—NIH-3T3 cells, obtained from ATCC and maintained as directed by the provider, were transduced with a retroviral vector encoding SHGFP-MUC5AC/CK essentially as described earlier for HT29–18N2 cells (26Perez-Vilar J. Olsen J.C. Chua M. Boucher R.C. J. Biol. Chem. 2005; 280: 16868-16881Abstract Full Text Full Text PDF PubMed Scopus (30) Google Scholar). After selection in puromycin-containing culture medium, the cells were maintained for at least 20 passages prior to use for these studies. At that point, this cell line did not differ from the parental cell line regarding morphologic features and growth rate. Confocal Microscopy and FRAP Analysis—Cells in confocal medium (Hanks' balanced solution salt plus 1% (v/v) fetal bovine serum, 5 mm l-glutamine, essential and nonessential amino acids, and 20 mm HEPES, pH 7.8) were observed with a Zeiss LSM 510 (University of North Carolina Michael Hooker Microscopy Facility) at 37 °C on the microscope stage, using 488 nm laser excitation for GFP. FRAP analysis of intragranular SHGFP-MUC5AC/CK in live mucous/goblet cells was carried out using a ×63 (NA 1.4) oil immersion objective as described before (26Perez-Vilar J. Olsen J.C. Chua M. Boucher R.C. J. Biol. Chem. 2005; 280: 16868-16881Abstract Full Text Full Text PDF PubMed Scopus (30) Google Scholar). Briefly, a spot the size of the laser beam diameter was irreversibly bleached and the fluorescence intensity in the bleached area monitored over time. From these raw data, the mobile fraction (Mf), i.e. the percent of intragranular SHGFP-MUC5AC/CK that diffuses, and the characteristic diffusion time (t½), i.e. the time required to reach 50% of the plateau fluorescence in the bleached area, were derived using standard procedures (26Perez-Vilar J. Olsen J.C. Chua M. Boucher R.C. J. Biol. Chem. 2005; 280: 16868-16881Abstract Full Text Full Text PDF PubMed Scopus (30) Google Scholar, 38Snapp E. Altan N. Lippincott-Schwartz J. Bonifacino J. Dasso M. Harford J. Lippincott-Schwartz J. Yamada K. Morgan K.S. Current Protocols in Cell Biolog. John Wiley & Sons, Inc., New York2003Google Scholar). In each case, vehicle- and compound-treated cells were analyzed in parallel. At least three different culture dishes from three independent experiments were processed. The statistical significance of differences between the means of specific parameters in specific pairs of vehicle- and compound-treated cells was assessed by student t tests. Caspase 3/7 Assays—To assess the cellular levels of caspases 3 and 7, cells grown in 4-well slides (Cultek, Inc.) were put on ice for 5 min, washed twice with cold Ca2+/Mg2+-free PBS, and lysed for 15 min at room temperature in 300 μl of 0.5% (v/v) Triton X-100 in PBS/well. The lysates were cleared by centrifugation at 16,000 × g for 1 min and then diluted 1:100 with sterile water. 1–10 μl of the latter solution was mixed with an equal volume of freshly made caspase 3/7-Glo reagent (Promega, Inc.). The mixture was incubated at room temperature for up to 1 h and light emission measured with a Turner Biosystem 20/20 luminometer. Analysis of Glycoproteins Metabolically Labeled with N-Acetyl-[3H]Glucosamine or 35SO4Na2—Mucous cells grown in 35-mm culture dishes were metabolically labeled at 37 °C with 25 μCi/ml N-acetyl-[3H]glucosamine ([3H]GlcNAc, Amersham Biosciences; 35 Bq/mmol) or 50 μCi/ml 35SO4Na2 (MPB; 74 MBq)) in PFHM-II culture medium (39Philips T.E. Ramos R. Duncan S.L. In Vitro Cell Dev. Biol. Anim. 1995; 31: 421-423Crossref PubMed Scopus (11) Google Scholar) for 24 h in the absence (control sample) or presence of the indicated compounds. At the end of the labeling, the cells were put on ice for 5 min and the culture medium collected and cleared from cell debris by centrifugation. The cell layer was rinsed with cold PBS and then extracted at 4 °C with 4 m guanidine HCl, 0.1 m sodium acetate, 4% (w/v) CHAPS, 100 μm phenylmethylsulfonyl fluoride, pH 5.8 (extraction buffer; 3 ml/dish), for 30 min with agitation. The lysates were cleared by centrifugation and the supernatant diluted with extraction buffer to a concentration of 0.5–0.7 mg of protein/ml. Radioactive proteins were separated from unincorporated radioactive probe by passing 500 μl of cell lysate through a 5-ml Sephadex G-50 (fine) column equilibrated in 6 m urea, 0.1 m Tris-HCl, 5 mm EDTA, 0.5% CHAPS (w/v), 100 μm phenylmethylsulfonyl fluoride, pH 8.0 (elution buffer). The proteins were eluted with elution buffer and 0.5-ml fractions collected. The radioactivity in each fraction was measured by scintillation counting. To determine the incorporation of the radioactive label into high molecular weight glycoproteins/mucins, cell lysates were dialyzed against 6 m urea, 0.1 m Tris-HCl, 5 mm EDTA, pH 8.0, for 18 h at 4 °C, then reduced with 20 mm DTT for 18 h at room temperature and carboxymethylated with freshly prepared 20 mm iodoacetamide for 30 min at 25 °C in the dark (40Thornton D.J. Howard M. Devine P.L. Sheehan J.K. Anal. Biochem. 1995; 227: 162-167Crossref PubMed Scopus (68) Google Scholar). Proteins (30 μg/well) were separated and visualized by standard gradient (4–20%) SDS-PAGE and fluorography. Alternatively, proteins were separated in 1% SDS-agarose gels as described previously (40Thornton D.J. Howard M. Devine P.L. Sheehan J.K. Anal. Biochem. 1995; 227: 162-167Crossref PubMed Scopus (68) Google Scholar), and equal square pieces (∼0.5 × 0.5 cm2) were cut from the top to the bottom of the agarose gels. The gel pieces were melted with guanidine thiocyanate, pH 8, at 65 °C for 10 min and mixed with 2.5 ml of Safety Solve prior to counting the total radioactivity in a scintillation counter. Protein Blotting of Endogenous MUC5AC Separated in Agarose Gels—Total cellular proteins were extracted, separated by SDS-agarose gel electrophoresis, transferred to nitrocellulose membranes, and MUC5AC detected with specific antibodies following published procedures (40Thornton D.J. Howard M. Devine P.L. Sheehan J.K. Anal. Biochem. 1995; 227: 162-167Crossref PubMed Scopus (68) Google Scholar). A mouse anti-human MUC5AC monoclonal antibody (2Q445; U. S. Biologicals) recognizing the unglycosylated tandem repeat region of MUC5AC (41Sheehan J.K. Brazeau C. Kutay S. Pigeon H. Kirkham S. Howard M. Thornton D.J. Biochemistry. 2000; 347: 37-44Crossref Google Scholar), and a specific rabbit antiserum, named MUC5AC-062, obtained against a peptide (TWTTWFDVDFPS) of the MUC5AC Cys-5 subdomain, respectively, were employed in these studies. As judged by protein blotting, this antiserum specifically detects the same molecular species of MUC5AC recognized by another well characterized rabbit anti-MUC5AC (41Sheehan J.K. Brazeau C. Kutay S. Pigeon H. Kirkham S. Howard M. Thornton D.J. Biochemistry. 2000; 347: 37-44Crossref Google Scholar), including cystine-reduced, unglycosylated, and O-glycosylated monomers, but not unreduced species of this mucin (not shown). Other Methods—Cellular DNA was purified from cell extracts using the DNeasy Tissue kit from Qiagen and its concentration estimated by absorbance at 260 nm. Protein concentration was determined by the bicinchoninic acid assay using reagents from Pierce. The sialic acid content in cultures of HT29-SHGFP-MUC5AC/CK cells was assessed by microscopy using SNA-biotin (Vector Laboratories) as the specific label. Thus, cells were fixed with methanol at -20 °C, nonspecific binding sites blocked with 10% (w/v) bovine serum albumin in PBS, and sialic acid residues located with SNA-biotin (0.05 μg/ml in PBS) and streptavidin-rhodamine. XY serial images of the cultures were obtained via confocal microscopy. Inhibition of Mucin-type O-Glycosylation Alters SHGFP-MUC5AC/CK Intragranular Mobile Fraction and Mobility—Mucin-type O-glycosylation is the major post-translational modification of gel-forming mucins, and it can represent up to 90% of their molecular weights (4Strous G.J. Dekker J. Crit. Rev. Biochem. 1992; 27: 57-92Crossref Scopus (781) Google Scholar). To assess the relative importance of this modification in intragranular organization, O-glycosylation was inhibited in HT29-SHGFP-MUC5AC/CK mucous intestinal adenocarcinoma cells (26Perez-Vilar J. Olsen J.C. Chua M. Boucher R.C. J. Biol. Chem. 2005; 280: 16868-16881Abstract Full Text Full Text PDF PubMed Scopus (30) Google Scholar). These cells stably express SHGFP-MUC5AC/CK, a fusion protein consisting of a secreted form of GFP and the CK domain of MUC5AC, one of the five human gel-forming mucins known. SHGFP-MUC5AC/CK does not covalently bind to endogenous MUC5AC, but it is stored inside the mucin granules (26Perez-Vilar J. Olsen J.C. Chua M. Boucher R.C. J. Biol. Chem. 2005; 280: 16868-16881Abstract Full Text Full Text PDF PubMed Scopus (30) Google Scholar). To inhibit O-glycosylation, cells were grown in the presence of compound 2-68A. This uridine derivative is a specific competitive inhibitor of the UDP-GalNAc:polypeptide α-GalNAc-transferase family of glycosyltransferases that initiate mucin-type O-glycosylation (31Hang H.C. Yu C. Ten Hagen K.G. Tian E. Winans K.A. Tabak L.A. Bertozzi C.R. Chem. Biol. 2004; 11: 337-345Abstract Full Text Full Text PDF PubMed Scopus (56) Google Scholar). Cultures incubated with vehicle (dimethyl sulfoxide) (Fig. 1A, a and b) or compound 2-68A (c and d) appeared to have comparable number of mucous cells with numerous mucin granules each (Fig. 1B), suggesting that compound 2-68A was not toxic under these conditions. Because it has been reported that inhibition of O-glycosylation by uridine derivatives triggers cellular apoptosis in different cell types (31Hang H.C. Yu C. Ten Hagen K.G. Tian E. Winans K.A. Tabak L.A. Bertozzi C.R. Chem. Biol. 2004; 11: 337-345Abstract Full Text Full Text PDF PubMed Scopus (56) Google Scholar, 42Tian E. Hagen K.G. Shum L. Hang H.C. Imbert Y. Young Jr., W.W. Bertozzi C.R. Tabak L.A. J. Biol. Chem. 2004; 279: 50382-50390Abstract Full Text Full Text PDF PubMed Scopus (14) Google Scholar), it was important to assess whether the degree of cellular apoptosis differed between control and 2-68A-incubated cultures. Thus, the activities of caspases 3 and 7, two well characterized apoptosis markers (43Wang Z.B. Liu Y.Q. Cui Y.F. Cell Biol. Int. 2005; 29: 489-496Crossref PubMed Scopus (188) Google Scholar), were determined in cell lysates of mucous cells incubated for 48 h in the presence of vehicle or compound 2-68A. As shown in Fig. 1C, the respective cellular levels of caspase 3/7 were very similar, suggesting that under these experimental conditions, compound 2-68A did not induce apoptosis. To validate these results, we generated NIH-3T3 cells stably expressing SHGFP-MUC5AC/CK, the same protein expressed by the mucous cell line. NIH-3T3 is a cell line known to undergo apoptosis upon inhibition of O-glycosylation with uridine derivatives (42Tian E. Hagen K.G. Shum L. Hang H.C. Imbert Y. Young Jr., W.W. Bertozzi C.R. Tabak L.A. J. Biol. Chem. 2004; 279: 50382-50390Abstract Full Text Full Text PDF PubMed Scopus (14) Google Scholar). Consistent with these reports, the addition of compound 2-68A to NIH-3T3-SHGFP-MUC5AC/CK fibroblasts resulted in increased caspase 3/7 activities within an hour (Fig. 1D) and widespread and extensive cellular apoptosis 24 h later (Fig. 1E). The effectiveness of compound 2-68A as an O-glycosylation inhibitor in mucous cells was assessed by determining the incorporation of a radioactive monosaccharide into cellular glycoproteins. In these studies, cells preincubated for 24 h with vehicle (dimethyl sulfoxide) or compound 2-68A were metabolically labeled with [3H]GlcNAc for another 24 h in the presence of dimethyl sulfoxide or compound 2-68A. 3H-Labeled proteins from cell lysates were separated from unincorporated radioactive monosaccharide by gel filtration and the radioactivity in each fraction quantified. Compared with control cells, cells incubated with compound 2-68A had a much smaller signal in the excluded volume fractions (Fig. 1F), which contain proteins eluted from the column. Similarly, compound 2-68A inhibited incorporation of [3H]GlcNAc into high molecular weight glycoproteins/mucins fractionated by SDS-agarose gel electrophoresis (see supplemental Fig. s1). Consistent with these results, the cellular binding of SNA, a lectin specific for sialic acid residues, was reduced in compound 2-68A-treated cells (see supplemental Fig. s2). These results are consistent with substantial inhibition of mucin type O-glycosylation by compound 2-68A in cultured mucous cells, as has been reported in other cell types treated with uridine derivatives in vitro and in vivo (31Hang H.C. Yu C. Ten Hagen K.G. Tian E. Winans K.A. Tabak L.A. Bertozzi C.R. Chem. Biol. 2004; 11: 337-345Abstract Full Text Full Text PDF PubMed Scopus (56) Google Scholar, 42Tian E. Hagen K.G. Shum L. Hang H.C. Imbert Y. Young Jr., W.W. Bertozzi C.R. Tabak L.A. J. Biol. Chem. 2004; 279: 50382-50390Abstract Full Text Full Text PDF PubMed Scopus (14) Google Scholar). Having provided experimental evidence that mucin-type O-glycosylation was inhibited efficiently, whereas the mucous cells were not apoptotic, we analyzed the intragranular FRAP parameters in granules from vehicle- or compound 2-68A-incubated living mucous cells processed in parallel. Because the inhibitor was added to cultures in which in vitro mucous cell differentiation was already in progress (26Perez-Vilar J. Olsen J.C. Chua M. Boucher R.C. J. Biol. Chem. 2005; 280: 16868-16881Abstract Full Text Full Text PDF PubMed Scopus (30) Google Scholar, 39Philips T.E. Ramos R. Duncan S.L. In Vitro Cell Dev. Biol. Anim. 1995; 31: 421-423Crossref PubMed Scopus (11) Google Scholar), we expected that after 2 days with the inhibitor, a significant fraction of underglycosylated mucins and other glycoproteins would be present within the granules. A spot
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