Noomi Asker

Normal blood microvessels are lined by pericytes, which contribute to microvessel development and stability through mechanisms that are poorly understood. Pericyte deficiency has been implicated in the pathogenesis of microvascular abnormalities associated with diabetes and tumors. However, the unambiguous identification of pericytes is still a problem because of cellular heterogeneity and few available molecular markers. Here we describe an approach to identify pericyte markers based on transcription profiling of pericyte-deficient brain microvessels isolated from platelet-derived growth factor (PDGF-B)-/- and PDGF beta receptor (PDGFRbeta)-/- mouse mutants. The approach was validated by the identification of known pericyte markers among the most down-regulated genes in PDGF-B-/- and PDGFRbeta-/- microvessels. Of candidates for novel pericyte markers, we selected ATP-sensitive potassium-channel Kir6.1 (also known as Kcnj8) and sulfonylurea receptor 2, (SUR2, also known as Abcc9), both part of the same channel complex, as well as delta homologue 1 (DLK1) for in situ hybridization, which demonstrated their specific expression in brain pericytes of mouse embryos. We also show that Kir6.1 is highly expressed in pericytes in brain but undetectable in pericytes in skin and heart. The three new brain pericyte markers are signaling molecules implicated in ion transport and intercellular signaling, potentially opening new windows on pericyte function in brain microvessels.

Pulse-chase experiments in the colon cell line LS 174T combined with subcellular fractionation by sucrose density gradient centrifugation showed that the initial dimerization of the MUC2 apomucin started directly after translocation of the apomucin into the rough endoplasmic reticulum as detected by calnexin reactivity. As the mono- and dimers were chased, O-glycosylated MUC2 mono- and dimers were precipitated using anO-glycosylation-insensitive antiserum against the N-terminal domain of the MUC2 mucin. These O-glycosylated species were precipitated from the fractions that comigrated with the galactosyltransferase activity during the subcellular fractionation, indicating that not only MUC2 dimers but also a significant amount of monomers are transferred into the Golgi apparatus. Inhibition ofN-glycosylation with tunicamycin treatment slowed down the rate of dimerization and introduced further oligomerization of the MUC2 apomucin in the endoplasmic reticulum. Results of two-dimensional gel electrophoresis demonstrated that these oligomers (putative tri- and tetramers) were stabilized by disulfide bonds. The non-N-glycosylated species of the MUC2 mucin were retained in the endoplasmic reticulum because no O-glycosylated species were precipitated after inhibition by tunicamycin. This suggests that N-glycans of MUC2 are necessary for the correct folding and dimerization of the MUC2 mucin. Pulse-chase experiments in the colon cell line LS 174T combined with subcellular fractionation by sucrose density gradient centrifugation showed that the initial dimerization of the MUC2 apomucin started directly after translocation of the apomucin into the rough endoplasmic reticulum as detected by calnexin reactivity. As the mono- and dimers were chased, O-glycosylated MUC2 mono- and dimers were precipitated using anO-glycosylation-insensitive antiserum against the N-terminal domain of the MUC2 mucin. These O-glycosylated species were precipitated from the fractions that comigrated with the galactosyltransferase activity during the subcellular fractionation, indicating that not only MUC2 dimers but also a significant amount of monomers are transferred into the Golgi apparatus. Inhibition ofN-glycosylation with tunicamycin treatment slowed down the rate of dimerization and introduced further oligomerization of the MUC2 apomucin in the endoplasmic reticulum. Results of two-dimensional gel electrophoresis demonstrated that these oligomers (putative tri- and tetramers) were stabilized by disulfide bonds. The non-N-glycosylated species of the MUC2 mucin were retained in the endoplasmic reticulum because no O-glycosylated species were precipitated after inhibition by tunicamycin. This suggests that N-glycans of MUC2 are necessary for the correct folding and dimerization of the MUC2 mucin. The mucus layer on the epithelial surface of the mucous membrane is mainly made up of water and the gel-forming components, the mucus glycoproteins, or mucins, consisting of more than 50%O-linked oligosaccharides (1Strous G.J. Dekker J. Crit. Rev. Biochem. Mol. Biol. 1992; 27: 57-92Crossref PubMed Scopus (752) Google Scholar, 2Forstner J.F. Forstner G.G. Johnson L.R. Physiology of the Gastrointestinal Tract. 3rd Ed. Raven Press, New York1994: 1255-1283Google Scholar). The peptide chain of mucins has domains with a high abundance of Ser, Thr, and Pro, usually in repetitive sequences (tandem repeats). The oligosaccharide chains are O-linked to Ser and Thr, thereby forming highly glycosylated domains or mucin domains. The apoprotein of the human intestinal MUC2 mucin, which is fully sequenced, contains two mucin domains with large amounts of the amino acids Thr, Pro, and Ser (3Gum Jr., J.R. Hicks J.W. Toribara N.W. Rothe E.-M. Lagace R.E. Kim Y.S. J. Biol. Chem. 1992; 267: 21375-21383Abstract Full Text PDF PubMed Google Scholar, 4Gum Jr., J.R. Hicks J.W. Toribara N.W. Siddiki B. Kim Y.S. J. Biol. Chem. 1994; 269: 2440-2446Abstract Full Text PDF PubMed Google Scholar); the larger of these domains consists of well conserved 23-amino acid repeated sequences. The mucin domains are flanked by Cys-rich domains; one C-terminal, one N-terminal, and one central domain. The carboxyl and amino termini of the human MUC2 mucin and the blood coagulation factor, the von Willebrand factor (vWF), 1The abbreviations used are: vWF, von Willebrand factor; NEM, N-ethylmaleimide; PAGE, polyacrylamide gel electrophoresis. 1The abbreviations used are: vWF, von Willebrand factor; NEM, N-ethylmaleimide; PAGE, polyacrylamide gel electrophoresis. show sequence similarities in the positions of the cysteines. The vWF forms disulfide-bonded dimers between two C termini, and the N termini mediate further oligomerization (5Ruggeri Z.M. Ware J. FASEB J. 1993; 7: 308-316Crossref PubMed Scopus (265) Google Scholar). We have earlier shown that the human MUC2 apomucin forms dimers before being O-glycosylated (6Asker N. Baeckström D. Axelsson M.A.B. Carlstedt I. Hansson G.C. Biochem. J. 1995; 308: 873-880Crossref PubMed Scopus (64) Google Scholar). To study the initial assembly of the human MUC2 mucin in more detail, pulse-chase labeling and subcellular fractionation has been performed on LS 174T cells. An early dimerization was observed in the endoplasmic reticulum; there was no further oligomerization, and the dimerization was followed by O-glycosylation of the mono- and dimer in the Golgi apparatus. Tunicamycin treatment slowed down the dimerization rate, introduced formation of putative tri- and tetramers, and prevented transfer of the apomucin into the Golgi apparatus. A rabbit antiserum α-MUC2TR (PH1900), against a synthetic peptide based on the tandem repeat region of the human MUC2 apoprotein, was raised (6Asker N. Baeckström D. Axelsson M.A.B. Carlstedt I. Hansson G.C. Biochem. J. 1995; 308: 873-880Crossref PubMed Scopus (64) Google Scholar). The rabbit antiserum, PH1491 (later referred to as α-MUC2N3), prepared against a synthetic peptide (CPKDRPIYEEDLKK) based on amino acids 1167–1180 on the N terminus of the human MUC2 apoprotein was prepared as follows. A New Zealand White rabbit was immunized once with 500 μg of peptide conjugated to 400 μg of keyhole limpet hemocyanin in Freund's complete adjuvant and then twice with 250 μg of peptide conjugated to 200 μg of keyhole limpet hemocyanin in Freund's incomplete adjuvant. The specificity of the α-MUC2N3 antiserum was tested by inhibition with the immunizing peptide. First, the optimum concentration for inhibition was tested by inhibiting binding to the immobilized peptide as described previously (6Asker N. Baeckström D. Axelsson M.A.B. Carlstedt I. Hansson G.C. Biochem. J. 1995; 308: 873-880Crossref PubMed Scopus (64) Google Scholar). One mg of specific and nonrelevant peptide/25 μl of antiserum was used for inhibiting immunoprecipitation, performed as described below. The colon adenocarcinoma cell line LS 174T (ATCC CL 188) was cultured as described (6Asker N. Baeckström D. Axelsson M.A.B. Carlstedt I. Hansson G.C. Biochem. J. 1995; 308: 873-880Crossref PubMed Scopus (64) Google Scholar). Cells were seeded at a concentration of about 50 × 106 cell/28-cm2 Petri dish the day before labeling. Cells were preincubated in methionine-free medium for 1 h followed by radiolabeling with 150 μCi 35S methionine (Redivue Promix [35S] labeling mix, Amersham Pharmacia Biotech)/dish. When cells were pulse-labeled for 2 min, 500 μCi of labeling mix/dish was added. In pulse-chase experiments cells were chased with culture medium supplemented by 15 μg of Met/ml of medium and 25 μg of Cys/ml of medium. Cells were washed and lysed as described (6Asker N. Baeckström D. Axelsson M.A.B. Carlstedt I. Hansson G.C. Biochem. J. 1995; 308: 873-880Crossref PubMed Scopus (64) Google Scholar), with the addition of 5 mm N-ethylmaleimide (NEM) in the lysis buffer. Inhibition ofN-glycosylation was performed by incubating cells in 20 μg of tunicamycin (Calbiochem)/ml of methionine-free medium 1 h prior to labeling, as well as during pulse and chase. Cells were washed twice with 5 ml of 250 mm sucrose and twice in 5 ml 50 mmsucrose. Cells were harvested in 500 μl of 50 mm sucrose including protease inhibitors (110 μg/ml phenylmethylsulfonyl fluoride, 20 μg/ml aprotinin, 60 μg/ml leupeptin, 3.8 μg/ml calpain inhibitor I) using a cell scraper. The cell suspension was homogenized with 15 strokes in a Dounce homogenizer using a tight pestle, and the sucrose concentration was adjusted to 250 mm followed by an additional 5 homogenization strokes. Washing and homogenization were performed on ice. The homogenized suspension was centrifuged in a microtube at 4200 rpm and +4 °C for 10 min, and the supernatant was recovered. The cell pellet was washed twice in 250 μl of 250 mm sucrose, and all supernatants were pooled and carefully placed on a sucrose gradient in a centrifuge tube containing a 4-ml gradient of 35–50% sucrose on top of a 400-μl 65% sucrose cushion. All sucrose solutions used contained 3 mm imidazole and were at pH 7.4. Ultracentrifugation was performed 50,000 rpm at 12 °C for 3 h in a Beckman vertical rotor (Vti, 65.2). Fractions were collected from the bottom. Phosphate-buffered saline was added to a final volume of 1.2 ml, and 300 μl of lysate buffer (250 mm Tris-HCl, pH 7.4, 750 mm NaCl, 25 mm EDTA, 5% (v/v) Triton X-100), including protease inhibitors (as above) and 5 mm NEM, was added. Samples were sonicated three times for 2 s each (intensity 15) on a MSE Soniprep 100. The fractions collected were analyzed for NADPH cytochrome c reductase activity and galactosyltransferase activity (7Borén J. Wettesten M. Sjöberg A. Thorlin T. Bondjers G. Wiklund O. Olofsson S.-O. J. Biol. Chem. 1990; 265: 10556-10564Abstract Full Text PDF PubMed Google Scholar, 8Boström K. Wettesten M. Borén J. Bondjers G. Wiklund O. Olofsson S.-O. J. Biol. Chem. 1986; 261: 13800-13806Abstract Full Text PDF PubMed Google Scholar). Fractions were also analyzed by 12% SDS-PAGE (9Laemmli U.K. Nature. 1970; 227: 680-685Crossref PubMed Scopus (205498) Google Scholar) followed by Western blot, and calnexin was visualized using a monoclonal α-calnexin antibody (Transduction Laboratories, Lexington, KY) and ECL (Amersham Pharmacia followed by was performed as described (6Asker N. Baeckström D. Axelsson M.A.B. Carlstedt I. Hansson G.C. Biochem. J. 1995; 308: 873-880Crossref PubMed Scopus (64) Google Scholar) using 25 μl of the α-MUC2TR μl of the α-MUC2N3 antiserum, or 1 μg of followed by 10 μl of a rabbit antiserum were washed and in μl of pH containing and 10 mm for 5 at Samples were and the concentration in the supernatant was adjusted to with buffer. was added at an amount than the amount of followed by the addition of 2 of Samples were at °C for 20 and was added at an amount than the amount of followed by for in the Samples were then with the α-MUC2TR as described were in or buffer at °C for 5 as described (6Asker N. Baeckström D. Axelsson M.A.B. Carlstedt I. Hansson G.C. Biochem. J. 1995; 308: 873-880Crossref PubMed Scopus (64) Google Scholar). Samples were analyzed on using gel of and a gel of and or was performed at 10 for h using a buffer (6Asker N. Baeckström D. Axelsson M.A.B. Carlstedt I. Hansson G.C. Biochem. J. 1995; 308: 873-880Crossref PubMed Scopus (64) Google Scholar, U.K. Nature. 1970; 227: 680-685Crossref PubMed Scopus (205498) Google Scholar). were in and in (Amersham Pharmacia and to at °C as described (6Asker N. Baeckström D. Axelsson M.A.B. Carlstedt I. Hansson G.C. Biochem. J. 1995; 308: 873-880Crossref PubMed Scopus (64) Google Scholar). gel electrophoresis was performed by the The was and the was for in Tris-HCl, pH containing 50 mm and then placed on top of The was with gel containing 10 mm LS 174T cells were with the α-MUC2TR antiserum, and were in saline containing at °C for 5 Samples were on top of a sucrose gradient containing Ultracentrifugation was performed at 50,000 rpm at 20 °C for h in a Beckman rotor Fractions were collected from the of the tube and analyzed by cells and or the and were and the cell supernatants were with an antiserum, to rate as described and analyzed by J. Olofsson J. Biol. Chem. 1994; 269: Full Text PDF PubMed Google Scholar, Z.M. J. Biol. Chem. Full Text PDF PubMed Google Scholar). The for the MUC2 mono- and dimer and were to the of Biochem. PubMed Scopus Google Scholar). of LS 174T cells using the α-MUC2TR antiserum against the large tandem repeat of the MUC2 mucin, have shown that the human MUC2 apomucin forms a that is as a dimer (6Asker N. Baeckström D. Axelsson M.A.B. Carlstedt I. Hansson G.C. Biochem. J. 1995; 308: 873-880Crossref PubMed Scopus (64) Google Scholar). To that the species was a of mono- and dimers were analyzed by rate The gradient was the of the tube into the sucrose concentration was and each was analyzed by The of the mono- and dimer was by and in The at sucrose, and the dimer at sucrose, of about and using the of Biochem. PubMed Scopus Google Scholar). The of the for the dimer and were to of species with by the showed a of for and a of of about 1.2 between and is to the one for the two MUC2 species suggests that the MUC2 forms a dimer and not a or larger species at the initial An antiserum raised against a peptide from the N terminus of has been used to study the MUC2 also after of LS 174T cells using antiserum followed by with gel electrophoresis two MUC2 with a than the and dimer antiserum MUC2 described in an M.A.B. N. Hansson G.C. J. Biol. Chem. Full Text Full Text PDF PubMed Scopus Google Scholar), to the α-MUC2N3 of the α-MUC2N3 antiserum with the peptide used for the of these two MUC2 species not to the positions were also the binding from was used for these were not precipitated with the antiserum, but with the that the two MUC2 The and of the two of the disulfide the at the A for the of the larger was that the was a dimer of the To further a two-dimensional gel electrophoresis was performed the in the and in the 2 The to the as the after the that the was a dimer of the The were on the two-dimensional a To the of the and O-glycosylated MUC2 subcellular fractionation by sucrose density gradient centrifugation was performed on LS 174T cells. The gradient was from the in which were analyzed for calnexin for the rough endoplasmic NADPH reductase activity for the endoplasmic and galactosyltransferase activity for the Golgi The sucrose gradient was adjusted to endoplasmic reticulum fractions that were of galactosyltransferase Fractions containing galactosyltransferase activity also contained NADPH reductase an incomplete of the Golgi from the endoplasmic reticulum. fractionation was performed on cells for 2 followed by sucrose density gradient as shown in The fractions were with the α-MUC2TR antiserum followed by the α-MUC2N3 antiserum and using gel electrophoresis of and The mono- and dimers were mainly in the sucrose containing the endoplasmic reticulum but also in fractions with sucrose The for is incomplete of the but also to of before of the O-glycosylated MUC2 species with the α-MUC2N3 antiserum showed that comigrated with the galactosyltransferase that O-glycosylated MUC2 species are to the Golgi as O-glycosylated mono- and dimers for were the O-glycosylated was in the Golgi that not only the MUC2 dimer but also was transferred into the Golgi apparatus. In of the O-glycosylated MUC2 in the fractions as the To study the of the MUC2 apomucin dimerization in more detail, LS 174T cells were pulse-labeled for 2 and chased for and 20 and followed by sucrose density gradient the described in The were into pooled in followed by using the α-MUC2TR antiserum and by gel electrophoresis. only 2 of labeling, amounts of the dimer were observed in the of the containing the for the endoplasmic reticulum. the amount of dimer and mono- and dimers were observed also in fractions with sucrose The of N-glycans in dimerization and further of the MUC2 mucin in LS 174T cells was Cells were with amounts of and an concentration inhibiting μg of of culture was for the experiments not LS 174T cells were with 2 and and 1 followed by using the α-MUC2TR and two were observed from cells When the from cells was analyzed only one was shown as the observed The as the with an of the N-glycans the This also made that the inhibition ofN-glycosylation by tunicamycin was The tunicamycin treatment was the non-N-glycosylated MUC2 and the showed a to the that was the non-N-glycosylated dimer To the of the two and to the of the two in two-dimensional gel electrophoresis was LS 174T cells were with the α-MUC2TR antiserum and analyzed in the and in the All to the on the gel after the of the non-N-glycosylated mono- and dimer and the formation not only of dimers but also of to putative and in cells. To the rate of further oligomerization, and O-glycosylation of the MUC2 apomucin in and LS 174T cells were for followed by a for were performed on the cell with the α-MUC2TR antiserum followed by with the α-MUC2N3 antiserum and by gel electrophoresis. In the the formation of mono- and dimers during the was followed by a in the amount during the As the mono- and dimers of MUC2 started to after about 3 the α-MUC2N3 antiserum started to O-glycosylated MUC2 mono- and In formation of non-N-glycosylated mono- and dimers was also but the rate was than in the cells. further oligomerization of the non-N-glycosylated MUC2 was were observed on the was performed using the α-MUC2N3 These that no non-N-glycosylated MUC2 were into the Golgi apparatus. times up to 10 h were tested and not show O-glycosylated species precipitated with the α-MUC2N3 antiserum not To the of the non-N-glycosylated MUC2 subcellular fractionation was performed cells were for and chased for 2 followed by subcellular fractionation to the tube was into pooled in followed by using the α-MUC2TR antiserum and by gel electrophoresis. the endoplasmic reticulum not only the non-N-glycosylated mono- and but also the to putative and The oligomerization early in the endoplasmic reticulum. The of the non-N-glycosylated and was the fractions to the Golgi that these were not to the Golgi apparatus. This was also shown by the to O-glycosylated from these fractions by using the α-MUC2N3 antiserum In earlier on the of the human MUC2 apomucin in the colon cell line LS 174T (6Asker N. Baeckström D. Axelsson M.A.B. Carlstedt I. Hansson G.C. Biochem. J. 1995; 308: 873-880Crossref PubMed Scopus (64) Google Scholar), showed that the apomucin forms a as by two-dimensional gel electrophoresis. This was as a dimer based on the by of the mono- and dimer by rate has been performed on The of the mono- and dimer were and These with of and only used to the between the two the of a is to the a of the and the between is to The of the for and a in of was also and to about a for a for These with the in SDS-PAGE and gel that the of the human MUC2 mucin forms a dimer in the initial of the To study the initial of the human MUC2 mucin in more detail, subcellular fractionation by sucrose gradient centrifugation on LS 174T cells has been Fractions containing the rough endoplasmic reticulum were well from the Golgi as from the of calnexin and the of galactosyltransferase activity the of the endoplasmic containing NADPH reductase were not from the fractions containing galactosyltransferase the of the MUC2 mono- and dimers were observed in the endoplasmic reticulum. This showed that the dimerization directly after of the the and dimer of the MUC2 apomucin are in fractions containing of endoplasmic that the of folding and dimerization MUC2 is the reticulum. on the of the mono- and dimers of the MUC2 apomucin showed that the amounts of these to 1 h after labeling and that as the This is in with the of the vWF, in which dimers are the only oligomers before to the Golgi J. Biol. PubMed Scopus Google Scholar). To the MUC2 apomucin into the Golgi anO-glycosylation-insensitive antiserum against the N-terminal of the MUC2 mucin was As the amount of mono- and dimers larger were precipitated with the α-MUC2N3 These two species were as O-glycosylated and in the subcellular fractions containing for the Golgi apparatus. The α-MUC2TR antiserum was not to these to of the by the added precipitated species to the on the The two O-glycosylated MUC2 species were as mono- and dimers because the mono- and dimer started to in the endoplasmic reticulum. was also by two-dimensional gel electrophoresis that O-glycosylated species to the positions 2 The to into two 2 a that has not been but that is to The the O-glycosylated is the with an on the gel as to the of species precipitated by The O-glycosylated monomers and dimers at the indicating that monomers and dimers from the endoplasmic reticulum are O-glycosylated in The of in the Golgi is The only of the MUC2 mucin, gel is to the of to the von Willebrand factor of that dimers are from monomers in the endoplasmic reticulum and in the Golgi but the of glycosylated monomers in the cells at a amount 3 after labeling M.A.B. N. Hansson G.C. J. Biol. Chem. Full Text Full Text PDF PubMed Scopus Google Scholar) the that a is of This also the that monomers in the Golgi were not for that monomers a specific from forming that monomers in the Golgi and that the from the endoplasmic reticulum the final A of the amount of mono- and dimer being transferred from the endoplasmic reticulum to the Golgi is to because the α-MUC2N3 antiserum is in MUC2 with the α-MUC2TR This that these not used to the amount of MUC2 apomucin into the Golgi further of the MUC2 apomucin assembly show the formation of larger and more forms of MUC2 in the Golgi M.A.B. N. Hansson G.C. J. Biol. Chem. Full Text Full Text PDF PubMed Scopus Google Scholar). The of the forms of the MUC2 apomucin precipitated by the α-MUC2N3 antiserum then not only on antiserum but also on the of O-glycosylated species into MUC2 species not using the LS 174T cells with the of addition of N-glycans to the MUC2 apomucin was the observed on the gel between the precipitated from and cells was that the of the MUC2 apomucin was on the of the human MUC2 apomucin after treatment with tunicamycin have been earlier Forstner J.F. Forstner G.G. Biochem. J. 1994; PubMed Scopus Google Scholar, J.R. Kim Y.S. Biochem. J. 1994; PubMed Scopus Google Scholar), but of showed in between the and the non-N-glycosylated MUC2 the the human MUC2 apomucin dimers in the endoplasmic reticulum. Pulse-chase experiments showed that dimerization was with dimerization in the cells. The rate of dimerization in cells to of the for the formation of disulfide bonds. As observed in large amounts of non-N-glycosylated MUC2 mono- and dimers after a as with a of mono- and dimer after a in cells. The human non-N-glycosylated MUC2 were not of the endoplasmic reticulum because MUC2 mono- and dimers were in the that the mono- and dimers were not and in the endoplasmic reticulum In a pulse-chase with a the amount of mono- and dimers was than was after a but no MUC2 species were precipitated with the α-MUC2N3 antiserum not These that non-N-glycosylated MUC2 in the endoplasmic reticulum and that were to the Golgi apparatus. The similarities in the in the of the vWF with mucins, including the human MUC2 and the mucin, suggests similarities in the dimer formation of these of the domain of the mucin in cells J. J. Biol. Chem. Full Text Full Text PDF PubMed Scopus Google Scholar) that the dimerization of the domain ofN-glycosylation The inhibition formation not further of the mucin dimers into the Golgi apparatus. Inhibition of of the human vWF in human cells dimerization and further into the Golgi T. J. Biol. 1986; PubMed Scopus Google Scholar). The for the non-N-glycosylated human MUC2 the vWF, and the mucin the of performed in cell in the folding and the of the the similarities in the of the disulfide bonds. of the amino acid sequences of the MUC2 the mucin, and the vWF to the positions of the domains in the vWF showed no in the of the to in in the was observed of the domain but all three sequences. This suggests that inhibition of N-glycans have during the of the three only but also two larger in than the were observed in the endoplasmic reticulum cells were precipitated using the α-MUC2TR These two were as disulfide putative tri- and of the non-N-glycosylated MUC2 apomucin as by with two-dimensional gel electrophoresis In the the mono- and dimers and the two were each In the all to the the lysis buffer contained NEM, inhibiting in disulfide the oligomers in the cells. The as putative tri- and also as the non-N-glycosylated mono- and dimers for and with the larger This is because the large amount of then the between and is not to to in 5% larger in than the dimer also shown tunicamycin but in amounts than in cells because these observed only after of the gel to the of the human MUC2 mucin with on mucins there is a for all The on mucins by Dekker J. G.J. J. Biol. Chem. Full Text PDF PubMed Google Scholar, J. G.J. J. Biol. Chem. 1990; 265: Full Text PDF PubMed Google Scholar) showed the formation of not only dimers but also and of the on human mucin and human mucin by G.J. Biochem. J. 1994; PubMed Scopus Google Scholar, G.J. Biochem. J. 1994; PubMed Scopus Google Scholar) showed that only one species in the endoplasmic as a tri- or based on the by of glycosylated monomers is also in to the of mucins, as described for the mucin J. G.J. J. Biol. Chem. Full Text PDF PubMed Google Scholar, J. G.J. J. Biol. Chem. 1990; 265: Full Text PDF PubMed Google Scholar). was as a for the of mucin into the Golgi apparatus. for the human MUC2 mucin, are as for the correct folding of MUC2 and and only these are to the endoplasmic reticulum The of J. J. Biol. Chem. Full Text Full Text PDF PubMed Scopus Google Scholar) of the of a C terminus of the mucin the of cells or cell specific for mucin with One then that there is not one for all gel-forming mucins, but between mucins, as well as between cells.

The MUC2 mucin is the major gel-forming mucin in the small and large intestine. Due to its sequence similarities with the von Willebrand factor, it has been suggested to dimerize in the endoplasmic reticulum and polymerize in the trans-Golgi network. Using an O-glycosylation-sensitive MUC2 antiserum, a dimerization has been shown to occur in the endoplasmic reticulum of LS 174T cells (Asker, N., Axelsson, M. A. B., Olofsson, S.-O., and Hansson, G. C. (1998) J. Biol. Chem. 273, 18857-18863). Using an antiserum immunoprecipitating O-glycosylated MUC2 mucin, monomers and dimers were shown to occur in soluble form in the lysate of LS 174T cells. The amount of O-glycosylated dimer was small, and no larger species were found even after long chase periods. However, most of the labeled MUC2 mucin was found in pelleted debris of the cell lysate. This insoluble MUC2 mucin was recovered by immunoprecipitation after reduction of disulfide bonds. Analysis by agarose gel electrophoresis revealed two bands, of which the smaller migrated as the O-glycosylated monomer and the larger migrated as the O-glycosylated dimer of the cell lysis supernatant. Mucins insoluble in 6 M guanidinium chloride could also be obtained from LS 174T cells. Such mucins have earlier been found in the small intestine (Carlstedt, I., Herrmann, A., Karlsson, H., Sheehan, J., Fransson, L. -A., and Hansson, G. C. (1993) J. Biol. Chem. 268, 18771-18781). Reduction of the mucins followed by purification by isopycnic density gradient ultracentrifugation and analysis by agarose gel electrophoresis revealed two bands reacting with an anti-MUC2 tandem repeat antibody after deglycosylation. These bands migrated identically to the bands shown by metabolic labeling, and they could also be separated by rate zonal ultracentrifugation. These results suggest that the MUC2 mucin is forming nonreducible intermolecular bonds early in biosynthesis, but after initial O-glycosylation.