The Glycosylation of Rat Intestinal Muc2 Mucin Varies between Rat Strains and the Small and Large Intestine
1997; Elsevier BV; Volume: 272; Issue: 43 Linguagem: Inglês
10.1074/jbc.272.43.27025
ISSN1083-351X
AutoresNiclas G. Karlsson, Annkatrin Herrmann, Hasse Karlsson, Malin Johansson, Ingemar Carlstedt, Gunnar C. Hansson,
Tópico(s)Proteoglycans and glycosaminoglycans research
ResumoThe large glycosylated domains obtained from the rat intestinal mucin Muc2 were isolated from the large and small intestine of the inbred rat strains GOT-W and GOT-BW. The expression of the rat Muc2 in the large intestine was confirmed immunochemically and by Northern blotting. Released oligosaccharides were structurally characterized by gas chromatography-mass spectrometry (neutral and sialylated species) or by tandem mass spectrometry (sulfated species), and a total of 63 structures was assigned. The large intestinal oligosaccharides were found to be identical between the strains, while the small intestinal glycosylation differed. Until now, detailed structural analysis of oligosaccharides isolated from a single mucin core or mucin domain with different origin have not been performed, and the information of different mucin glycoforms has been limited to immunochemistry. Blood group A-determinants (GalNAcα1–3(Fucα1–2)Galॆ1-, and structures related to the blood group Sda/Cad-related epitope NeuAc/NeuGcα1–3(GalNAcॆ1–4)Galॆ1-, were found in GOT-BW small intestine, and also in both large intestines. Blood group H-determinants and NeuAc/NeuGcα1–3Galॆ1- were found in all samples. Core 1 (Galॆ1–3GalNAcα1-), core 2 (Galॆ1–3(GlcNAcॆ1–6)GalNAcα1-), core 3 (GlcNAcॆ1–3GalNAcα1-), and core 4 (GlcNAcॆ1–3(GlcNAcॆ1–6)GalNAcα1- were also found in all the samples. The large intestine were enriched in sulfated oligosaccharides and the small intestine contained higher amounts of sialylated species. Sulfation were found exclusively on C-6 of GlcNAc. The large glycosylated domains obtained from the rat intestinal mucin Muc2 were isolated from the large and small intestine of the inbred rat strains GOT-W and GOT-BW. The expression of the rat Muc2 in the large intestine was confirmed immunochemically and by Northern blotting. Released oligosaccharides were structurally characterized by gas chromatography-mass spectrometry (neutral and sialylated species) or by tandem mass spectrometry (sulfated species), and a total of 63 structures was assigned. The large intestinal oligosaccharides were found to be identical between the strains, while the small intestinal glycosylation differed. Until now, detailed structural analysis of oligosaccharides isolated from a single mucin core or mucin domain with different origin have not been performed, and the information of different mucin glycoforms has been limited to immunochemistry. Blood group A-determinants (GalNAcα1–3(Fucα1–2)Galॆ1-, and structures related to the blood group Sda/Cad-related epitope NeuAc/NeuGcα1–3(GalNAcॆ1–4)Galॆ1-, were found in GOT-BW small intestine, and also in both large intestines. Blood group H-determinants and NeuAc/NeuGcα1–3Galॆ1- were found in all samples. Core 1 (Galॆ1–3GalNAcα1-), core 2 (Galॆ1–3(GlcNAcॆ1–6)GalNAcα1-), core 3 (GlcNAcॆ1–3GalNAcα1-), and core 4 (GlcNAcॆ1–3(GlcNAcॆ1–6)GalNAcα1- were also found in all the samples. The large intestine were enriched in sulfated oligosaccharides and the small intestine contained higher amounts of sialylated species. Sulfation were found exclusively on C-6 of GlcNAc. The family of highly glycosylated glycoproteins found at the mucosal surfaces are known as mucins (1Gendler S.J. Spicer A.P. Annu. Rev. Physiol. 1995; 57: 607-634Crossref PubMed Scopus (854) Google Scholar). Characteristic for mucins is the high degree of O-linked glycosylation known as the mucin-type, where α-N-acetylgalactosamine is linked to serine or threonine of the protein backbone. These amino acids together with proline are mainly found in long and often repeated protein sequences, called mucin domains, that become a scaffold for the glycosylation. The mucins are largely responsible for the physical properties of mucus that serves as a lubricant for the mucosal surface and protects the underlying epithelium from mechanical and chemical stress. However, the identification of several different encoded mucins, and an enormous repertoire of possible mucin oligosaccharides indicates that the tasks for these glycoproteins may be more subtle than the macroscopic properties suggest. Mucins produced in the intestine are mainly derived from the goblet cells. The major part (at least 807) of the rat intestinal mucins, measured as protease-resistant mucin domains, can be recovered as an 舠insoluble舡 glycoprotein complex in as denaturating conditions as 6.0 m guanidinium chloride (2Carlstedt I. Herrmann A. Karlsson H. Sheehan J. Fransson L.-Å. Hansson G.C. J. Biol. Chem. 1993; 268: 18771-18781Abstract Full Text PDF PubMed Google Scholar), providing that shear homogenization is avoided. Two highly glycosylated peptides, named glycopeptide A (gpA) 1The abbreviations used are: gpA, mucin glycopeptide A; gpB, mucin glycopeptide B; GSL, glycosphingolipid; TBS, Tris-buffered saline; LNF-1, lacto-N-fucopentaose 1 (Fucα1–2Galॆ1–3GlcNAcॆ1–3Galॆ1–4Glc); GC, gas chromatography; MS, mass spectrometry; FAB, fast atom bombardment; CID, collision induced dissociation; Hex, hexose; HexNAc,N-acetylhexosamine; HexNAcol,N-acetylhexosaminitol; mAb, monoclonal antibody. 1The abbreviations used are: gpA, mucin glycopeptide A; gpB, mucin glycopeptide B; GSL, glycosphingolipid; TBS, Tris-buffered saline; LNF-1, lacto-N-fucopentaose 1 (Fucα1–2Galॆ1–3GlcNAcॆ1–3Galॆ1–4Glc); GC, gas chromatography; MS, mass spectrometry; FAB, fast atom bombardment; CID, collision induced dissociation; Hex, hexose; HexNAc,N-acetylhexosamine; HexNAcol,N-acetylhexosaminitol; mAb, monoclonal antibody.and B (gpB), 650 kDa and 335 kDa, respectively, were isolated by trypsin digestion of subunits from this insoluble mucin complex. A polyclonal antiserum raised against the deglycosylated gpA was used to isolate a cDNA clone (3Hansson G.C. Baeckström D. Carlstedt I. Klinga-Levan K. Biochem. Biophys. Res. Commun. 1994; 198: 181-190Crossref PubMed Scopus (41) Google Scholar). This sequence together with two other partial sequences (4Xu G. Huan L. Khatri I.A. Wang D. Bennick A. Fahim R.E.F. Forstner G.G. Forstner J.F. J. Biol. Chem. 1992; 267: 5401-5407Abstract Full Text PDF PubMed Google Scholar, 5Xu G. Khatri I.A. Sajjan U.S. McCool D. Wang D. Jones C. Forstner G. Forstner J. Biochem. Biophys. Res. Commun. 1992; 183: 821-828Crossref PubMed Scopus (30) Google Scholar, 6Ohmori H. Dohrman A.F. Gallup M. Tsuda T. Kai H. Gum J.R. Kim Y.S. Basbaum C.B. J. Biol. Chem. 1994; 269: 17833-17840Abstract Full Text PDF PubMed Google Scholar) have been shown to have homology within separate regions of the intestinal human mucin MUC2 (7Gum 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), proposing that all three are parts of the rat Muc2 gene. The organization of rat Muc2 mucin has recently been further explored and compared with its human homologue (8Karlsson N.G. Johansson M.E.V. Asker N. Karlsson H. Gendler S.J. Carlstedt I. Hansson G.C. Glycoconj. J. 1996; 13: 823-831Crossref PubMed Scopus (22) Google Scholar). The Muc2 mucin has two large domains of sequences rich in serine, threonine, and proline residues, with a small cysteine-rich region in between. It was concluded that the gpB was the smaller domain at the N-terminal side, while gpA was the larger one at the C-terminal side. The two highly glycosylated mucin domains are flanked by less glycosylated cysteine-rich regions. The N- and C-terminal of the human MUC2 and rat Muc2 are believed to be responsible for oligomerization via intramolecular disulfide bridges. Recent biosynthetic studies in the colon cancer cell line LS 174T have shown that a disulfide-stabilized dimer of MUC2 is formed (9Asker N. Baeckström D. Axelsson M.A.B. Carlstedt I. Hansson G.C. Biochem. J. 1995; 308: 873-880Crossref PubMed Scopus (64) Google Scholar), similar to the initial biosynthesis of the von Willebrand factor. Several reports elucidating the enormous diversity of mucin oligosaccharides do not provide any further information about possible heterogeneity due to the presence of different mucin subpopulations (for example, Refs. 10Klein A. Carnoy C. Lamblin G. Roussel P. vanKuik J.A. Vliegenthart J.F.G. Eur. J. Biochem. 1993; 211: 491-500Crossref PubMed Scopus (36) Google Scholar and 11Lo-Guidice J.-M. Wieruszeski J.-M. Lemoine J. Verbert A. Roussel P. Lamblin G. J. Biol. Chem. 1994; 269: 18794-18813Abstract Full Text PDF PubMed Google Scholar). The glycosylation of separate mucins or parts of mucins is rarely described, but there is a growing interest due to the fact that the presentation of oligosaccharides will most likely be dependent on the protein backbone (12Varki A. Proc. Natl. Acad. Sci. U. S. A. 1994; 91: 7390-7397Crossref PubMed Scopus (950) Google Scholar). We have previously reported a detailed structural characterization of the oligosaccharides from gpA and gpB from an inbred white rat strain (GOT-W), known to express H-determinants but not A-determinants in the small intestine (2Carlstedt I. Herrmann A. Karlsson H. Sheehan J. Fransson L.-Å. Hansson G.C. J. Biol. Chem. 1993; 268: 18771-18781Abstract Full Text PDF PubMed Google Scholar, 8Karlsson N.G. Johansson M.E.V. Asker N. Karlsson H. Gendler S.J. Carlstedt I. Hansson G.C. Glycoconj. J. 1996; 13: 823-831Crossref PubMed Scopus (22) Google Scholar). It was concluded that the glycosylation of the two domains was similar. To further explore the glycosylation of a mucin domain from Muc2, we studied the glycosylation of the small intestinal gpA from a black-and-white inbred rat strain (GOT-BW). This strain has been shown to express blood group A positive glycosphingolipids in the small intestine (13Hansson G.C. J. Biol. Chem. 1983; 258: 9612-9615Abstract Full Text PDF PubMed Google Scholar). The analysis was also extended by the identification of oligosaccharides from gpA of rat large intestine of both rat strains, where both strains express blood group A positive glycosphingolipids (14Hansson G.C. Karlsson K.-A. Thurin J. Biochim. Biophys. Acta. 1984; 792: 281-292Crossref PubMed Scopus (24) Google Scholar). The question addressed was to what extent a single mucin core exhibit a tissue-dependent glycosylation and if it is influenced by the variation of the glycosylation between individuals. Detailed qualitative and quantitative information from GC-MS, FAB-MS, and MS/MS revealed that the Muc2-derived mucin domain gpA existed as different glycoforms in the small and large intestine. It was further concluded that the small intestinal glycosylation of this domain differed between the two inbred rat strains. The mucin gpA was prepared from mucosal scrapings essentially as described (2Carlstedt I. Herrmann A. Karlsson H. Sheehan J. Fransson L.-Å. Hansson G.C. J. Biol. Chem. 1993; 268: 18771-18781Abstract Full Text PDF PubMed Google Scholar) from GOT-W and GOT-BW rats small intestines (17 and 9 animals, respectively) and large intestines (7 and 5 animals, respectively). Shortly the procedure involved extracting insoluble material with 957 ethanol, followed by reduction and alkylation with dithiothreitol and iodoacetamide in 6.0 m guanidinium chloride. The samples were digested with RNase and DNase and two highly glycosylated domains (gpA and gpB) were purified on Sephacryl S-200 and S-500 after trypsin digestion. GpA (0.1 mg/ml, 50 ॖl) were coated in soft polystyrene microtiter wells at room temperature overnight, blocked with 27 bovine serum albumin for 1 h, incubated with the antibodies serially diluted (1:2) for 2 h, washed, and detected with 125I-labeled (IODO-GEN, Pierce) anti-mouse antibodies (DAKO, Copenhagen, Denmark). After washing and drying, the wells were cut out and radioactivity measured in a γ-counter. The primary antibodies used were anti-blood group H (type 2) (starting dilution 1:25) (DAKO), anti-blood group A (starting dilution 1:100) (DAKO), anti-blood group B (starting dilution 1:50) (DAKO), anti-blood group Lewis b and H type 1 (NS10cl17) (starting dilution 1:50) (The Wistar Institute, Philadelphia, PA), and anti-blood group A type 2 (A005) (starting dilution 1:50) (Monocarb, Lund, Sweden). The reactivity was measured against glycosphingolipids isolated in our department: A6–1 (A active type 1 hexaglycosylceramide), A6–2 (A active type 2 hexaglycosylceramide), B6–2 (B active type 2 hexaglycosylceramide), H5–1 (H active type 1 pentaglycosylceramide), and H5–2 (H active type 2 pentaglycosylceramide). The large and small intestine were removed and thoroughly washed with phosphate-buffered saline. A 40-cm piece of the small intestine was removed 40 cm from the distal end and mucosal scrapping was collected. Mucosal scrapping was also collected from the entire large intestine. The samples were homogenized with a Dounce homogenizer (tight piston, 25 strokes) in 35 ml of phosphate-buffered saline and centrifuged at 25,000 × g for 20 min. The pellet was reconstituted in phosphate-buffered saline (2 ml for the small intestine and 1 ml for the large) and stored frozen at −20 °C. For the enzyme assay, 20 ॖg of LNF-1 (H type 1 pentasaccharide) in 10 ॖl of cacodylate buffer, 0.50 m, with 1.5 m NaCl, pH 6.8, was used as substrate. After the addition of 10 ॖl of ATP (100 mm), 10 ॖl of MnCl2 (150 mm) with 100 mm NaN3 and 1.07 Triton X-100, 5 ॖl of UDP-[14C]GalNAc (0.12 ॖCi), and 65 ॖl of the redissolved pellet, the mixture was incubated for 18 h at 37 °C and the reaction stopped by heating to 90 °C for 30 min. The reaction mixture was desalted on a column with a mixture of AG3-X4A and AG 50W-X8 (2 ml) and eluted with 5 ml of water, lyophilized, and redissolved in 100 ॖl of TBS. An affinity column (3 mm × 10 cm) was prepared as described (15Dakour J. Lundblad A. Zopf D. Anal. Biochem. 1987; 161: 140-143Crossref PubMed Scopus (33) Google Scholar) after washing concanavalin A-Sepharose for 1.5 h (6 ml/h) with Tris-HCl-buffered saline (50 mm) (TBS). The monoclonal anti-blood group A antibody A003 (Monocarb) (10 mg) was added at 20 °C, followed by 3 h washing with TBS (6 ml/h). An aliquot (25 ॖl) of the reaction mixture from above was applied to the affinity column at 37 °C and fractions of 7 drops (approximately 250 ॖl) were collected. After the addition of 3 ml of liquid scintillation mixture (Ready Safe, Beckman Instruments, Fullerton, CA) the radioactivity was measured by a Beckman LS6000TA liquid scintillator. GpA (0.4 ॖg) was coated into microtiter plates (Maxisorb, Nalge Nunc, Roskilde, Denmark) by slow evaporation of a water solution at 37 °C for 12 h. The plates were further dried in an exicator for 2 h and treated with gaseous hydrogen fluoride in an HF-apparatus (Peptide Institute, Tokyo, Japan) for 18 h. The samples were recoated in their original wells by adding 100 ॖl of phosphate-buffered saline and incubating for 24 h at 37 °C. The strips were washed twice with 5 mm Tris-HCl buffer, pH 8.0, containing 0.15 mNaCl, 0.0057 Tween 20, and 0.027 NaN3 and once with 0.15m NaCl followed by blocking with 0.17 bovine serum albumin and 67 sorbitol in 50 mm Tris-HCl buffer, pH 8.0, with 0.15 m NaCl, 90 ॖm CaCl2, 4 ॖm EDTA, and 0.02 ॖm NaN3 for 6 h at 37 °C. The dissociation enhanced lanthanide fluoroimmunoassay was performed as described (16Baeckström D. Hansson G.C. Nilsson O. Johansson C. Gendler S. Lindholm L. J. Biol. Chem. 1991; 266: 21537-21547Abstract Full Text PDF PubMed Google Scholar), using a rabbit polyclonal antibody against the deglycosylated gpA from GOT-W small intestine (α-gpA, PH497) (3Hansson G.C. Baeckström D. Carlstedt I. Klinga-Levan K. Biochem. Biophys. Res. Commun. 1994; 198: 181-190Crossref PubMed Scopus (41) Google Scholar) or serum from unimmunized rabbit. As secondary antibody a goat anti-rabbit antiserum (Jackson Immunoresearh, West Grove, PA) labeled with europium was used. mRNA (from approximately 50 ॖg of total RNA) from GOT-W large and small intestine were prepared from mucosal scrapping, electrophoresed, blotted, and probed with the VR-1A probe as described (3Hansson G.C. Baeckström D. Carlstedt I. Klinga-Levan K. Biochem. Biophys. Res. Commun. 1994; 198: 181-190Crossref PubMed Scopus (41) Google Scholar, 8Karlsson N.G. Johansson M.E.V. Asker N. Karlsson H. Gendler S.J. Carlstedt I. Hansson G.C. Glycoconj. J. 1996; 13: 823-831Crossref PubMed Scopus (22) Google Scholar). Oligosaccharides from gpA of the various preparations (4–20 mg) were released by ॆ-elimination in 0.05 KOH with 1.0 m NaBH4 for 45 h at 45 °C (1 ml/mg glycopeptide). The reactions were quenched by adding acetic acid, followed by desalting using an AG 50W-X8 column (1.5 ml of resin/ml reaction solution) eluted with water (5 ml/ml resin), and repeated treatment with acetic acid in methanol with subsequent evaporation. Oligosaccharides were applied to DE23 cellulose (Ac− form, 1–2 g), and neutral oligosaccharides were eluted with 50–100 ml of water (containing 17 1-butanol), followed by elution of the acidic ones with 1.0 m pyridinium acetate, pH 5.4, and the fractions were lyophilized. An aliquot of the acidic oligosaccharides, or alternatively an aliquot of the total fraction of mucin oligosaccharides (from 2 to 10 mg of glycoprotein), was applied to DEAE-Sephadex A-25 (Ac− form, 0.5 ml of packed resin/mg of glycopeptide). Neutral oligosaccharides (if present) were eluted with methanol (5 ml/ml resin), sialylated oligosaccharides were methyl esterified as described (17Karlsson N.G. Karlsson H. Hansson G.C. Glycoconj. J. 1995; 12: 69-76Crossref PubMed Scopus (30) Google Scholar) by loading 200 ॖl of dimethyl sulfoxide/MeI (5:1) per ml of DEAE-Sephadex resin and incubating for 5 min. This procedure was repeated 3 times, and the sialylated oligosaccharides were eluted with 5 ml of dry methanol/ml resin. Finally, the sulfated oligosaccharides were eluted with 1.0m pyridinium acetate, pH 5.4. Solvents were removed by rotary evaporation and lyophilization. Sulfated oligosaccharides were desalted by a G-10 column (Pharmacia, Sweden) 16 m × 400-mm eluted with water containing 17 1-butanol. Sialylated species (from 2 to 10 mg of gpA) were converted from their methylesters intoN-methylamides by stirring for 10 min in methanol containing 6–127 methylamine. Analyses of neutral monosaccharides (1/100–1/20 of either the neutral, sialylated, or sulfated species) were performed after acidic hydrolysis in 4.0m trifluoroacetic acid followed by either conversion into alditol acetates (18Yang H. Hakomori S. J. Biol. Chem. 1971; 246: 1192-1200Abstract Full Text PDF PubMed Google Scholar), and analysis by GC, or by re-N-acetylation followed by analyzes using high performance anion exchange chromatography-pulsed amperometric detection (19Karlsson N.G. Hansson G.C. Anal. Biochem. 1995; 224: 538-541Crossref PubMed Scopus (40) Google Scholar). The amino acid composition of the glycopeptides was determined as described (20Spackman D.H. Stein W.H. Moore S. Anal. Chem. 1958; 30: 1190-1206Crossref Scopus (6576) Google Scholar) with an Alpha Plus amino acid analyzer (Pharmacia). The neutral oligosaccharides and theN-methylamide derivative of sialylated ones were permethylated with methyl iodide in a slurry of NaOH in dimethyl sulfoxide as described (21Ciucano I. Kerek F. Carbohydr. Res. 1984; 131: 207-217Google Scholar, 22Larson G. Karlsson H. Hansson G.C. Pimlott W. Carbohydr. Res. 1987; 161: 14430-14437Crossref Scopus (96) Google Scholar). Samples were analyzed by GC and GC-MS after dissolving in ethyl acetate (25–100 ॖl) and 0.5–1 ॖl were injected on-column at 70 °C. Permethylated N,N-dimethyl amides of the sialylated species were purified on a Sephadex LH-20 column (7 × 500 mm) eluted with methanol (0.25 ml/min), before being analyzed by GC and GC-MS. High-temperature capillary columns for GC were prepared as described (17Karlsson N.G. Karlsson H. Hansson G.C. Glycoconj. J. 1995; 12: 69-76Crossref PubMed Scopus (30) Google Scholar) from fused silica capillaries (11–12 m × 0.25 mm, inner diameter, HT-polyimide coated, Chrompack, Middelburg, The Netherlands) which were coated with 0.02–0.04 ॖm of PS264 or SE-54 and cross-linked. Capillary GC was performed on a Hewlett-Packard 5890A gas chromatograph with hydrogen as carrier gas (0.7 bar, linear gas velocity of 114 cm/s at 70 °C) including an oxygen trap (Oxypurge, Alltech) in the carrier gas line. The flame ionization detector was kept at 390 or 395 °C. Sialylated oligosaccharides were analyzed by a temperature program from 70 °C (1 min) to 200 °C by 50 °C/min and then by 10 °C/min to 390 °C (5 min). Neutral oligosaccharides were analyzed by a linear temperature program from 70 °C (1 min) up to 395 °C (5 min) at 10 °C/min. For GC-MS, helium was used as carrier gas (0.2 bar, linear gas velocity of 75 cm/s at 70 °C) and a Hewlett-Packard 5890A-II gas chromatograph working in a constant flow mode. The gas chromatograph was coupled to a JEOL SX-102A mass spectrometer (JEOL, Tokyo, Japan). The conditions for the mass spectrometer: interface temperature, 385 °C; ion source temperature, 370 °C; electron energy, 70 eV; trap current, 300 ॖA; acceleration voltage, +10 kV; mass range scanned, m/z 100–1600; total cycle time, 1.4–1.8 s; resolution, 1400 (m/Δm 107 valley); pressure in the ion source region, 5 × 10−4 pascal. Sulfated species were peracetylated with 100–400 ॖl of pyridine and 50–200 ॖl of acetic acid anhydride-d 6 for 12 h in room temperature before analyzing the sample with negative ion FAB-MS and MS/MS. Peracetylated sulfated oligosaccharides were dissolved in 50–100 ॖl of methanol and 1–2 ॖl was mixed with triethanolamine and ionized by fast atom bombardment for analyzing negative ions. The FAB-MS of the mixture of peracetylated sulfated mucin oligosaccharides was done on MS1 of a JEOL HX/HX 110A four sector tandem mass spectrometer scanning m/z from 100 to 3000 with a cyclic time of 37 s and a linear magnet scan. The ions were accelerated to 10 keV. MS/MS was performed as described (23Karlsson N.G. Karlsson H. Hansson G.C. J. Mass Spectrom. 1996; 31: 560-572Crossref PubMed Scopus (46) Google Scholar) by selecting [M-H]− of the sulfated oligosaccharides as primary ions for CID. In the field free region between the mass spectrometers the primary ion was attenuated by approximately 707 in a collision cell (floating at −8 kV) with helium as collision gas and a collision energy of 2 keV. CID spectra were obtained fromm/z 70 up to 30 atomic mass units below the precursor ions. The resolution was set to 1000 in both MS1 and MS2 and the daughter ions were detected with a JEOL MS-ADS II focal plane array detector. By reduction and alkylation of mucosa from the large intestine of the GOT-W strain and the small and large intestine of the GOT-BW strain followed by nuclease and trypsin digestion and Sephacryl S-200 and S-500 gel filtration, two highly glycosylated mucin domains (gpA and gpB, respectively) were isolated (Fig. 1). Isolation of the two mucin domains from GOT-W small intestine and their relation to the rat Muc2 mucin have already been reported (2Carlstedt I. Herrmann A. Karlsson H. Sheehan J. Fransson L.-Å. Hansson G.C. J. Biol. Chem. 1993; 268: 18771-18781Abstract Full Text PDF PubMed Google Scholar, 8Karlsson N.G. Johansson M.E.V. Asker N. Karlsson H. Gendler S.J. Carlstedt I. Hansson G.C. Glycoconj. J. 1996; 13: 823-831Crossref PubMed Scopus (22) Google Scholar). The reactivity of the polyclonal antiserum (α-gpA, reacting with rat Muc2) with the hydrogen fluoride deglycosylated gpA isolated from the various sources (Table I) it could be concluded that they all contain the same major peptide part. The lower reactivity of the large intestinal glycopeptides could reflect difficulties in purifying the significantly smaller amounts of material from this tissue.Table IReactivities of rat intestinal gpA with antibodies against blood group determinants and mucin coreSmall intestineLarge intestineGOT-WGOT-BWGOT-WGOT-BWRecovered amount of gpA (mg/animal)3.67.50.90.6Blood group activity of gpA (relative reactivity (7))1-aValues given are percent of the maximum reactivity of the antisera with 50 ng of standard glycosphingolipids. Values below 5 were considered as negative. Blood group A1-bMAb DAKO A581, starting dilution 1:100, GSL standard A6–1.0110123159 Blood group A type 21-cMAb A005 specific to A type 2 (Hansson, G. C., unpublished data), starting dilution 1:50, GSL standard A6–2.0543652 Blood group B1-dMAb DAKO A582, starting dilution 1:50, GSL standard B6–2.0000 Blood group H type 11-eMAb cl10–17, directed against Lewis a (rat is negative) and H-type 1, starting dilution 1:50, GSL standard H5–1.58712120488 Blood group H type 21-fMAb DAKO A583, starting dilution 1:25, GSL standard H5–2.118813921Apomucin activity of gpA (relative activity (7))1-gValues given are reactivity of 400 ng of HF-deglycosylated gpA with polyclonal antibody raised against HF-deglycosylated gpA from GOT-W small intestine (α-gpA, PH497). α-Large mucin domain of rat Muc210010276601-a Values given are percent of the maximum reactivity of the antisera with 50 ng of standard glycosphingolipids. Values below 5 were considered as negative.1-b MAb DAKO A581, starting dilution 1:100, GSL standard A6–1.1-c MAb A005 specific to A type 2 (Hansson, G. C., unpublished data), starting dilution 1:50, GSL standard A6–2.1-d MAb DAKO A582, starting dilution 1:50, GSL standard B6–2.1-e MAb cl10–17, directed against Lewis a (rat is negative) and H-type 1, starting dilution 1:50, GSL standard H5–1.1-f MAb DAKO A583, starting dilution 1:25, GSL standard H5–2.1-g Values given are reactivity of 400 ng of HF-deglycosylated gpA with polyclonal antibody raised against HF-deglycosylated gpA from GOT-W small intestine (α-gpA, PH497). Open table in a new tab So far, rat Muc2 has only been known to be expressed in the rat lung and small intestine (3Hansson G.C. Baeckström D. Carlstedt I. Klinga-Levan K. Biochem. Biophys. Res. Commun. 1994; 198: 181-190Crossref PubMed Scopus (41) Google Scholar, 4Xu G. Huan L. Khatri I.A. Wang D. Bennick A. Fahim R.E.F. Forstner G.G. Forstner J.F. J. Biol. Chem. 1992; 267: 5401-5407Abstract Full Text PDF PubMed Google Scholar, 5Xu G. Khatri I.A. Sajjan U.S. McCool D. Wang D. Jones C. Forstner G. Forstner J. Biochem. Biophys. Res. Commun. 1992; 183: 821-828Crossref PubMed Scopus (30) Google Scholar, 6Ohmori H. Dohrman A.F. Gallup M. Tsuda T. Kai H. Gum J.R. Kim Y.S. Basbaum C.B. J. Biol. Chem. 1994; 269: 17833-17840Abstract Full Text PDF PubMed Google Scholar). To verify the observation that Muc2 is present in the large intestine, mRNA was isolated from GOT-W large intestine and Northern blot was performed using the VR-1A clone from rat Muc2 (3Hansson G.C. Baeckström D. Carlstedt I. Klinga-Levan K. Biochem. Biophys. Res. Commun. 1994; 198: 181-190Crossref PubMed Scopus (41) Google Scholar, 8Karlsson N.G. Johansson M.E.V. Asker N. Karlsson H. Gendler S.J. Carlstedt I. Hansson G.C. Glycoconj. J. 1996; 13: 823-831Crossref PubMed Scopus (22) Google Scholar) as probe. A single band was detected with identical size (>12 kilobases) as for Muc2 in the GOT-W small intestine (Fig.2). The amino acid analysis of GOT-W large intestinal gpA showed a high content of proline, serine, and threonine, with a similar distribution as the GOT-W small intestinal gpA. However, an increased level of especially glycine, glutamine/glutamate, and lysine was found. This result together with the slightly lower reactivity of the gpA from large intestine with the α-gpA antiserum (Table I), indicated that there may be additional unidentified biomolecules within these samples. Alternatively, this is a reflection of the variation in glycosylation between the large and small intestine, causing a difference in the size of the tryptic gpA fragments isolated from different sources. The different Muc2 content of the gpA preparations from GOT-W and GOT-BW large intestine, measured as the reactivity with α-gpA antiserum, did not render in different glycosylation (described below). This indicated that glycosylation of the large intestinal gpA as presented here reflected the glycosylation of the rat Muc2 large mucin domain from this tissue, even though small amounts of other glycosylated molecules could be present within the samples. From the reactivity with blood group specific antibodies with gpA from the small and large intestine of the GOT-W and GOT-BW rat strains it was concluded that the glycosylation was different both between the tissues and strains (TableI). Blood group A-determinants were found in all of the gpA except for the small intestinal gpA from GOT-W. This correlated with the previously analyzed expression of blood group A active glycosphingolipids (24Hansson G.C. Wu A.M. The Molecular Immunology of Complex Carbohydrates. Plenum Press, 1988: 465-494Google Scholar). To confirm that the presence of blood group A epitopes was due to the action of an GalNAcα1–3 transferase using blood group H epitopes as substrate, an in vitro enzyme assay was used. The LNF-1 oligosaccharide was used as substrate and UDP-[14C]GalNAc as sugar nucleotide. After incubation with tissue homogenates the mixtures were analyzed using a blood group A affinity column, where oligosaccharides containing A-determinants are retarded (15Dakour J. Lundblad A. Zopf D. Anal. Biochem. 1987; 161: 140-143Crossref PubMed Scopus (33) Google Scholar). Indeed, the assay specifically excluded the GOT-W small intestine from having A-transferase activity, while the other samples all had an active enzyme, as shown by the retarded signal from [14C]GalNAc-labeled oligosaccharides (Fig.3). The oligosaccharides were released and fractionated into neutral, sialylated, and sulfated species. The number of GalNAcol in each of the neutral (not retarded), sialylated (esterifyable), and sulfated (high salt buffer eluted) fractions is a measure of the number of oligosaccharide chains recovered in each fraction. From Table II it can be concluded that while the distribution within the three oligosac
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