Purification and characterization of the MUC1 mucin-type glycoprotein, epitectin, from human urine: structures of the major oligosaccharide alditols (original) (raw)
Related papers
Lectin cytochemical characterization of the N- and O-linked oligosaccharides in the human rectum
Histochemical Journal, 2000
The oligosaccharides of the mucus glycoproteins of the human rectum are important for the lubricant and protective role suggested for the rectal mucus. Changes in oligosaccharide composition are observed in several colon diseases, and some of these changes could be used as diagnostic and prognostic indicators. Thus, a previous knowledge of the normal mucus glycoproteins is necessary. The aim of the present study is the characterization of the oligosaccharides of the goblet cells and enterocytes of the human rectum. For this, a battery of 15 lectins, in combination with chemical and enzymatic deglycosylation procedures, was used. Our results suggest the presence of N-acetylglucosamine (GlcNAc), Man, Glc, N-acetylneuraminic acid (Neu5Ac)(α2-6)-and Neu5Ac(α2-3)-linked, N-acetylgalactosamine (GalNAc) and Gal(β1-3)GalNAc in the oligosaccharides of the goblet cells. Moreover, N-linked oligosaccharides specifically contained Gal(β1-4)GlcNAc, while AAA-positive Fuc was only detected in O-linked oligosaccharides. Some of these carbohydrates were only visualized after removal of N-or O-linked oligosaccharides, suggesting a high level of approximation between the oligosaccharide chains, that render the carbohydrate inaccessible to the lectins. Differences in the labelling pattern between the goblet cells of the surface epithelium and the upper half of the crypts, and those of the lower half of the crypts suggests a maturation process for the goblet cells, which modifies the oligosaccharide composition of the secreted glycoproteins, as they ascend throughout the crypts. This maturation process includes the incorporation of new carbohydrates (GlcNAc), and the masking (Neu5Ac(α2-3)-linked) or unmasking (Glc and GalNAc) of others.
European Journal of Biochemistry, 2005
Glycopeptides and oligosaccharides of either the N-acetyllactosaminic or the oligomannosidic type derived from glycoproteins containing the N-glycosylamine linkage were used to define the specificity of different lectins (concanavalin A, Lens culinaris agglutinin, Vicia faba agglutinin, Pisum sativum agglutinin, Ricinus communis agglutinins, soybean agglutinin, wheat germ agglutinin, Solanum tuberosum agglutinin, Datura stramonium agglutinin, Lotus tetragonolobus agglutinin, Ulex europeus agglutinin) by studying the inhibition of human red blood cell agglutination by these structures. The results obtained show that lectins considered 'identical' in terms of monosaccharide specificity, possess the ability to recognize fine differences in more complex structures. In fact, different lectins are able to recognize different saccharidic sequences on the same glycan structure. As these sequences are likely to be common to numerous glycoproteins, including cell membrane glycoproteins, the results obtained with lectins in the study of cell surface carbohydrates have to be very carefully interpreted. Moreover, our results confirm previous data on the spatial configuration of the glycan moiety of glycoproteins deduced from the construction of molecular models: the fact that oligosaccharides bearing an alpha-NeuAc-(2 leads to 6)-Gal unit are more powerful inhibitors than oligosaccharides bearing an alpha-NeuAc-(2 leads to 3)-Gal unit could be related to the high rotational freedom of alpha-2,6 linkage; the observation that glycoasparagines, glycopeptides and glycoproteins possess a higher affinity for lectins than the related oligosaccharides could be explained by the fact that the glycan--amino acid linkage leads to structures more rigid than those of the oligosaccharides themselves.
Method for the Comparative Glycomic Analyses of O-Linked, Mucin-Type Oligosaccharides
Analytical Chemistry, 2004
A method is presented for the direct relative quantitation of distinct O-linked mucin-type oligosaccharides. Mucintype oligosaccharides are found in a host of tissues from anuran to humans. Because they are often associated with extracellular matrix, they play important roles in cell-cell recognition. Changes in glycosylation of O-linked oligosaccharides are associated with diseases such as cancer. In fertilization, they play an active role in sperm-egg recognition. We describe a method for observing changes in glycosylation of mucin-type oligosaccharides by incorporating deuterium in the release procedure. Oligosaccharides from two different sources are released separately by sodium tetrahydroborate and sodium tetradeuterioborate. The oligosaccharides are combined and separated into components by HPLC. By observing the ratio of deuterated and undeuterated species, changes in glycosylation are precisely quantified. This method is illustrated with mucin-type oligosaccharides from the egg jelly coat of the anuran Xenopus laevis.
Glycoconjugate Journal, 2008
Transposition of intestinal segments is frequently used for bladder reconstruction. Following transposition, bowel segments continue to produce mucus and a correlation between excessive mucus production and complications such as urinary tract infection or catheter blockage has been observed for a long time. However, no information is currently available on the change of mucin expression and glycosylation under these abnormal conditions. In this study, the variable number tandem repeat region and the irregular repeat domain of human MUC2 were isolated as two glycopeptide populations after reduction and trypsin digestion followed by gel chromatography from urine of patients transposed with urinary bladders. After alkaline borohydride treatment, the oligosaccharides released from the whole MUC2 mucin and the two glycosylated domains were investigated by nanoESI Q-TOF MS/MS (electrospray ionization quadrupole time-of-flight tandem mass spectrometry). More than 60 different glycans were identified, mainly based on sialylated core 3 structures. Some core 1, 2 and 4 oligosaccharides were also found. Most of the structures were acidic with NeuAc residues mainly alpha2-6 linked to the N-acetylgalactosaminitol and sulphate residues exclusively 3-linked to galactose. No expression of blood group A and B or Sda/Cad determinants was observed. Similar patterns of glycosylation were found in the tandem repeat region and the irregular repeat domain and the level of expression of the major oligosaccharides were in the same order of magnitude. The most interesting feature of this study was that sialyl-Tn antigen, which is considered as a tumour antigen, was the oligosaccharide most highly expressed. This result suggests that mucins from intestinal transposed segments are abnormally glycosylated.
2002
Glycoproteins are generally composed by a population of closely related glycosylation variants having in common the same polypeptide chain but varying by thei r carbohydrate moiety. This carbohydrate-mediated heterogeneity is often called the microheterogeneity of glycoproteins and the different variants referred to as glycoforms . This heterogeneity arises from the presence of multiple glycannic structures attached a t one glycosylation site . The carbohydrate groups are covalently attached to the polypeptidic backbone through the amide nitrogen of an asparagine residue (N-glycosylation) o r through an 0-linkage with a serine, threonine and sometimes a hydroxyproline residu e (O-glycosylation) . N-linked oligosaccharides may be classified on the basis of th e nature of monosaccharides which constitute the oligosaccharides . High-mannose-type oligosaccharides only consist in mannose and N-acetylglucosamine residues and ar e not charged . Complex-type oligosaccharides may be negatively charged and contai n galactose, fucose and sialic acid residues in addition to the monosaccharides foun d in the high-mannose chains . Hybrid structures are intermediate structures between th e high-mannose and the complex-type oligosaccharides . The three families of oligosaccharides share the same pentasaccharide structure (GlcNac-GlcNac-Man 3 ) by whic h the oligosaccharide is attached to the protein .
N-Glycosylation of the MUC1 mucin in epithelial cells and secretions
Glycobiology, 2006
The MUC1 mucin is an important tumor-associated antigen that shows extensive glycosylation in vivo. The O-glycosylation of this molecule, which has been well characterized in many cell types and tissues, is important in conferring the unusual biochemical and biophysical properties on a mucin. N-Glycosylation is crucial to the folding, sorting, membrane trafficking, and secretion of many proteins. Here, we evaluated the N-glycosylation of MUC1 derived from two sources: endogenous MUC1 isolated from human milk and a recombinant epitope-tagged MUC1F overexpressed in Caco2 colon carcinoma cells. N-Glycans on purified MUC1F/MUC1 were analyzed by matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF-MS), gas chromatography-mass spectrometry (GC-MS), and CAD-ESI-MS/MS. The spectra indicate that MUC1F N-glycans have compositions consistent with highmannose structures (Hex 5-9 HexNAc 2 ) and complex/hybridtype glycans (NeuAc 0-3 Fuc 0-3 Hex 3-8 HexNAc 3-7 ). Many of the N-glycan structures are identical on MUC1F and native MUC1; however, a marked difference is seen between the N-glycans on membrane-bound and secreted forms of the native molecule.
Biochemical Journal, 2004
Purified human mucins from different parts of the intestinal tract (ileum, cecum, transverse and sigmoid colon and rectum) were isolated from two individuals with blood group ALe b (A-Lewis b ). After alkaline borohydride treatment the released oligosaccharides were structurally characterized by nano-ESI Q-TOF MS/MS (electrospray ionization quadrupole time-of-flight tandem MS) without prior fractionation or derivatization. More than 100 different oligosaccharides, with up to ten monosaccharide residues, were identified using this technique. Oligosaccharides based on core 3 structures, GlcNAc(β1-3)GalNAc (where GlcNAc is N-acetyl-D-glucosamine and GalNAc is N-acetylgalactosamine), were widely distributed in human intestinal mucins. Core 5 structures, GalNAc(α1-3)GalNAc, were also recovered in all fractions. Moreover, a comparison of the oligosaccharide repertoire, with respect to size, diversity and expression of glycans and terminal epitopes, showed a high level of mucin-specific glycosyl-ation: highly fucosylated glycans, found specifically in the small intestine, were mainly based on core 4 structures, GlcNAc-(β1-3)[GlcNAc(β1-6)]GalNAc, whereas the sulpho-Le X determinant carrying core 2 glycans, Gal(β1-3)[GlcNAc(β1-6)]-GalNAc (where Gal is galactose), was recovered mainly in the distal colon. Blood group H and A antigenic determinants were present exclusively in the ileum and cecum, whereas blood group Sd a /Cad related epitopes, GalNAc(β1-4)[NeuAc(α2-3)]Gal (where NeuAc is N-acetylneuraminate), were found to increase along the length of the colon. Our findings suggest that mucins create an enormous repertoire of potential binding sites for microorganisms that could explain the regio-specific colonization of bacteria in the human intestinal tract.
Carbohydrate Research, 1991
Glycosylated Wins represent a series of glycoproteins with related activities and, in the case of the Leguminosae, related amino acid sequences. Therefore, they offer a model system in which to study the diversity of N-linked oligosaccharide structures of plant glycoproteins. The influence of the polypeptide on the type of oligosaccharide substitution and the problem of inter-and intra-genus variation in glycosylation can also be addressed. Analysis of the glycosylation of 18 lectins has shown that they can be classified into four qualitatively similar groups on the basis of the Bio-Gel P-4 elution protiles of the oligosaccharides released by hydrazinolysis: (a) The Erythrina cristagalli profile, with a major component at 8.8 glucose units (gu) and minor components at 8.0,7.2, and 5.8 gu. The major component is the heptasaccharide, a-o-Manp-