Characterization of the rabbit homolog of human MUC1 glycoprotein isolated from bladder by affinity chromatography on immobilized jacalin

Tsuyoshi Higuchi1, Ping Xin, Melissa S. Buckley, Deborah R. Erickson3 and V.P. Bhavanandan2

Department of Biochemistry and Molecular Biology and 3Surgery, Milton S. Hershey Medical Center, The Pennsylvania State University, Hershey, PA 17033, USA

Received on September 3, 1999; revised on January 2, 2000; accepted on January 18, 2000.


    Abstract
 Top
 Abstract
 Introduction
 Results
 Discussion
 Materials and methods
 Acknowledgments
 Abbreviations
 References
 
The urinary bladder is lined by transitional epithelium, the glycocalyx on the luminal surface has interesting properties and is implicated in protective functions. Glycoconjugates are major components of the glycocalyx, but their biochemical nature is not well understood. Previous studies on rabbit bladder indicated the presence of significant levels of sialoglycoproteins compared to glycosaminoglycans in the epithelium. In this study, rabbit explant cultures were radiolabeled by precursor sugars or amino acids and a major lectin-reactive glycoprotein of rabbit bladder mucosa was isolated by affinity chromatography on jacalin-agarose. The radiolabeled glycoprotein was purified to homogeneity by a second cycle on the lectin column, followed by gel filtration and density gradient centrifugation. The average molecular mass of the glycoprotein was estimated to be 245 kDa and 210 kDa by gel filtration and SDS–PAGE, respectively. Its buoyant density was 1.40 g/ml, suggesting a carbohydrate content of ~50%. The percent distribution of glucosamine-derived tritium label in sialic acid, galactosamine, and glucosamine was 30, 52, and 18, respectively. The glycoprotein consisted entirely of small sialylated and neutral oligosaccharides O-glycosidically linked to serine and threonine residues. The same glycoprotein could be immunoprecipitated with an antibody against the carboxy terminal 17 amino acid peptide of human MUC1 mucin glycoprotein. This suggests that this mucin glycoprotein is the rabbit homolog of MUC1 glycoprotein, which has been previously established to be a component of human bladder urothelium and has been purified from human urine and biochemically characterized.

Key words: bladder epithelium/mucin glycoprotein/MUC1/jacalin/interstitial cystitis/urinary tract infection


    Introduction
 Top
 Abstract
 Introduction
 Results
 Discussion
 Materials and methods
 Acknowledgments
 Abbreviations
 References
 
Glycoconjugates are important contributors to the protective properties of the delicate mucosa of the bladder. The mucosa provides the first line of defense against noxious agents in the urine and assault by environmental pathogens (Parsons et al., 1977Go; Svanborg Eden et al., 1990Go; Badalament et al., 1992Go; Lanne et al., 1995Go). Defects or abnormalities in bladder glycoconjugates are suggested to play critical roles in pathogenesis of urinary tract infections (Parsons et al., 1978Go; Badalament et al., 1992Go; Ruggieri et al., 1992Go), bladder carcinoma (Langkilde et al., 1992Go; Bergeron et al., 1997Go), and interstitial cystitis (Moskowitz et al., 1994Go; Erickson et al., 1996Go, 1998; Hurst et al., 1996Go; Parsons, 1997Go; Byrne et al., 1999Go).

We are interested in carrying out detailed biochemical characterization of the important glycoconjugates of the mammalian bladder mucosa. Our long-term objective is to elucidate the roles of glycoconjugates in the development of interstitial cystitis and other bladder pathologies. Due to the limited availability of human bladders for research purposes, the initial studies are being conducted on rabbit bladders. Previously, we demonstrated by metabolic labeling and biochemical analysis that glycosaminoglycans are minor components of the bladder epithelium in comparison to sialoglycoproteins, which are much more abundant (Buckley et al., 2000Go). In order to obtain information on the glycoproteins of rabbit bladder, paraffin sections were stained with biotinylated lectins with specificities for a variety of saccharides. Thirteen of the 17 lectins tested showed positive reactivity of varying intensity with the epithelium (Buckley et al., 2000Go). Several lectins gave strong unequivocal staining of the epithelium, which was most intense on the luminal surface. This is in striking contrast to the very weak or absent staining of the epithelium by antibodies against heparan- and chondroitin-sulfates and hyaluronic acid binding protein (Buckley et al., 2000Go). One of the major glycoproteins of the rabbit bladder mucosa was purified and characterized as a mucin, based on its behavior on gel filtration, ion exchange chromatography, density gradient centrifugation under dissociative conditions, resistance to GAG-degrading enzymes, and the presence of O-linked oligosaccharides (Buckley et al., 2000Go).

In the present study, another major mucin glycoprotein of the rabbit bladder demonstrating reactivity with several lectins was purified by affinity chromatography on jacalin-agarose. It migrated on SDS–PAGE as a diffuse band of apparent average molecular size of 210 kDa. The same glycoprotein could be immunoprecipitated with an antibody against a 17 amino acid peptide found in the carboxy terminal of human MUC1 mucin glycoprotein (Pemberton et al., 1992Go). These results indicate that the 210 kDa glycoprotein is the rabbit homolog of epitectin, which we have previously established to be a component of human urothelium (Bramwell et al., 1983Go), and recently purified from human urine and biochemically characterized (Bhavanandan et al., 1998Go).


    Results
 Top
 Abstract
 Introduction
 Results
 Discussion
 Materials and methods
 Acknowledgments
 Abbreviations
 References
 
Examination of rabbit bladder glycoproteins binding to immobilized lectins
Figure 1 illustrates SDS–PAGE of the radiolabeled glycoproteins of rabbit bladder, precipitated by various immobilized lectins. The predominant glycoprotein, which interacted with several lectins, appeared on SDS–PAGE as a doublet with mobilities close to that of the 200 kDa standard protein. Jacalin, Maackia amurensis lectin (MAL–II), and WGA which recognize {alpha}-Gal/GalNAc and sialic acids reacted strongly with both components of the doublet. But Amaranthus caudatus (ACL) and Lycopersicon esulentum (LEL), lectins which recognizes ß-Gal- and chitiooligo-saccharides, appear to react preferentially with the lower and higher molecular size species, respectively (Figure 1). Jacalin and WGA also precipitated other glycoproteins that moved as a diffuse band in the 65 kDa to 98 kDa molecular weight range. In contrast, Solanum tuberosum (STL) and Datura stramonium (DSL), which primarily recognize ß-GlcNAc terminating saccharides, precipitated the low molecular weight glycoproteins, but not the high molecular weight glycoproteins.



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Fig. 1. SDS gel electrophoresis of radiolabeled rabbit bladder glycoproteins precipitated by immobilized lectins. Extracts of mucosa from rabbit bladder explants cultured in the presence of [3H]glucosamine were treated with various immobilized lectins (lane 1, ACL; lane 2, STL; lane 3, jacalin; lane 4, DSL; lane 5, MAL-II; lane 6, WGA; and lane 8, LEL) as described in Materials and methods. The lectin-bound [3H]-glycoproteins were dissociated by heating in SDS-buffer and subjected to SDS–PAGE on a 4–15% gel followed by fluorography. Lane 7, 14C-labeled protein standards.

 
Isolation of the major lectin-reactive glycoprotein by affinity chromatography on jacalin-agarose
The results of affinity chromatography on jacalin-agarose of an extract of in vitro [3H] glucosamine-labeled rabbit bladder mucosa and rechromatography of the material specifically bound to the lectin are illustrated in Figure 2. The elution profile on Sepharose CL-4B of the bladder mucosa [3H]-glycoprotein recovered after two cycles on jacalin-agarose is illustrated in Figure 3 (upper panel). The high molecular weight material was recovered and rechromatographed on the Sepharose CL-4B column. The material recovered after the second chromatography gave a single, albeit broad peak on Sepharose CL-4B (Figure 3, lower panel). In comparison to the elution of nonglycosylated protein calibration standards, the molecular weight of the glycoprotein was estimated to be about 245 kDa (Figure 3 inset). [3H]Proline-labeled glycoprotein was also similarly purified from bladder mucosa, whereas explant bladders cultured in the presence of [3H]mannose or [35S]sulfate did not incorporate significant label into the high molecular weight glycoproteins that bound to jacalin. Chromatography of the [3H]glucosamine-labeled glycoprotein isolated from rabbit bladder mucosa on a column of DEAE-Sephacel gave a single peak eluting before reference chondroitin sulfate and at a NaCl concentration of about 0.4 M (not illustrated). SDS–PAGE of the [3H]glucosamine- or [3H]proline-labeled purified glycoprotein on 6% (Figure 4) and 4–12.5% gradient gels (not illustrated) gave single, but diffuse, bands. The average molecular weight of material based on its mobility in the 6% gel, again based on nonglycosylated protein standards, was estimated to be 210 kDa (Figure 4, lane 1). The lower molecular weight glycoproteins (fractions 49–80) from the Sepharose CL-4B fractionation (Figure 3, upper) showed a long streak extending from the 200 kDa region to the bottom of the gel on SDS–PAGE (not illustrated). The purified [3H]glucosamine-labeled mucosal glycoprotein, when subjected to density gradient centrifugation in CsTFA under dissociative conditions, was found to have a high density (peak density about 1.40 g/ml). Based on the density of mucins and other mucin glycoproteins of known carbohydrate composition it can be concluded that the carbohydrate content is high (~50% by weight) (Bhaskar and Reid, 1981Go; Devaraj et al., 1992Go).



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Fig. 2. Affinity chromatography of radiolabeled rabbit bladder glycoconjugates on jacalin-agarose. Rabbit bladder mucosa extract prepared as described in Materials and methods was chromatographed on a jacalin-agarose column (1 x 8 cm) (solid circles). The column was eluted with PBS –0.1% NP-40, followed by PBS (I), 0.1 M melibiose in PBS (II), and finally 0.5 M galactose in PBS (III). One milliliter fractions were collected and aliquots analyzed for radioactivity. The material eluting with 0.1 M melibiose and 0.5 M galactose were pooled, dialyzed against PBS, and rechromatographed on the same column (x). The material that was eluted with melibiose was recovered by dialysis followed by lyophilization.

 


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Fig. 3. Gel filtration of jacalin-binding glycoproteins on Sepharose CL-4B. Upper panel: The jacalin affinity purified rabbit bladder mucosa glycoprotein was applied on a Sepharose CL-4B column (1.3 x 100 cm) and eluted with 50 mM Tris/HCl buffer, pH 8.0, containing 0.1% CHAPS and protease inhibitors. Fractions of 1 ml each were collected, and aliquots analyzed for radioactivity. The material eluting in fractions 37–48 was recovered and rechromatographed on the same column. Lower panel: Elution profiles of the glycoproteins recovered after the second chromatography (solid circles) and of asialoglycoprotein prepared from the purified material by treatment with neuraminidase as described in the text (open circles). Inset, Calibration curve generated by plotting Kave values against log molecular weight of protein standards chromatographed on the same column.

 


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Fig. 4. SDS gel electrophoresis of purified jacalin-binding glycoproteins from rabbit bladder. The [3H]-labeled glycoprotein samples were subjected to SDS–PAGE on a 6% gel under reducing conditions and detected by fluorography. Lane 1, [3H]glucosamine-labeled glycoprotein purified from bladder mucosa; lane 2, [3H]glucosamine-labeled glycoprotein purified from culture media; lane 3, asialoglycoprotein prepared from the mucosa derived material shown in lane 1; lane 4, [3H]proline-labeled glycoproteins purified from bladder mucosa; lane 5, [3H]proline-labeled glycoprotein after deglycosylation by treatment with neuraminidase followed by TFMS. The positions of molecular weight marker proteins are indicated.

 
The secreted/shed glycoprotein was similarly purified by affinity chromatography of the spent culture media on immobilized-jacalin columns followed by chromatography on Sepharose CL-4B. On SDS–PAGE the glycoprotein purified from media revealed a diffuse band with an average molecular weight of 218 kDa. (Figure 4, lane 2).

Susceptibility of the purified jacalin-binding glycoprotein to glycosidases and analysis of the carbohydrate composition
Exhaustive V.cholerae or A.ureafaciens neuraminidase treatment of [3H]GlcNH2-labeled mucosa glycoprotein released 28.4 % of the tritium activity, which co-migrated with authentic N-acetyl neuraminic acid on Bio Gel P-2. Mild acid hydrolysis with 0.1N H2SO4 (80°C, 1 h) also released an identical amount of sialic acid indicating that the sialic acids in the glycoprotein are fully susceptible to the above neuraminidases. Further, when the sialic acid released by the A.ureafaciens enzyme was recovered and examined by HPAEC (Rohrer et al., 1998Go), a single component coeluting with reference N-acetyl neuraminic acid was detected. This confirmed the absence of N-glycolyl or O-acetylated sialic acids in the jacalin-binding rabbit bladder glycoprotein. The molecular weight of the asialoglycoprotein, obtained by treatment with A.ureafaciens neuraminidase, was estimated to be 230 kDa and 180 kDa as determined by gel filtration on Sepharose CL-4B (Figure 3, lower panel) and SDS–PAGE (Figure 4, lane 3), respectively. A portion of the asialoglycoprotein was treated with endo-{alpha}-N-acetyl galactosaminidase and the products analyzed on a Bio Gel P-4 column. About 28.5% of the radioactivity eluted in a peak that coincided with the elution position of reference Galß1 -> GalNAc. The [3H]glucosamine-labeled glycoprotein was also treated with O-sialoglycoprotein endopeptidase (50 mM hydroxyethylpiperazine ethanesulfonic acid, pH 7.4, 37°C, 18 h), and the reaction mixture chromatographed on a Sepharose column as described in Figure 3. The radiolabeled material now eluted as a very broad peak over the range from fraction 45 to 85, indicating susceptibility of the glycoprotein to this enzyme.

The percent of radioactivity in galactosamine and glucosamine in the purified [3H]glucosamine labeled rabbit bladder mucosa 210 kDa glycoprotein was found to be 52 and 18, respectively.

Deglycosylation of the jacalin-binding glycoprotein
SDS–PAGE of the [3H]proline-labeled glycoprotein deglycosylated by treatment with neuraminidase, in the presence of protease inhibitors, followed by TFMS (Edge et al., 1981Go; Woodward et al., 1987Go) revealed streaks extending from ~100 kDa to the bottom of the gel (~45 kDa) (Figure 4, lane 5). We have previously established that the conditions used (TFMS, 25°C, 3 h) result in 90–95% deglycosylation of mucins without detectable polypeptide degradation (Bhavanandan and Hegarty, 1987Go; Woodward et al., 1987Go). A portion of the [3H]proline-labeled asialoglycoprotein was also subjected to exhaustive treatment with a mixture of endo-{alpha}-N-acetyl galactosaminidase, ß-hexosaminidase, and exo-{alpha}-N-acetyl galactosaminidase in the presence of a mixture of protease inhibitors. SDS–PAGE of the enzymatically deglycosylated material also showed no distinct band; instead the disappearance of the native glycoprotein was accompanied by appearance of faint streaks (not illustrated). These results are typical of deglycosylated mucin glycoproteins as observed by several investigators (Marianne et al., 1986Go; Byrd et al., 1989Go; Gerken et al., 1992Go).

Action of alkaline borohydride on the purified 210 kDa glycoprotein
The products of the mild alkaline borohydride treatment were desalted by gel filtration on a column of BioGel P-6 and the recovered saccharide alditols fractionated on a column of AG1 (acetate). One neutral fraction (I) and two acidic fractions (II, III) containing 30.8, 48.7, and 17.0 % of the radiolabel, respectively, were recovered. Each of these fractions was size fractionated on a (2.5 x 100 cm) column of BioGel P4 (–400 mesh) as illustrated in Figure 5. Six of the major fractions, numbered I-8, I-7, I-6, II-4, II-3, and III-2, were recovered in pure form and in sufficient quantities for structural analysis.



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Fig. 5. Fractionation of 3H-labeled oligosaccharide alditols on Bio Gel P-4. The neutral (top panel) and the two acidic fractions (middle and lower panels) from DEAE anion exchange chromatography were chromatographed individually on a preparative Bio Gel P-4 (minus 400 mesh) column (2.5 x 100 cm) and eluted at 55°C with 0.1 M pyridine/acetic acid (Yamashita et al., 1982). Fractions of 2 ml were collected and aliquots analyzed for radioactivity. The saccharides eluting in peaks I-6, I-7, I-8, II-3, II-4, and III-2 were recovered by pooling appropriate fractions and lyophilization. The peak elution positions of Neu5Ac->Gal->(Neu5Ac)->GalNAc(OH) (Tetra), Neu5Ac->Gal->GalNAc(OH) (Tri), Neu5Ac->GalNAc(OH) (Di-A), GlcNAc->GlcNAc(OH) ( Di-B), Gal->GalNAc(OH) (Di-C), and glucose (Glc) are indicated.

 
Structural determination of the major saccharide alditols
Each of the above six fractions was rechromatographed on a pre-calibrated analytical column of Bio Gel P-4 and the recovered homogenous material analyzed for [3H]hexosaminitol and [3H]hexosamine content after acid (4N HCl, 100°C, 8h) hydrolysis. The acidic fractions were treated with A.ureafaciens neuraminidase and the products applied on a AG1 (formate) column (1 ml resin) and eluted with water followed by 0.3N formic acid to separate the released sialic acid from the neutral component by chromatography. The neutral component in the water eluate was recovered and further examined by chromatography on the above Bio Gel P-4 column.

Fraction I-8.
This saccharide had mobility identical to reference GalNAc(OH) on Bio Gel P-4 column. Acid hydrolysis followed by analysis of the products by cation exchange chromatography yielded [3H]-galactosaminitol as the only product.

Fraction I-7.
This oligosaccharide had mobility identical to reference Gal ß1->3 GalNAc(OH) on both Bio Gel P-4 and P-6 columns. Acid hydrolysis and treatment with bovine testicular ß-galactosidase yielded [3H]-galactosaminitol and [3H]GalNAc(OH), respectively, as the only radioactive products.

Fraction I-6.
This oligosaccharide coeluted with [3H] di-N-acetyl chitobiositol on Bio Gel P-2 and P-4 columns. Acid hydrolysis followed by cation exchange chromatography yielded both [3H]GlcNH2 and [3H]-galactosaminitol.

Fraction II-4.
This acidic oligosaccharide had mobility identical to reference Neu5Ac 2->6GalNAc(OH), isolated from ovine submaxillary mucin, on both Bio Gel P-4 and P-6 columns. Hexosamine and hexosaminitol analysis revealed only [3H]-galactosaminitol. Treatment with A.ureafaciens neuraminidase released about 36% of the radioactivity as sialic acid and the balance in a neutral product that coeluted with GalNAc(OH) on a Bio Gel P-4 column.

Fraction II-3.
This acidic oligosaccharide had mobility identical to reference trisaccharide, Neu5Ac2->3Gal1-> 3GalNAc(OH) isolated from fetuin, on both Bio Gel P-4 and P-6 columns. Hexosamine and hexosaminitol analysis revealed only 3H-galactosaminitol. Treatment with A.ureafaciens neuraminidase yielded sialic acid containing 42% of the radioactivity and a neutral disaccharide (containing 58% of the radioactivity) that had mobility identical to Galß1-> 3GalNAc(OH).

Fraction III-2.
This acidic oligosaccharide had mobility ­identical to the sialyl tetrasaccharide, Neu5Ac-> Gal->(Neu5Ac)->GalNAc(OH) derived from fetuin, on pre-calibrated Bio Gel P-4 and P-6 columns. Hexosamine and hexosaminitol analysis revealed only 3H-galactosaminitol. Treatment with A.ureafaciens neuraminidase yielded sialic acid containing 60% of the radioactivity and the disaccharide Galß1->3GalNAc(OH) containing the balance of the radioactivity. Based on these results the structures of these six saccharides were deduced as summarized in Table I.


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Table I. Structures of the major neutral and acidic oligosaccharide alditols derived by alkaline borohydride treatment of the 210 kDa mucin glycoprotein from rabbit bladder
 

Interaction of the 210 kDa glycoprotein with anti-MUC1 antibodies
Portions of the rabbit bladder mucosal extracts from the [3H]glucosamine-labeled explants were subjected to affinity chromatography on columns of jacalin-agarose or immobilized chicken anti-CTP IgY antibodies. The material specifically bound to the column and eluted with 0.1 M galactose or 3M KSCN, respectively, was recovered and subjected to SDS–PAGE. The electrophoretic mobility of the jacalin and antibody bound materials was identical, as illustrated in Figure 6. Further, when the material purified by affinity chromatography on jacalin-agarose was applied on the immobilized CTP antibody, about 68% of the radiolabel bound and could be eluted with 3 M KSCN. The staining of rabbit bladder sections by the chicken anti-CTP IgY and mouse monoclonal Ca2 IgG antibodies was also examined. The bladder epithelium stained positively with both antibodies but not with control mouse IgG as illustrated (Figure 7). The staining with the IgY antibody was abolished when the incubation was done in the presence of the peptide antigen (Figure 7D). In other experiments, the chicken anti-CTP antibody was used to precipitate specifically the 400 and 350 kDa bands of the human MUC1 glycoprotein, epitectin, from extracts of human laryngeal carcinoma H.Ep.2 cells cultured in the presence of [3H]glucosamine (not illustrated). This confirmed the specificity of the anti-CTP antibody for the carboxy terminal sequence of human MUC1 glycoprotein and its ability to immunoprecipitate the target molecules. Taken together, these results strongly suggest that the 210 kDa mucin glycoprotein purified from rabbit bladder is the rabbit homolog of the human MUC1 mucin glycoprotein. The reactivity of the CTP antibody is not surprising, since it has been demonstrated that the carboxy terminal segment of the MUC1 glycoprotein, on the cytoplasmic side of the plasma membrane, is conserved among species (Pemberton et al., 1992Go; Patton et al., 1995Go).



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Fig. 6. SDS–PAGE of [3H]glycoproteins that bound to anti-CTP antibody and jacalin. Extracts of mucosa from rabbit bladder explants cultured in the presence of [3H]glucosamine were subjected to affinity chromatography on columns (1 ml gel) of immobilized chicken anti-CTP antibody or jacalin. The material eluted from the CTP-antibody column with 3 M KSCN (lane 1) and from the jacalin column with 0.1 M galactose (lane 2) were recovered and subjected to SDS–PAGE on a 7.5% gel followed by fluorography. The positions of molecular weight protein markers are indicated.

 


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Fig. 7. Immunohistochemical staining of normal rabbit bladder sections for MUC1 glycoprotein. The deparaffinized and rehydrated sections were first treated with hydrogen peroxide-methanol for 4 min to quench endogenous peroxidase activity and then with normal horse (for Ca2) or goat (for anti-CTP) serum to block nonspecific binding. The tissue sections were then treated with 100 µl of 4 µg/ml of mouse monoclonal Ca2 antibody (a); 4 µg/ml of normal mouse IgG (b); 1 µg/ml of chicken anti-CTP IgY antibody (c); 1 µg/ml of chicken anti-CTP antibody and 20 µg/ml of CTP peptide (d). The slides were washed with PBS and incubated with biotinylated horse anti-mouse IgG (a, b), or biotinylated goat anti-chicken IgY (c, d). The slides were again washed with PBS and incubated with the ABC Vectastain reagent followed by the substrate solution containing equal volume of 0.1% diaminobenzidine tetrahydrochloride and 0.02% hydrogen peroxide.

 

    Discussion
 Top
 Abstract
 Introduction
 Results
 Discussion
 Materials and methods
 Acknowledgments
 Abbreviations
 References
 
Previously, we demonstrated that about 80% of the tritium-labeled glucosamine incorporated into rabbit bladder glycoconjugates is found in glycoproteins, with the balance in glycosaminoglycans (Buckley et al., 1996Go). This finding is in agreement with the results of histochemical studies revealing very intense staining of the epithelium by several biotinylated lectins (Buckley et al., 2000Go). The results of the precipitation of rabbit bladder extracts by immobilized lectins confirmed the presence of a glycoprotein of about 200 kDa that strongly interacted with jacalin, Mal-II, and WGA (Figure1).

Affinity chromatography on immobilized jacalin served as the first step in the purification of this glycoprotein. Jacalin specifically binds {alpha}-galactopyranosides and {alpha}-2-acetamido-2-deoxygalactopyranosides and particularly the T-antigen (Gal ß->3 GalNAc {alpha}->) (Mahanta et al., 1990Go). Therefore, for eluting the bound glycoproteins we tested solutions of galactose (0.1 and 0.5 M) and melibiose (an {alpha}-galactoside disaccharide). Elution with galactose, after first eluting with 0.1 M melibiose, displaced an additional small quantity (~4%) of glycoprotein (Figure 2). However, qualitatively, there was no difference when the materials were recovered and compared by SDS–PAGE (not illustrated). The practically quantitative binding of the galactose-eluted glycoproteins on rechromatography confirmed the specificity and reproducibility of their interaction with the lectin (Figure 2). The high molecular weight glycoprotein recovered after two cycles of gel filtration on Sepharose CL-4B was free of contaminants as judged by SDS–PAGE, gel filtration, and DEAE ion exchange chromatography of the native and desialylated molecules. The elution position of the glycoprotein and specifically the asialoglycoprotein on DEAE-Sephacel suggests the absence of sulfation of this molecule. This was confirmed by the absence of [35S]-label in the glycoprotein purified from mucosa of rabbit bladder cultured in the presence of [3H]glucosamine and Na235SO4 by affinity chromatography on jacalin-agarose.

The purified jacalin-binding glycoprotein exhibited polydispersity as judged by the diffuse bands on SDS–PAGE and broad peaks in molecular sieve and ion exchange chromatography and density gradient centrifugation. This behavior is typical of mucin-type glycoproteins and mucins and is at least partly due to microheterogeneity of the O-linked saccharide chains. The purified glycoprotein showed diffuse bands on SDS–PAGE (Figure 4) in contrast to the doublets seen for the material precipitated by the immobilized lectins (Figure 1). This is probably due to the fact that the purified glycoproteins were derived from a pool of several radiolabeled and unlabeled bladders, while a single bladder was used for the precipitation experiment. The variability of the glycoproteins in pooled rabbit specimens is consistent with our knowledge of the human homolog, the MUC1 glycoproteins. Human bladder MUC1 glycoproteins have been shown to exhibit genetic polymorphism leading to multiple forms (Swallow et al., 1987Go). Thus, average molecular mass of the purified glycoprotein was estimated to be 245 kDa and 210 kDa by gel filtration and SDS–PAGE, respectively. These values are considered apparent molecular weights, because the standards used for calibrations are proteins and not mucin-type glycoproteins. The apparent molecular mass of the asialoglycoprotein was estimated by gel filtration to be about 180 kDa. Based on these values, sialic acid would constitute ~25% by weight of the molecule, all of which is present as N-acetyl neuraminic acid, as determined by HPAEC. The approximate total carbohydrate content of the glycoprotein is calculated to be about 50% based on the density of 1.40 g/ml for the molecule. This could be verified by isolating the core protein of the glycoprotein and estimating its size, but unfortunately, deglycosylation did not yield an intact product. However, the streaky material seen on SDS–PAGE extends from just above the 97.4 kDa molecular weight marker to the bottom of the gel (Figure 4, lane 5), which suggests that the largest deglycosylated fragment is about 100 kDa.

The elution profile of the mild alkaline borohydride degradation products of the glycoprotein did not reveal any material eluting near the void volume of Bio Gel P-6. The quantitative beta-elimination of the saccharides, indicating the absence of N-linked saccharides in the glycoprotein, is in agreement with the failure to incorporate [3H]mannose into this glycoprotein during radiolabeling of rabbit bladder explant cultures. Fractionation gave evidence for the presence of at least 20 different saccharide alditols, many of which are in very small quantities (Figure 5). Of the major components, we were able to obtain sufficient quantities of six for elucidation of structures (Table I). Three classes of saccharides are evident: those based on (1) the linkage sugar GalNAc (I-8 and II-4), (2) the core 1 structure (I-7, II-3, and III-2), and (3) the core 3 structure (I-6). Of these, the core 1 disaccharides (Gal ß1->3 GalNAc) are the ones that were released on treatment of the asialoglycoprotein with endo-{alpha}-N-acetyl galactosaminidase (Umemoto et al., 1977Go). The presence of these small O-linked saccharides is consistent with the jacalin-binding characteristics of the glycoprotein (Mahanta et al., 1990Go). The saccharides listed in Table I are widely distributed in epithelial mucins for example, ovine submaxillary mucin) and cell membrane mucin-type glycoproteins such as glycophorin, leukosialin, and mouse and human melanoma glycoproteins (Bhavanandan, 1991Go). Of particular interest is the presence of such saccharides in the MUC1 glycoprotein (epitectin) associated with human bladder epithelium. The recently elucidated structures of the oligosaccharides isolated from human urine epitectin (Bhavanandan et al., 1998Go) include all those listed in Table 1, except I-6. Immunochemical studies demonstrated that the relationship of the human MUC1 glycoprotein and the 210 kDa jacalin-binding glycoprotein of rabbit bladder extend beyond similarities of saccharide structures. An antibody generated in chicken against the conserved C-terminal end of this transmembrane glycoprotein specifically interacted with the 210 kDa glycoprotein of rabbit bladder epithelium. In addition, the rabbit jacalin-binding 210 kDa glycoprotein was susceptible to O-sialoglycoprotein endopeptidase like the human MUC1 glycoprotein, as previously demonstrated by us (Hu et al., 1994Go).


    Materials and methods
 Top
 Abstract
 Introduction
 Results
 Discussion
 Materials and methods
 Acknowledgments
 Abbreviations
 References
 
Materials
Fresh bladders were obtained from rabbits that were sacrificed for various experiments at the Animal Facility at the Pennsylvania Sate University College of Medicine (Hershey, PA). Rabbit bladders "snap frozen" in liquid nitrogen were purchased from Pel-Freez (Rogers, AR). Components of the culture media were obtained from Grand Island Biological Co (Grand Island, NY). D-[6-3H] glucosamine hydrochloride (60 Ci/mmol), L-[2,3,4,5–3H] proline (100 Ci/mmol), D-[2,6–3H]mannose (44 Ci/mmol), and Na2 35SO4 (carrier free; 885 mCi/mmol) were purchased from American Radiolabeled Chemicals (St. Louis, MO). Bio Gels P-2, P-4, P-6, and AG1 (acetate) resin were from Bio-Rad Laboratories (Richmond, CA). Sepharose CL-6B and CL-4B and cesium trifluoroacetate were from Pharmacia (Piscataway, NJ). DEAE-Sephacel was obtained from Sigma Chemical Co. (St. Louis, MO). Immobilized jacalin (4–5 mg/ml) as well as all other lectin-gels were purchased from E-Y Laboratories. Vibrio cholerae and Arthrobacter ureafaciens neuraminidases were from Calbiochem (San Diego, CA). Endo-{alpha}-N-acetyl galactosaminidase was purified from culture filtrates of Diplococcus pneumoniae as described previously (Umemoto et al., 1977Go). Exo-{alpha}-N-acetyl galactosaminidase (Patella vulgata) and ß-hexosaminidase (jack bean) were from V-Labs (Covington, LA). Pasteurella haemolytica O-sialoglycoprotein endopeptidase (Abdullah et al., 1992Go) was purchased from Accurate Chemicals (Westbury, NY). Reference Galß1->3GalNAc was purchased from Toronto Research Chemicals (North York, Ontario, Canada). The tritium labeled oligosaccharides Neu5Ac->Gal->(Neu5Ac)->GalNAc(OH), Neu5Ac->Gal->GalNAc(OH), Neu5Ac->GalNAc(OH), Gal-> GalNAc(OH) and GalNAc(OH) for use as reference standards were prepared from fetuin, glycophorin, and ovine submaxillary mucin as described previously (Bhavanandan and Katlic, 1979Go). [3H]-GlcNAc->GlcNAc(OH) was prepared by reduction of di-N-acetylchitobiose (Bhavanandan and Katlic, 1979Go) with sodium borotritide and purification on a column of Bio Gel P-2. The mouse monoclonal Ca2 antibody against MUC1 glycoprotein was a gift from Prof. Henry Harris, University of Oxford (Bramwell et al., 1985Go). The biotinylated second antibodies, the Vectasatin ABC peroxidase kit and the Vectastain VIP substrate kit were purchased from Vector Laboratories (Burlingame, CA). Pefabloc was from Roche Molecular Biochemicals, (Indianapolis, IN). Amplify for fluorography was purchased from Amersham (Arlington Heights, IL). Ready-Safe scintillation counting fluid was obtained from Beckman Instruments (Fullerton, CA).

Organ culture, radiolabeling of rabbit bladder, and extraction of mucosal glycoconjugates
Bladders, removed immediately after sacrifice of rabbits, were rinsed with ice-cold PBS containing fungizone and penicillin–streptomycin, transported to the laboratory on ice, and established in culture within about 30 min. One or two bladders were segmented into quarters, and each piece placed mucosal side up in a scored Nunc culture dish (15 x 60 mm). CMRL 1066 media (4–5 ml), prewarmed to 37°C, was added so that the mucosa was not submerged, and it was then incubated in a 5% CO2 incubator at 37°C. For radiolabeling, the bladder was incubated in medium containing [3H]glucosamine (20 µCi/ml) and [35S]sulfate (100 µCi/ml), but no inorganic sulfate and one-third the usual glucose concentration (Bhavanandan, 1981Go), for up to 48 h to obtain equilibrium labeling. Bladders also were radiolabeled by incubating with medium containing with [2,6 3H]mannose (10 µCi/ml) or [2,3,4,5–3H]proline (20 µCi/ml). The bladder epithelium was carefully separated using a Teflon spatula (Buckley et al., 1996Go) and extracted at 4°C by stirring in PBS, pH 7.0, containing 0.1% NP-40, 0.02% sodium azide, 1 mM phenyl methyl sulfonyl fluoride, 2 mM ethylmaleimide, 5 mM EDTA, 0.01% PefablocR, leupeptin (0.5 µg/ml), and pepstatin (0.7 µg/ml). The bladder mucosa extract was centrifuged (10,000 x g, 30 min) at 4°C, and the supernatant used for affinity chromatography.

Precipitation of bladder glycoproteins using immobilized lectins
Aliquots of the above PBS-0.1% NP-40 extracts of radiolabeled rabbit bladder mucosa containing about 106 d.p.m. in 250 µl were added to 100 µl packed lectin-gels. The suspensions were incubated at 4°C with end-over-end shaking for 18 h. After centrifugation in a Microfuge for 5 min, the supernatant was discarded, and gel washed three times each with 1 ml of 20 mM Tris–HCl buffer, pH 8.0, containing 0.1 M NaCl, 0.5% NP-40, and 1 mM phenyl methyl sulfonyl fluoride followed by 10 mM Tris–HCl, pH 8.0 (to remove NP-40). The gel pellet was mixed with 30 µl of 50 mM Tris–HCl, pH 8.4, containing 2% SDS, 10% glycerol, 0.1 M DTT, and 0.1% bromophenol blue and heated at 100°C for 5 min. After centrifugation, 25 µl of the supernatant was subjected to SDS–PAGE followed by fluorography.

Column chromatography
Columns of Bio Gel P-2, P-4 (–400 mesh), and P-6 (200–400 mesh), were equilibrated and eluted with 0.1 M pyridine acetate, pH 5.0. Sepharose CL-4B column was equilibrated and eluted with 50 mM Tris–HCl, pH 8.0, containing 0.1% CHAPS or deoxycholate and 0.01% PefablocR. The jacalin-agarose column was equilibrated with PBS/0.1% NP-40, and the bound glycoproteins were eluted with 0.1 M galactose in PBS.

Determination of sialic acid and hexosamines
[3H]Sialic acid in isotopically labeled glycoprotein and oligosaccharides was determined by either acid hydrolysis (0.1N H2SO4, 80°C, 1 h) followed by neutralization or by treatment with Arthrobacter ureafaciens, addition of carrier sialic acid (1 mg), and fractionation on a Bio Gel P-2 column. Aliquots of the fractions were analyzed for sialic acid (Bhavanandan and Sheykhnazari, 1993Go) and radioactivity. Hexosamines and hexosaminitol in labeled glycoproteins and oligosaccharides were estimated after acid hydrolysis (4 N HCl, 100°C, 8h). Standard hexosamines, hexosaminitols, and glycine were added to the dried hydrolysates, and the mixture chromatographed on a AG 50W(H+) cation exchange resin column (Cheng and Boat, 1977Go; Bardales et al., 1989Go). Aliquots of the fractions were analyzed by a ninhydrin assay to detect the unlabeled reference standards and by liquid scintillation counting for the radioactive hexosamines.

Alkaline-borohydride treatment to release Ser/Thr-linked saccharides
Glycoproteins were treated with freshly prepared 0.1 M NaOH containing 1.0 M NaBH4 at 37°C for 72 h in an atmosphere of nitrogen. The reaction mixture was cooled in an ice bath, neutralized by dropwise addition of 4 M acetic acid, passed through a column of AG 50 (H+) resin, and the water eluate evaporated to dryness in a rotary evaporator. The residue was treated by repeated addition of methanol:HCl (1000:1) and evaporation to remove borate.

Fractionation of the oligosaccharide-alditols
The mixture was chromatographed on AG1 (acetate) and eluted with a linear gradient of 0.01 M-0.5 M pyridine acetate. Further fractionation of the neutral and the two sialylated oligosaccharide-alditol fractions was performed on a preparative column of Bio Gel P-4 (–400 mesh) (Yamashita et al., 1982Go). The major fractions were rechromatographed on an analytical column of Bio Gel P-4 that had been precalibrated with reference standards.

Antibody against the carboxy-terminal peptide sequence of human MUC1 glycoprotein
The 17 amino acid (SSLSYTNPAVAATSANL) peptide was synthesized in the Macromolecular Core Facility of this institution and further purified by HPLC. The purified peptide (20 mg) was mixed with Keyhole limpet hemocyanin (KLH, 20 mg), dissolved in 8 ml of PBS, and an equal volume of 0.2% glutaraldehyde was added with stirring and incubated for 1 h at 20°C. The reaction was stopped by addition of 1 M glycine in PBS to a final concentration of 200 mM and incubation for an additional hour. The reaction mixture was dialyzed extensively against PBS and concentrated by ultrafiltration. The peptide-KLH conjugate was submitted to Lampire Biological Labs (Pipersville, PA) for immunization of hens (Carroll and Stollar, 1983Go). Eggs were collected beginning week 6 after initial intramuscular injection of the antigen. IgY was prepared from yolks of batches of eggs by differential polyethylene glycol precipitation and purified by chromatography on a column of a DEAE-Sephacel as described by Gassmann et al. (1990)Go. The antibody was demonstrated to react specifically with the above synthetic peptide, but not other unrelated peptides. The antibody also reacted with epitectin purified from both human laryngeal carcinoma H Ep-2 cells and human urine (unpublished observations). The IgY specific for synthetic peptide was isolated by affinity chromatography of the total IgY on a column of immobilized peptide prepared by conjugating the peptide to AH-Sepharose (3 mg/ml).


    Acknowledgments
 Top
 Abstract
 Introduction
 Results
 Discussion
 Materials and methods
 Acknowledgments
 Abbreviations
 References
 
We thank Mr. Mostafa Sheykhnazari, Ms. Sharlene Washington, and Ms. Nancy Herb for their technical support and Ms. Nancy Herb for assistance in the preparation of the manuscript. This investigation was supported by USPHS Grant DK 47511.


    Abbreviations
 Top
 Abstract
 Introduction
 Results
 Discussion
 Materials and methods
 Acknowledgments
 Abbreviations
 References
 
ACL, Amaranthus caudatus lectin; Con A, Concanavalin A; CTP-, carboxy terminal peptide; CsTFA, cesium trifluoroacetic acid; DEAE, diethylaminoethyl; DBA, Dolichos biflorus agglutinin; DSL, Datura stramonium lectin; ECA, Erythrina cristagalli agglutinin; GAG, glycosaminoglycans; Gal, D-galactose; GalNAc, D-N-acetyl galactosamine: GlcNAc, D-N-acetyl glucosamine: GalNAc(OH), D-N-galactosaminitol; HPA, Helix pomatia agglutinin; HPAEC, high pH anion exchange chromatography; KLH, Keyhole limpet hemocyanin; LEL, Lycopersicon esculentum lectin; MAL-II, Maackia amurensis lectin II; NP-40, Nonidet P-40; Neu5AC, D-N-acetyl neuraminic acid; PAGE, polyacrylamide gel electrophoresis; PBS, phosphate-buffered saline; PNA, peanut agglutinin; RCA, Ricinus communis agglutinin; SBA, soybean agglutinin; SDS, sodium dodecyl sulfate; STL, Solanum tuberosum lectin; TFMS, trifluoromethane sulfonic acid; UEA-II, Ulex europaeus agglutinin II; WGA, wheat germ agglutinin.


    Footnotes
 
1 Present address: Department of OB/GYN, Hirosaki University School of Medicine, Hirosaki 036, Japan Back

2 To whom correspondence should be addressed Back


    References
 Top
 Abstract
 Introduction
 Results
 Discussion
 Materials and methods
 Acknowledgments
 Abbreviations
 References
 
Abdullah,K.M., Udoh,E.A., Shewen,P.E. and Mellors,A. (1992) A neutral glycoprotease of Pasteurella haemolytica Al specifically cleaves O-sialoglycoproteins. Infect. Immun., 60, 56–62.[Abstract]

Bardales,R., Bhavanandan,V.P., Wiseman,G. and Bramwell,M.E. (1989) Purification and characterization of the epitectin from human laryngeal carcinoma cells. J. Biol. Chem., 264, 1980–1987.[Abstract/Free Full Text]

Badalament,R.A., Franklin,G.L., Page,C.M., Dasani,B.M., Wientjes,M.G. and Drago,J.R. (1992) Enhancement of Bacillus Calmette-Guerin attachment to the urothelium by removal of the rabbit bladder mucin layer. J. Urol., 147, 482–485.[ISI][Medline]

Bergeron,A., LaRue H. and Fradet,Y. (1997) Biochemical analysis of a bladder-cancer-associated mucin: structural features and epitope characterization. Biochem. J., 321, 889–895.[ISI][Medline]

Bhaskar,K.R. and Reid,L. (1981) Application of density gradient methods for the study of mucus glycoprotein and other macromolecular components of the sol and gel phases of asthmatic sputa. J. Biol. Chem., 256, 7583–7589.[Free Full Text]

Bhavanandan,V.P. (1981) Glycosaminoglycans of cultured human fetal uveal melanocytes and comparison with those produced by cultured human melanoma cells. Biochemistry 20, 5595–5602.[ISI][Medline]

Bhavanandan,V.P. (1991) Cancer-associated mucins and mucin-type glycoproteins. Glycobiology 1, 493–503.

Bhavanandan,V.P. and Hegarty,J.D. (1987) Identification of the mucin core protein by cell-free translation of messenger RNA from bovine submaxillary glands. J. Biol. Chem., 262, 5913–5917.[Abstract/Free Full Text]

Bhavanandan,V.P. and Katlic,A.W. (1979) The interaction of wheat germ agglutinin with sialoglycoproteins. J. Biol. Chem., 254, 4000–4008.[ISI][Medline]

Bhavanandan,V.P. and Sheykhnazari,M. (1993) Adaptation of the periodate-resorcinol method for determination of sialic acids to a microassay using microtiter plate reader. Anal. Biochem., 213, 438–440.[ISI][Medline]

Bhavanandan,V.P., Zhu,Q., Yamakami,K., Dilulio,N.A., Nair,S., Capon,C., Lemoine,J. and Fournet,B. (1998) Purification and characterization of the MUC1 mucin-type glycoprotein, epitectin, from human urine: structures of the major oligosaccharide alditols. Glycoconj J., 15, 37–49.[ISI][Medline]

Bramwell,M.E., Bhavanandan,V.P., Wiseman,G. and Harris,H. (1983) Structure and function of the Ca antigen. Br. J. Cancer, 48, 177–183.[ISI][Medline]

Bramwell,M.E., Ghosh,A.K., Smith,W.D., Wiseman,G., Spriggs,A.I. and Harris,H. (1985) New monoclonal antibodies evaluated as tumor markers in serous effusions. Cancer, 56, 105–110.[ISI][Medline]

Buckley,M.S., Washington,S., Laurent,C., Erickson,D.R. and Bhavanandan,V.P. (1996) Characterization and immunohistochemical localization of the glycoconjugates of the rabbit bladder mucosa. Arch. Biochem. Biophys., 330, 163–173.[ISI][Medline]

Buckley,M., Xin,P., Washington,S., Herb,H., Erickson,D. and Bhavanandan, V.P. (2000) Lectin histochemical examination of rabbit bladder glyco­proteins and characterization of a mucin isolated from the bladder mucosa. Arch. Biochem. Biophys., 375, 270–277.[ISI][Medline]

Byrd,J.C., Lamport,D.T.A., Siddiqui,B., Kuan, S-F., Erickson,R., Itzkowitz,S.H. and Kim,Y.S. (1989) Deglycosylation of mucin from LS174T colon cancer cells by hydrogen fluoride treatment. Biochem. J., 261, 617–625.[ISI][Medline]

Byrne,D.S., Sedor,J.F., Estojak,J., Fitzpatrick,K.J., Chiura,A.N. and Mulholland,S.G. (1999) The urinary glycoprotein GP51 as a clinical marker for interstitial cystitis. J. Urol., 161, 1786–1790.[ISI][Medline]

Carroll,S.B. and Stollar,B.D. (1983) Antibodies to calf thymus RNA polymerase II from egg yolks of immunized hens. J. Biol. Chem., 258, 24–26.[Abstract/Free Full Text]

Cheng, P-W. and Boat,T.F. (1977) An improved method for the determination of galactosaminitol, glucosaminitol, glucosamine and galactosamine on an amino acid analyzer. Anal. Biochem., 85, 276–282.[ISI]

Devaraj,N., Devaraj,H. and Bhavanandan,V.P. (1992) Purification of mucin glycoproteins by density gradient centrifugation in cesium trifluoroacetate. Anal. Biochem., 206, 142–146.[ISI][Medline]

Edge,A.S.B., Faltynek,C.R., Hof,L., Reichert, Jr.,L.E. and Weber,P. (1981) Deglycoslyation of glycoproteins by trifluoromethanesulfonic acid. Anal. Biochem., 118, 131–137.[ISI][Medline]

Erickson,D.R., Mast,S., Ordille,S. and Bhavanandan,V.P. (1996) Urinary epitectin (MUC-1 glycoprotein) in the menstrual cycle and interstitial cystitis. J. Urol., 156, 938–942.[ISI][Medline]

Erickson,D.R., Sheykhnazari,M., Ordille,S. and Bhavanandan,V.P. (1998) Increased urinary hyaluronic acid and interstitial cystitis. J. Urol., 160, 1282–1284.[ISI][Medline]

Gassmann,M., Thommes,P., Weiser,T. and Hubscher,U. (1990) Efficient production of chicken egg yolk antibodies against a conserved mammalian protein. FASEB J., 4, 2518–2532.[Abstract/Free Full Text]

Gerken,T.A., Gupta,R. and Jentoft,N. (1992) A novel approach for chemically deglycosylating O-linked glycoproteins. The deglycoslyation of submaxillary and respiratory mucins. Biochemistry, 31, 639–648.[ISI][Medline]

Hu,R.-H., Mellors,A. and Bhavanandan,V.P. (1994) Cleavage of epitectin, a mucin-type sialoglycoprotein, from the surface of human laryngeal carcinoma cells by a glycoprotease from Pasteurella haemolytica. Arch. Biochem. Biophys., 310, 300–309.[ISI][Medline]

Hurst,R.E., Roy,J.B., Min,K.W., Veltri,R.W., Marley,G., Patton,K., Shackelford,D.L., Stein,P. and Parsons,C.L. (1996) A deficit of chondroitin sulfate proteoglycans on the bladder uroepithelium in interstitial cystitis. Urology, 48, 817–821.[ISI][Medline]

Langkilde,N.C., Wolf,H., Clausen,H. and Orntoft,T.F. (1992) Human urinary bladder carcinoma glycoconjugates expressing T-(Galß (1–3)GalNAc{alpha}1-O-R) and T-like antigens: a comparative study using peanut agglutinin and poly- and monoclonal antibodies. Cancer Res., 52, 5030–5036.[Abstract]

Lanne,B., Olsson,B.M., Jovall,P.A., Angstrom,J., Linder,H., Marklund,B.I., Bergstrom,J. and Karlsson,K.A. (1995) Glycoconjugate receptors for p-fimbriated Escherichia coli in the mouse. An animal model of urinary tract infection. J. Biol. Chem., 270, 9017–9025.[Abstract/Free Full Text]

Mahanta,S.K., Sastry,M.V.K. and Surolia,A. (1990) Topography of the combining region of a Thomsen-Friedenreich-antigen-specific lectin jacalin. Biochem. J., 265, 831–840.[ISI][Medline]

Marianne,T., Perini,J.-M., Houvenaghel,M.-C., Tramu,G., Lamblin,G. and Roussel,P. (1986) Action of trifluoromethanesulfonic acid on highly glycosylated regions of human bronchial mucins. Carbohydr. Res., 151, 7–19.[ISI][Medline]

Moskowitz,M.O., Byrne,D.S., Callahan,H.J. Parsons,C.L., Valderrama,E. and Moldwin,R.M. (1994) Decreased expression of a glycoprotein component of bladder surface mucin (GP1) in interstitial cystitis. J. Urol., 151, 343–345.[ISI][Medline]

Patton,S., Gendler,S.J. and Spicer,A.P. (1995) The epithelial mucin, MUC1, of milk, mammary gland and other tissues. Biochim. Biophys. Acta, 1241, 407–424.[ISI][Medline]

Parsons,C.L. (1997) Epithelial coating techniques in the treatment of interstitial cystitis. Urology, 49, 100–104.[ISI][Medline]

Parsons,C.L., Greenspan,C., Moore,S.W. and Mulholland,S.G. (1977) Role of surface mucin in primary antibacterial defense of bladder. Urology, 9, 48–51.[Medline]

Parsons,C.L., Shrom,S.H., Hanno,P.M. and Mulholland,S.G. (1978) Bladder surface mucin: Examination of possible mechanisms for its antibacterial effect. Invest. Urol., 16, 196–200.[ISI][Medline]

Pemberton,L., Taylor-Papadimitriou,J. and Gendler,S.J., (1992) Antibodies to the cytoplasmic domain of the MUC1 mucin show conservation throughout mammals. Biochem. Biophys. Res. Commun., 185, 167–175.[ISI][Medline]

Rohrer,J.S., Thayer,J., Weitzhandler,M. and Avdalovic,N. (1998) Analysis of the N-acetylneuraminic acid and N-glycolylneuraminic acid contents of glycoproteins by high-pH anion-exchange chromatography with pulsed amperometric detection (HPAEC/PAD). Glycobiology 8, 35–43.[Abstract/Free Full Text]

Ruggieri,M.R., Balagani,R.K., Rajtar,J.-J. and Hanno,P.M. (1992) Characterization of bovine bladder mucin fractions that inhibit Escherichia coli adherence to the mucin deficient rabbit bladder. J. Urol., 148, 173–178.[ISI][Medline]

Svanborg Eden,C., Andersson,B., Aniansson,G., Lindstedt,R., de Man,P., Nielsen,A., Leffler,H. and Wold,A. (1990) Inhibition of bacterial attachment: examples from the urinary and respiratory tracts. Curr. Top. Microbiol. Immunol., 151, 167–184.[ISI][Medline]

Swallow,D.M., Gendler,S., Griffiths,B., Corney,G., Taylor-Papadimitriou,J. and Bramwell,M.E. (1987) The human tumour-associated epithelial mucins are coded by an expressed hypervariable gene locus PUM. Nature, 328, 82–84.[ISI][Medline]

Thompson,A.C. and Christmas,T.J. (1996) Interstitial cystitis—an update. Br. J. Urol., 78, 813–820.[ISI][Medline]

Umemoto,J., Bhavanandan,V.P.,and Davidson,E.A. (1977) Purification and properties of an endo-{alpha}-N-acetyl-D-galactosaminidase from Diplococcus pneumoniae. J. Biol. Chem., 252, 8609–8614.[Abstract]

Woodward,H.D., Ringler,N.J., Selvakumar,R., Simet,I.M., Bhavanandan,V.P. and Davidson,E.A. (1987) Deglycosylation studies on tracheal mucin glycoproteins. Biochemistry, 26, 5315–5322.[ISI][Medline]

Yamashita,K., Mizuochi,T. and Kobata,A. (1982) Analysis of oligosaccharides by gel filtration. Methods Enzymol., 83, 105–126.[ISI][Medline]





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