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.
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Abstract |
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Key words: bladder epithelium/mucin glycoprotein/MUC1/jacalin/interstitial cystitis/urinary tract infection
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Introduction |
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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., 2000). 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., 2000
). 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., 2000
). 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., 2000
).
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 SDSPAGE 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., 1992). 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., 1983
), and recently purified from human urine and biochemically characterized (Bhavanandan et al., 1998
).
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Results |
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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., 1998), 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 SDSPAGE (Figure 4, lane 3), respectively. A portion of the asialoglycoprotein was treated with endo-
-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
SDSPAGE of the [3H]proline-labeled glycoprotein deglycosylated by treatment with neuraminidase, in the presence of protease inhibitors, followed by TFMS (Edge et al., 1981; Woodward et al., 1987
) 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 9095% deglycosylation of mucins without detectable polypeptide degradation (Bhavanandan and Hegarty, 1987
; Woodward et al., 1987
). A portion of the [3H]proline-labeled asialoglycoprotein was also subjected to exhaustive treatment with a mixture of endo-
-N-acetyl galactosaminidase, ß-hexosaminidase, and exo-
-N-acetyl galactosaminidase in the presence of a mixture of protease inhibitors. SDSPAGE 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., 1986
; Byrd et al., 1989
; Gerken et al., 1992
).
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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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 ß13 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 26GalNAc(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, Neu5Ac23Gal1
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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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 SDSPAGE. 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., 1992; Patton et al., 1995
).
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Discussion |
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Affinity chromatography on immobilized jacalin served as the first step in the purification of this glycoprotein. Jacalin specifically binds -galactopyranosides and
-2-acetamido-2-deoxygalactopyranosides and particularly the T-antigen (Gal ß
3 GalNAc
) (Mahanta et al., 1990
). Therefore, for eluting the bound glycoproteins we tested solutions of galactose (0.1 and 0.5 M) and melibiose (an
-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 SDSPAGE (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 SDSPAGE, 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 SDSPAGE 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 SDSPAGE (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., 1987). Thus, average molecular mass of the purified glycoprotein was estimated to be 245 kDa and 210 kDa by gel filtration and SDSPAGE, 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 SDSPAGE 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 ß13 GalNAc) are the ones that were released on treatment of the asialoglycoprotein with endo-
-N-acetyl galactosaminidase (Umemoto et al., 1977
). The presence of these small O-linked saccharides is consistent with the jacalin-binding characteristics of the glycoprotein (Mahanta et al., 1990
). 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, 1991
). 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., 1998
) 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., 1994
).
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Materials and methods |
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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 penicillinstreptomycin, 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 (45 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, 1981), 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 [2,3,4,53H]proline (20 µCi/ml). The bladder epithelium was carefully separated using a Teflon spatula (Buckley et al., 1996
) 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 TrisHCl buffer, pH 8.0, containing 0.1 M NaCl, 0.5% NP-40, and 1 mM phenyl methyl sulfonyl fluoride followed by 10 mM TrisHCl, pH 8.0 (to remove NP-40). The gel pellet was mixed with 30 µl of 50 mM TrisHCl, 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 SDSPAGE followed by fluorography.
Column chromatography
Columns of Bio Gel P-2, P-4 (400 mesh), and P-6 (200400 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 TrisHCl, 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, 1993) 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, 1977
; Bardales et al., 1989
). 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., 1982). 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, 1983). 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)
. 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).
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Acknowledgments |
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Abbreviations |
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Footnotes |
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2 To whom correspondence should be addressed
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References |
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