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Isolation and characterization of peptides from the protein core of bovine gallbladder mucin
GASTROENTEROLOGY 1990;98:1633-1641
Isolation and Characterization of Peptides From the Protein Core of Bovine Gallbladder Mucin NEZAM H. AFDHAL, GWYNNETH D. OFFNER, FRANCIS E. MURRAY, ROBERT F. TROXLER, and BERNARD F. SMITH Section of Gastroenterology and Hepatology, Boston City Hospital, and Department Biochemistry, Boston University School of Medicine, Boston, Massachusetts
Gallbladder mucin may promote cholesterol gallstone formation by accelerating cholesterol monohdrate crystal nucleation in supersaturated bile. In this study, peptides were isolated from the mucin protein core by protease digestion and molecularsieve high-performance liquid chromatography. Tryptic peptides were purified by anion exchange or reverse-phase high-performance liquid chromatography, and amino acid compositions were determined. Tryptic peptides were (a) nonglycosylated, (b) selectively enriched in serine, glutamic acid plus glutamine, and glycine, and (c) depeleted in threonine and proline compared with native gallbladder mutin. Bilirubin derivatized with Woodward’s reagent K covalently bound to purified mucin. Tryptic digestion of the mucin-bilirubin complex yielded lowmolecular-weight nonglycosylated peptides with covalently bound bilirubin. These data indicate that the mucin protein core contains at least two distinct domains. One domain is rich in threonine and proline and contains the majority of covalently bound carbohydrate. A second domain, possibly internally located, is nonglycosylated, enriched in serine, glutamic acid plus glutamine, and glycine, and binds hydrophobic ligands such as bilirubin and l-anilino8-naphthalene sulfonate. Hydrophobic domains on the mucin protein core may contribute to the pathogenesis of cholesterol cholelithiasis.
M
ucin is the major secretory product of the gallbladder epithelium and is the principal organic constituent of mucous gel. Recent evidence from this laboratory (l-31 and others (4-6) indicates that gallbladder mucin is a critical element in the pathogenesis of cholesterol cholelithiasis. Mucin hypersecretion precedes gallstone formation in both
of
experimental animals (4-6) and humans (7,8) and results in the accumulation of a thick mucous gel adherent to the gallbladder epithelium. Nucleation of cholesterol monohydrate crystals, the initial event in cholesterol stone formation, occurs within the mucous gel (1,8,9). Moreover, inhibition of mucin secretion with aspirin prevents gallstone formation in experimental animals (5). It has been shown that gallbladder mucin can bind biliary lipids and accelerate the nucleation of cholesterol monohydrate crystals in supersaturated model bile (2,3). The structural integrity of the protease-sensitive areas on the mucin peptide core is essential for the acceleration of cholesterol crystal nucleation by mucin in model bile (2). Mucin is a densely glycosylated macromolecule that consists of a central peptide core accounting for 16% of its weight and a covalently bound carbohydrate accounting for approximately 76% of its weight (10). Extensive posttranslational glycosylation of mucin results in molecular heterogeneity with respect to size and polydispersity with respect to charge. Mucin monomers aggregate in aqueous solution and polymerize via both electrostatic and hydrophobic interactions 111). Mucin viscosity increases above a concentration of 2.5 mg/ml, and gel formation occurs at concentrations of 20-30 mg/ml (11). In this study, the structure of the protein core of bovine gallbladder mucin has been examined. The
Abbreviations used in this paper: ANS, I-anilino-g-naphthalene sulfonate; BW, bilirubin-Woodward’s reagent K; cDNA, complementary deoxyribonucleic acid; HPLC, high-performance liquid chromatography; RP-HPLC, reverse-phase high-performance liquid chromatography; SDS-PAGE, sodium dodecyl sulfate-polyacrylamide gel electrophoresis; TFA, trifluoroacetic acid. 0 1990 by the American Gastroenterological Association 0016-5065/90/$3.00
GASTROENTEROLOGY Vol. 98,No. 6
1634 AFDHAL ET AL.
results indicate that nonglycosylated areas hydrophobically bind bilirubin and the fluourescent ligand l-anilino-8-naphthalene sulfonic acid (ANS) (10). These areas are internally located on the mucin peptide core and have an amino acid composition that is distinct from the glycosylated segments of the mucin peptide core. Materials and Methods Purification
of Gallbladder
Mucin
Mucin was purified from bovine gallbladders obtained fresh at a local abbatoir (A. Arena and Sons, Hopkinton, Mass.]. Gallbladder mucus was scraped from the epithelial surface with a glass slide and homogenized for 3 min in a Waring blender in 0.2 M NaCl. Following centrifugation at 50,000 xg for 30 min, soluble mucin was purified as previously described (3,10,11] by Sepharose CL-4B (Sigma Chemica! Co., St. Louis, MO.) exclusion gel chromatography and two sequential equilibrium-density gradient ultracentrifugations in 60% cesium chloride. Purified mucin is free of low-molecular-weight protein, glycoprotein, and lipid contaminants (lo], but remains polydisperse with respect to size and charge.
Proteolytic
Digestion
For trypsin and chymotrypsin digestion, lyophilized mucin (10mg] was dissolved in 2 ml of 0.05 mM ammonium bicarbonate, 0.01 M CaCI,, and 0.04% sodium azide (pH 8.0) containing 0.1 ml of penicillin-streptomycin (1000 U + 1000 for 12 h at 37°C and wg-' * ml-‘). Mucin was incubated vortexed periodically to solubilize the mucin beffre digestion. Trypsin [type II, treated with phenylmethyl-sulfonylfluoride to inactivate chymotrypsin; Sigma Chemical Co., St. Louis, MO.) was added to mucin in a ratio of 1:lOO (enzymemucin, wt/wt] and incubated for either 24, 48, or 72 h at 37°C. Trypsin activity was terminated by adding 0.1% trifluoroacetic acid (TFA] to obtain pH 1.0 and freezing at -20°C. Chymotrypsin [type VII, treated with Na-p-tosyl+lysine chloromethyl ketone to inactivate trypsin; Sigma] was added to mucin (10 mg) in 0.08 M Tris, 0.01 M CaCl, (pH 7.8) at a ratio of 1:lOO (enzyme-mucin, wt/wt), and digestion was performed under conditions identical to those described for trypsin. For digestion with pepsin [Sigma], lyophilized mucin (10 mg) was reconstituted in 2 ml of a buffer of 0.08 M Tris HCl, 0.01 M CaCl, (pH 1.0) and incubated with pepsin (pepsin A, Sigma) at an enzyme-mucin ratio of 1:lOO (wt/wt) for either 24, 48, or 72 h at 37°C. Pepsin activity was terminated by the addition of 0.1 M sodium hydroxide to pH 8.0 and freezing at -20°C. Leucine aminopeptidase (5 mg; Worthington Biochemical, Freehold, N.J.) was dissolved in 1 ml of 0.14 M trimethylamine, 0.002 M MgCl, (pH 8.5) and incubated for 12 h at 4°C with diisopropyl-fluorophosphate to remove contaminating tryptic or chymotryptic activity. Enzyme activity was confirmed by the spectrophotometric method of Mitz and
Schlueter (12). Lyophilized mucin (10 mg] was reconstituted in 5 ml of the same buffer and incubated at an enzymemucin ratio of 1:20 (wt/wt] for either 24, 48, or 72 h at 37°C. Leucine aminopeptidase activity was terminated by acidification to pH 1.5 and freezing at -20°C. Carboxypeptidase A [Worthington) was prepared as a 1 x lo-‘-g/ml stock solution, and enzyme activity was confirmed by the reaction-velocity method of Folk and Schirmer (13) using hippuryl-L-phenylalanine as substrate. Lyophilized mucin (10 mg) was reconstituted in 10 mM Tris (pH 8.0) and incubated as previously described. Carboxypeptidase Y (Worthington] was prepared as a 2 x IO-‘-g/ml stock solution, and activity was confirmed using the enzymatic hydrolysis method of Kuhn (14). Lyophilized mucin (10 mg] was dissolved in 10 mM Tris (pH 6.0) and incubated as previously described.
High-Performance
Liquid Chromatography
Gallbladder mucin-derived peptides were analyzed by high-performance liquid chromatography (HPLC) using a Waters system with monitoring of column eluates at 229 nm (for peptides) an in some experiments at 436 nm [for bilirubin). Analytic separation of mucin peptides by molecular sieve was performed either on Bio-Gel TSK-60 and Bio-Sil TSK-400 columns connected in series or on a Bio-Sil TSK-125 column (Bio-Rad, Richmond, Calif.). Following preparative molecular-sieve HPLC, mucin tryptic peptides were dialyzed against deionized water at 4’C for 48 h, and anion exchange chromatography was performed on a Protein Pak DEAE 5PW anion-exchange column (Waters, Milford, Mass.]. Selected fractions were collected and pooled for subsequent amino acid analysis and sodium dodecyl sulfate-polyacrylamide gel electrophoresis [SDS-PAGE]. Mucin tryptic peptides were also analyzed directly without prior dialysis by reverse-phase HPLC (RP-HPLC) using a Bio-Rad Hi-Pore C-18 column. Selected peptides were further purified by RP-HPLC run isocratically at a concentration of the eluting solvent (0.01% TFA in 80% acetonitrile and 20% water) that was 16% less than the concentration at which the peptide originally eluted from the reverse-phase column.
l-Aniline-8-naphthalene Studies
Sulfonate-Binding
The hydrophobic binding properties of mucin were studied by fluorescence spectroscopy, which examined the binding of the hydrophobic fluorescent probe ANS to native and protease-digested mucin as previously described (10). After protease digestion, mucin glycopeptides were purified by gel-filtration chromatography using a Sepharose 2B column. Glycoprotein in the column effluent was detected by the periodic acid-Schiff assay (15], and glycopeptide fractions in the included volume were collected, pooled, dialyzed against deionized water for 48 h at 4”C, and lyophilized. Fluorescence spectoscopy was performed on a Perkin-Elmer Model LS-3B fluorimeter by mixing mucin glycopeptides (400 wg) with increasing concentrations of ANS (2-40 wM) in a final volume of 1 ml in 0.01 M Tris (pH
GALLBLADDER
June 1990
7.0). Relative fluorescence was measured wavelength of 365 nm and an emission nm.
PoJyacryJamide
using an excitation wavelength of 475
Gel Electrophoresis
Peptides separated by anion-exchange chromatography were analyzed by SDS-PAGE according to the method of Laemmli (IS] using a Bio-Rad Protean II slab-gel system with a acrylamide stacking gel and a 5%-30% acrylamide separating gel. Protein was detected by silver staining. 4%
Amino
Acid Analysis
Peptides were hydrolyzed in 6 N HCl in vacua at for 22 hours and analysed on a Beckman System 6300 amino acid analyzer. 110°C
Covalent
Binding
of Bilirubin
to Mucin
Bilirubin (Porphyrin Products, Logan, Utah] was purified to isolate the IXa isomer (17) and then derivatized with N-ethyl-5-phenylisoxazlium-3’-sulfonate (Woodward’s reagent K] according to the method of Kuenzle et al. (18). Bilirubin (116.8 mg; 0.2 mM] was incubated with Woodward’s reagent K (151.8 mg; 0.6 mM) and stirred at 20°C for 45 min in 20 ml of acetonitrile containing triethylamine (0.3 ml; 2.0 mM]. The mixture was evaporated to dryness at 3O”C, resuspended in 5 ml water, and chromatographed on a Sephadex G-25 column (50 cm x 1.0 cm] eluted with water at 40 ml/min. The bilirubin-Woodward’s reagent K complex (BW) was recovered in the third peak to elute from the column that was orange. The BW was lyophilized and stored in the dark at -20°C until further use. The BW was covalently bound to purified mucin by a modification of the method described by Kuenzle et al. for (18) and by Boyer for glutathione serum albumin S-tran:sferase (19). Lyophilized mucin (90 mg) was reconstituted in 23 ml of 0.01 M sodium phosphate and 0.145 M NaCl (pH 7.4). The BW was suspended in 5 ml water, and 1.6 ml was mixed with the mucin solution and incubated at 20°C for 2 h under N, in the dark. Ethylenediaminetetraacetic acid was added to obtain a final concentration of 1 mM, and the pH was brought to 9.4 by the addition of 700 mg of imidazole. After an additional 8 h of incubation in the dark at 20”C, the solution was chromatographed on a Sepharose CL-2B column (86.0 cm x 2.5 cm) and eluted with 0.1 M sodium phosphate with 0.145 M NaCl (pH 7.4) at a flow rate of 42 ml/h. Fractions (12 ml] were collected and monitored at A,,,, for protein and A,,, for bilirubin. Void volume fractions containing mucin and covalently bound BW were dialyzed against water in the dark at 4°C for 48 h and lyophilized. The mucin-BW complex (10 mg] was digested with trypsin for 24 h, and mucin peptides were separated by molecular sieve and RP-HPLC as previously described. Selected peptides were further fractionated by molecular-sieve HPLC using a Bio-Rad TSK-125 column.
MUtXN
PEPTIDES
1635
Results Protease Digestion Chromatography
and Molecular-Sieve
Endopeptidase digestion of purified bovine gallbladder mucin with trypsin, chymotrypsin, and pepsin yielded similar elution profiles through molecularsieve HPLC (Figure 1). Proteolysis resulted in the appearance of a second, well-defined, large-molecularweight peak that eluted just after the void volume peak. In addition, small poorly defined peaks were present in the intermediateand low-molecularweight portions of the included volume. Column fractions of the trypsin digest [Figure 1B) were pooled as indicated, and the peptides in these fractions were examined by anion-exchange HPLC and RP-HPLC. The large peak at the bed volume represents absorbance at 229 nm by sodium azide, which was added to all digests at a concentration of 0.04%. The time course of enzymatic digestion was examined for trypsin at 2, 24, and 48 h (not shown]. Digestion of mucin was apparent by molecular-sieve HPLC as early as 2 h and was essentially complete by 48 h.
l-Aniline-&naphthaJene
Protease-Digested
Sulfonate
Binding
to
Mucin
Digestion of mucin with trypsin, chymotrypsin, and pepsin resulted in a shift of the elution of glycoprotein from the void volume to the included volume of the Sepharose CL-2B column following proteolysis (not shown). After exopeptidic digestion with leucine amino-peptidase and carboxypeptidases A and Y, no alteration in the elution profile of glycoprotein from the Sepharose CL-2B column was noted (not shown). Digestion of bovine gallbladder mucin with trypsin, chymotrypsin, and pepsin resulted in a >7O% decrease in the binding of ANS to mucin as gauged by the decrease in relative fluorescence of ANS compared with the native mucin [Figure 2). Carboxypeptidases A and Y caused no change in ANS-mucin binding as gauged by ANS fluorescence. Leucine aminopeptidase digestion, on the other hand, caused an increase in ANS-mucin binding compared with native mucin (Figure 2).
Anion-Exchange
Chromatography
The elution of mucin tryptic peptides contained in peak III (Figure 1B) from the Protein Pak DEAE5PW is shown in Figure 3. Fractions A-G were collected and pooled as indicated. Peak II (Figure 1B) gave an identical elution profile but yielded a considerably lower quantity of peptides as gauged by absorbance at 229 nm.
1636 AFDHAL
GASTROENTEROLOGYVol.98.No.6
ET AL.
A
0.1~
0.05
-
I 0
/
I
I
I
I
I
I
I
I
B
O.l-
0.05
-
0 -C
ure 4). Each fraction from the anion-exchange column contained large-molecular-weight aggregates, a predominant low-molecular-weight peptide, and multiple intermediate-sized peptides. The electrophoretic pattern of the peptides from each anion-exchange fraction was nearly identical. The similarity of the peptides in anion-exchange fractions A-G was also reflected in the amino acid composition of these peptides [Table 1). Fractions A and B contained amino sugars, whereas the remainder did not. The absence of amino sugars in fractions C-F suggests that trypsin cleaved these peptides from nonglycosylated segments of the mucin peptide core. Compared with the amino acid composition of native mucin, the nonglycosylated peptides were significantly enriched in serine, glutamic acid plus glutamine, and glycine, and significantly depleted in threonine, proline, valine, leucine, and arginine. Reverse-Phase High-Performance Chromatography
Liquid
Given the apparent difficulty in peptide separation using anion exchange chromatography, peptide separation by RP-HPLC using a C-18 stationary
0.1
phase was attempted and Figure 5 shows the chromatogram obtained for fractions II, III, and IV (Figure 1B)
0.05 :
L
nl
I
I
I
I
I
I
I
I
D
O.l-
P
Aminopeptadase
0.05Carboxypeptldase
O-
I 10
I 20
A
Carboxypeptfdase
30
40
50
60
70
80
Time (minutes)
Figure 1. Molecular-sieve HPLC of protease-digested bovine gallbladder mucin. Purified bovine gallbladder mucin was digested with either trypsin, chymotrypsin, or pepsin for 72 h as described. After proteolytic digestion, mucin structure was analyzed by molecular-sieve HPLC using Bio-Gel TSK-60 (7.5 mm x 300 mm) and Bio-Sil TSK-400 (7.5 mm x 600 mm) columns connected in series. Chromatography was performed in 0.01 M Tris HCI with 0.05 M NaCl (pH 6.6) at a flow rate of 1 ml/min. Absorbance of the column effluent was monitored at 226 nm, and l-ml fractions were collected. A, native mucin; B, trypsin-digested mucin; C, chymotrypsin-digested mucin; D, pepsin-digested mucin. Column fractions of trypsin-digested mucin were pooled as indicated in B (fractions I-IV) and stored at -20% for further analysis.
Sodium DodecyJ Sulfate-PoJyacryJamide Electrophoresis
Gel
The tryptic peptides in fractions A-G (Figure 3) showed anomalous electrophoretic behavior through SDS-PAGE, each fraction showing multiple peptides on silver staining that appeared as a stepladder (Fig-
10
20
WW.
30
40
PM
Figure 2. Binding of ANS to protease-digested mucin. After protease digestion, mucin glycopeptides were repurified by gelfiltration chromatography using a Sepharose 2B column (80 cm x 1.5 cm] eluted with 0.2 M NaCl at a flow rate of 2 ml/min. Glycoprotein in the column effluent was detected by the periodic acid-Schiff assay (15), and glycopeptide fractions were collected, pooled, dialyzed against deionized water for 48 h at 4%, and lyophilized. Fluorescence spectoscopy was performed by mixing mucin glycopeptides (400 ag] with increasing concentrations of ANS (2-40 PM) in a final volume of 1 ml 0.01 M Tris buffer (pH 7.0). Relative fluorescence was measured at an excitation wavelength of 365 nm and an emission wavelength of 475 nm.
GALLBLADDER
Tune 19!)0
MUCIN
PEPTIDES
1637
Covalent Binding of Bilirubin to Mucin I
i
fi a
0.04
I
oL-=-7
10
I
Bilirubin was covalently bound to purified mutin after derivitization with Woodward’s reagent K. Sepharose CL-2B chromatography separated the mutin-bilirubin complex from unbound bilirubin (Figure 7). The mucin-bilirubin complex was recovered in the void volume of the Sepharose CL-2B column with an apparent mol wt >2 x 106. Tryptic digestion of the mucin-bilirubin complex was performed exactly as described for native gallbladder mucin. The elution profile of the trypsin-digested mucin-bilirubin complex after molecular-sieve HPLC was identical to that shown in Figure 1B. Mucin-bilirubin peptides that
/’ ,’
-
I----_ 20
/’
, 30
’
,
I
40
50
’
Mr
A
B
C
D
E
F
Time (minutes) Figure 3. Anion-exchange chromatography of tryptic mucin peptides. Mucin tryptic peptides in peak III of Figure 1B were dialyzed against deionized water for 48 h at 4OC. Anion-exchange HPLC was performed with a Protein Pak DEAE 5PW anion exchange column (7.5 mm x 75 mm]. Samples were loaded in 30 mM KH,PO, (pH 7.0) buffer and eluted with a curvilinear gradient over 1 h at a flow rate of 1 ml/min. The elution gradient was from 100% 30 mM KH,PO, (pH 7.0) to 100% 30 mM KH,PO, and 1 M NaCl (pH 3.0). Absorbance was monitored at 229 nm, and fractions (1 ml) were collected as indicated (fractions A-G) and pooled for subsequent amino acid analysis and SDS-PAGE.
from t‘he C-18 column. As with anion exchange chromatography, very similar elution profiles of these fractions were obtained with RP-HPLC. The major peaks designated as A, B, and c in Figure 5B were collected, pooled, and subjected to amino acid analysis (Table 2). Amino sugars were not present in the peptides purified by RP-HPLC, and these peptides were significantly enriched in serine, glutamic acid plus glutamine, and glycine, and depleted in threonine, proline, valine, and arginine. Attempts to further purify these peptides to homogeneity by molecularsieve IHPLC or by repeated RP-HPLC under isocratic conditions were unsuccessful. Figure 6 shows the molecular-sieve HPLC elution profile of fraction c from F:igure 5B after this fraction had further purified by RP-HPLC using isocratic elution. The major peptide elutes with an apparent molecular weight of approximately 19,000, but several smaller peptides were resolved as well. Because of the persistent aggregation of these peptides in a variety of solvents and the small quantities of peptide available, no tryptic peptide was purified to homogeneity in sufficient quantities to obtain an amino acid sequence from the nonglycosylated portion of the mucin protein core.
92,500
66,200
45,000
31,000
i
Figure 4. Polyacrylamide-gel electrophoresis of tryptic mucin peptides purified by anion-exchange chromatography. Fractions A-F from the anion exchange column (Figure 3) were diluted 1:4 in a sample buffer containing 0.125 M Tris-HCl (pH 6.8),10% glycerol (wt/vol), 2% SDS (wt/vol], 0.1 M 2-mercaptoethanol, and 0.004% bromophenol blue (wt/vol) and analyzed by SDSPAGE according to the method of Laemmli (16). Electrophoresis was performed using a stacking gel of 4% acrylamide and 1% SDS in 0.125 M Tris (pH 6.8) and a separating gradient gel that was 5%-30% acrylamide and 1% SDS in 0.375 M Tris (pH 8.8). Peptides were detected by silver staining. The positions of molecular weight standards are shown at left.
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Table 1. Amino Acid Composition of Mucin Tryptic Peptides Purified by Anion-Exchange Chromatography Residues Amino Asx Thr Ser Glx Pro GIY Ala CYS Val Met Be Leu Tyr Phe His LYS Arg Peptides Peptides
acid
per 1000 amino
acids
A
B
C
D
E
F
73 45 200 158 30 200 77 6 32 4 21 36 21 17 30 28 20
75 49 197 156 29 195 75 7 34 5 22 38 27 17 29 29 20
78 45 211 167 25 203 68 0 31 5 21 35 23 17 25 26 16
78 44 201 165 25 210 70 0 30 4 22 37 23 18 25 27 19
95 53 260 203 27 219 84 0 40 4 22 34 25 18 28 26 14
80 43 206 179 24 217 78 0 30 4 16 32 10 16 21 23 22
isolated by anion exchange chromatography are identified by same letters as in Figure 3.
as described.
eluted in peak III were further examined by RPHPLC under operating conditions identical to those shown in Figure 5. The major bilirubin-binding peptide eluted with a similar retention time to peak C in Figure 5B. Figure 8 shows the elution profile of this peptide from a Bio-Sil TSK 125 column. Identical aliquots were chromatographed, and column eluates were monitored either at A,,, for the detection of protein [Figure 8A) or at A,,, for the detection of bilirubin (Figure 8B). The elution of the mucinbilirubin peptide was retarded from the Bio-Sil TSK 125 column compared with the elution of similar peptides in Figure 6. Nonspecific interaction of the bilirubin with the column may account for the difference in apparent molecular weight as determined by chromatography. The amino acid composition of the peptides that bind bilirubin are shown in Table 2 and are similar to that of the nonglycosylated tryptic peptides that were purified by anion-exchange chromatography (Table 1) and RP-HPLC (Table 2) after trypsin digestion of native mucin. Discussion
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mucus is believed to provide a barrier that retards the diffusion of detergents such as bile salts to the mucosal surface of the gallbladder. Current knowledge of gallbladder mucin structure is limited. In this study, gallbladder mucin structure was examined by the isolation and characterization of tryptic peptides from the protein core. Endopeptidases with different peptide-bond specificities disaggregated the mucin polymer (Figure 1) into large-molecular-weight glycopeptides and lowmolecular-weight nonglycosylated peptides. Furthermore, endopeptidase digestion of mucin decreased the hydrophobic binding of the fluorescent ligand ANS to mucin (Figure 2), indicating that the hydrophobic domains that bind ANS are internally located on the mucin peptide core. Digestion of mucin with carboxypeptidases A and Y did not alter the mucin polymer structure or decrease the binding of ANS. Leucine aminopeptidase, an amino-terminal-specific peptidase, caused an increase in the binding of ANS to mucin. A definitive interpretation of the effect of exopeptidase digestion on ANS binding cannot be made based on the current data, but the findings are consistent with the location of hydrophobic domains on an interior segment of the mucin peptide core. The increase in ANS-mucin binding observed after leutine aminopeptidase digestion suggests that aminoterminal digestion of mucin facilitates ANS binding by removal of the N-terminus of the molecule. Previous attempts to identify the N-terminal amino acid in Table 2. Amino Acid Composition
of Native Mucin, Tryptic Peptides Purified by Reverse-Phase High-Performance Liquid Chromatography, and the Mucin-Bilirubin Peptide Residues
Amino Asx Thr Ser Glx Pro GIY Ala Val CYS Met Ile Leu
The structure of gallbladder mucin is of interest because of the physiologic functions of mucus in health and the pathophysiologic role of mucus in
‘W
gallstone disease. The gallbladder epithelium is exposed to potentially cytotoxic detergents during the interprandial state, and the mechanisms responsible for mucosal protection are unknown. Gallbladder
A%
Phe LYS His
Peptides described in Figure
acid
per 1000 amino
acids
NM
A
B
C
MB
97 147 101 126 76 127 76
73 45 155 138
82 45 183 149
73 38 176 146
68 42 205 134
220 71 96
214 73 42
146 68 27
206 86 27
40 25 51
47 25 59
85
51 28
28 20 41 22 22 38 28 28
21 24 55 19 23 46 24 38
33 18 31 18 ‘18 57 18
isolated by RP-HPLC are identified as A, B, and C as in Figure 5, and MB is the mucin-bilirubin peptide shown 6. NM, native mucin.
]une
1990
GALLBLADDER MUCIN PEPTIDES
1639
-A
0.04
_
0.03, a
Figure 5. Tryptic-mucin peptides after RPHPLC. After preparative molecular-sieve HPLC, mucin tryptic peptides in fractions II (A), III (B), and IV(C) from the chromatograph shown in Figure 1B were analyzed by RPHPLC using a Bio-Rad Hi-Pore C-18 column (7.5 mm x 150 mm). Mucin tryptic peptides were not dialysed before RP-HPLC. Samples were loaded in 0.01% TFA in water and eluted with a linear gradient over 90 min at a flow rate of 1 ml/min. The elution gradient went from 100% 0.01% TFA in water to 100% 0.01% TFA in 80% acetonitrile and 20% water (vol/ vol). Absorbance was monitored at 229 nm, and fractions (1 ml) A, B, and c were collected and pooled for further analysis.
w 0.02
-
0
bovine gallbladder mucin by dansylation have been unsuccessful, suggesting that the amino terminus of gallbladder mucin is blocked (Yee and Smith, unpublished observations]. The low-molecular-weight tryptic peptides were predominantly nonglycosylated, and apparent aggregation prevented characterization of their size and primary structure. The similarity of the gel-electrophoretic pattern and amino acid composition of the peptides fractionated by anion-exchange chromatography suggests that they aggregate under both native and denaturing conditions. The aggregation of these peptides was also evident after RP-HPLC (Figure 5) and analytic molecular-sieve HPLC (Figure 6). Compositional analysis of the tryptic peptides fractionated by both anion-exchange chromatography and RP-HPLC showed an enrichment of serine, glutamate plus glutamine, and glycine, and a relative depletion in threonine and proline compared with native mucin (10). The relative depletion of the tryptic peptides of gall-
!I I,10
20
30
40 lime
I 50
I
60
i--L 70
80
.ti
90
100
(minutes)
bladder mucin in threonine and proline suggests that these amino acids reside primarily on nonglycosylated segments of the protein core. A recently published partial sequence of a human intestinal mucin complementary deoxyribonucleic acid (cDNA) showed tandem repeating sequences of 23 amino acids on the glycosylated portion of the molecule containing 14 threonine and 5 proline residues in each repeat sequence (20). In addition, the 3’terminus of intestinal mucin contained a nonrepeating sequence that had a significantly different amino acid composition than the tandem repeats on the glycosylated segment of the molecule. Although the full primary structure of intestinal and gallbladder mucin have not been determined, there seems to be significant heterogeneity in the structure and amino acid composition of their protein cores. It is attractive to speculate that nonglycosylated peptides such as those resulting from trypsin digestion separate the repetitive glycosylated regions of the mucin molecule. Based on
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0-J
10
20
30
40
50
Time (minutes) Figure 6. Tryptic peptide C (Figure 5B) was further purified by isocratic RP-HPLC and eluted as a single peak from the C-16 column. This peptide was then analyzed by molecular-sieve HPLC using a Bio-Sil TSK-125 column (7.5 mm x 900 mm) eluted with 0.01M Tris HCl, 0.05 M NaCl (pH 6.5) at a flow rate of 1 ml/min. Absorbance was monitored at 229 nm. Arrows indicate elution of molecular weight standards.
quantitative amino acid analysis, it can be estimated that protease-sensitive, nonglycosylated segments account for approximately 10% of the gallbladder mucin protein core.
An additional aim of the present study was to identify the areas on the mucin molecule that bind the hydrophobic fluor ANS (lo), bilirubin (Zl), and biliary lipids (23). Previous reports from this laboratory had indicated that these ligands bound to proteasesensitive areas of the mucin core because the large glycopeptides resulting from proteolytic digestion of mucin were devoid of the hydrophobic-binding properties of the native molecule. Bilirubin was derivatized with Woodward’s reagent K and covalently bound to mucin at its presumed native binding domain in a manner similar to that used to study bilirubin binding to albumin (18) and glutathione-S-transferase (19). After partial purification by preparative molecular-sieve HPLC and RP-HPLC, the major tryptic peptide that had covalently bound bilirubin eluted from an analytical molecular-sieve column as a lowmolecular-weight peptide (Figure 81. The amino acid composition (Table 2) and the lack of amino sugars in this peptide indicate that it was derived from the nonglycosylated portion of the mucin protein core. The hydrophilic mucous gel acts as a diffusion barrier by creating an unstirred water layer at the epithelial surface. The present data suggest that nonglycosylated portions of the mucin protein core may nonspecifically absorb hydrophobic substances that are potentially toxic to the underlying epithelial cell
Vol. 98. No. 6
and, thereby, provide an additional protective function for mucous gel. The hydrophobic-binding properties of gastric mucin have also been described recently, and they were linked to the protective function of gastric mucus (22,231. Further elucidation of gallbladder mucin structure and function requires detailed structural knowledge of the mucin momomer. Attempts to obtain primary structure data on the mucin core have been unsuccessful because of the small quantities of nonglycosylated peptide that could be recovered and the tendency of these peptides to aggregate during both chromatography and gel electrophoresis. Detailed study of mucin structure by the standard techniques of protein chemistry is hampered by the unique structural and physical properties of mucin. An alternate strategy will be the use of nonglycosylated tryptic peptides to raise antibodies to the mucin protein core. The predominant antigenicity of native mucin resides in oligosaccarhide side chains that constitute the bulk of the molecule. Antibodies raised against native mucin have not identified the mucin protein core in in vitro translation studies using gallbladder epithelial cell messenger RNA (Turner and LaMont, personal communication, July 1989). This finding indicates that polyclonal antibodies raised against the densely glycosylated native mucin may lack reactivity toward the mucin protein core. Antibodies raised against nonglycosylated tryptic peptides should be directed against epitopes present on the
0.2
I aP
0.2
%
t
4
0.1
0.1
10
20
30
d
40
Fractions
Figure 7. Covalent binding of bilirubin to mucin. Bilirubin was derivatized with N-ethyl&phenylisoxazlium-3’-sulfonate (Woodward’s reagent K) as described. Lyophilized mucin (60 mg] was reconstituted in 23 ml of 0.01 M sodium phosphate, 0.145 M NaCl (pH 7.4). The BW complex was suspended in 5 ml water, and 1.6 ml was mixed with the mucin solution and incubated at 20% for 2 h under N, in the dark. Ethylenediaminetetraacetic acid was added to obtain a final concentration of I mM, and the pH was brought to 6.4 by the addition of 700 mg of imidazole. After an additional 6 h of incubation in the dark at 2o°C, the solution was chromatographed on a Sepharose 2B-CL column (66.0 cm x 2.5 cm) eluted with 0.1 M sodium phosphate, 0.145 M NaCl (pH 7.4) at a flow rate of 42 ml/h. Fractions (12 ml) were collected and monitored at A,,, for protein and at A,,, for bilirubin.
June 1990
oi
GALLBLADDER MLJCIN PEPTIDES
I
I
’
10
20
I
30
40
50
IL60
Time (minutes) Figure 5. Isolation of a bilirubin-binding tryptic mucin peptide. The mucin-BW complex that eluted in the void volume of a Sepbarose ZB-CL column (Figure 7) was digested with trypsin for 24 b as described and subjected to molecular-sieve HPLC as described in legend to Figure 1. Tryptic peptides eluting in the same position as those indicated as fraction III in Figure 1B were collected and analyzed by RPNPLC as described in Figure 5 legend. A peptide eluting with a retention time identical to that of peak c in Figure 58 was collected and analyzed by molecular-sieve HPLC using a Bio-Rad TSK-125 column (7.5 mm x 300 mm) eluted with 0.01 M Tris Hcl and 0.05 M NaCl (pH 6.5) at a flow rate of 1 ml/min. Fractions were monitored at A,,, (A] for the detection of protein and at A,,,(B) for detection of bilirubin.
mucin
protein
cDNA library
core
and
may
of the gallbladder
be useful
to screen
a
epithelium.
References Levy PF, Smith BF, LaMont JT. Human gallbladder mucin accelerates in vitro nucleation of cholesterol in artificial bile. Gastroenterology 1984;87:270-275. Smith BF. Human gallbladder mucin binds biliary lipids and promotes cholesterol crystal nucleation in model bile. J Lipid Res 1987;28:1088-1097. Lee TJ. Smith BF. Bovine gallbladder mucin accelerates cholesterol crystal nucleation from cholesterol enriched vesicles in supersaturated model bile. J Lipid Res 1989;30:491-498.
1641
4. Lee SP, LaMont JT, Carey MC. The role of gallbladder mucus hypersecretion in the evolution of cholesterol gallstones: studies in the prairie dog. J Clin Invest 1981;67:1712-1723. 5. Lee SP, Carey MC, LaMont JT. Aspirin prevention of cholesterol gallstone formation in prairie dogs. Science 1981;211:14291432. 6. MacPherson BR, Pemsingh RS, Scott GW. Experimental cholelithiasis in the ground squirrel. Lab Invest 1987;56:138-145, 7. Messing B, Boires C, Ku&linger F, Bernier JJ. Dooes total parenteral nutrition induce gallbladder sludge formation and lithiasis? Gastroenterology 1983;84:1012-1019. 8. Hulten 0. Formation of gallstones. II. Acta Chir Stand 1968;134: 557-560. 9. Lee SP, Nichols JF. The nature and composition of biliary sludge. Gastroenterology 1986;90:677-686. 10. Smith BF, LaMont JT. Hydrophobic binding properties of bovine gallbladder mucin. J Biol Chem 1984;259:12170-12177. 11. Smith BF, Peetermans JA, Tanaka T, LaMont JT. Subunit interactions and physical properties of bovine gallbladder mutin. Gastroenterology 1989;97:179-187. 12. Mitz MA, Schlueter RJ. Direct spectrophotometric measurement of the peptide bond: applications to the determination of acylase I. Biochem Biophys Acta 1958;27:168-177. 13. Folk JE, Schirmer EW. The porcine pancreatic carboxypeptidase A system. J Biol Chem 1963;238:3884-3889. 14. Kuhn RW. Reaction of yeast carboxypeptidase C with groupspecific reagents. Biochemistry 1976;15:4881. 15. Mantle M, Allen A. A colometeric assay for glycoproteins based on the periodic acid/Schiff stain. Biochem Sot Trans 1978:6:607609. 16. Laemmli UK. Cleavage of the structural proteins during assembly of the head of bacteriophage T,. Nature 1970;227:680-685. 17. McDonagh AF, Assisi F. The ready isomerization of bilirubin IXa in aqueous solution. Biochem J 1972;129:797-800, N, Wilson KJ. Affinity 18. Kuenzle CC, Gitzelmann-Cumarasamy labeling of the primary bilirubin binding site of human serum albumin. J Biol Chem 1976;251:801-807. ligand19. Boyer TD. Covalent labeling of the non-substrate binding site of glutathione-S-transferase with bilirubin-woodward’s reagent K. J Biol Chem 1986;261:5363-5367. 20. Gum JR, Byrd JC, Hicks JW, Toribara NW, Lamport DTA, Kim YS. Molecular cloning of human intestinal mucin cDNAs. Sequence analysis and evidence for genetic polymorphism. J Biol Chem 1989; 264:6480-6487. 21. Smith BF, LaMont JT. Bovine gallbladder mucin binds bilirubin in vitro. Gastroenterology 1983;85:707-712. 22. Sarosiek J, Piotrowski J, Gabryelewicz A, Slomiany A, Slomiant BL. Alterations in mucin hydrophobicity and molecular form distribution with peptic ulcer (abstr). Gastroenterology 1989;96: A441. 23. Slomiany BL, Nishikawa, Slomiany A. Gastric mucin hyrophobicity: effects of proteolysis, reduction and lipid removal (abstr]. Gastroenterology 1989;96:A478.
Received August 2,1989. Accepted November 30,1989. Address requests for reprints to: Bernard F. Smith, M.D., Section of Gastroenterology, Thorndike 503. Boston City Hospital, 818 Harrison Avenue, Boston, Massachusetts 02118. Dr. Smith was supported by National Institutes of Health research grant DK39017, and Dr. Afdal was supported by NIH BRSQ RR 0556924.
Isolation and Characterization of Peptides From the Protein Core of Bovine Gallbladder Mucin NEZAM H. AFDHAL, GWYNNETH D. OFFNER, FRANCIS E. MURRAY, ROBERT F. TROXLER, and BERNARD F. SMITH Section of Gastroenterology and Hepatology, Boston City Hospital, and Department Biochemistry, Boston University School of Medicine, Boston, Massachusetts
Gallbladder mucin may promote cholesterol gallstone formation by accelerating cholesterol monohdrate crystal nucleation in supersaturated bile. In this study, peptides were isolated from the mucin protein core by protease digestion and molecularsieve high-performance liquid chromatography. Tryptic peptides were purified by anion exchange or reverse-phase high-performance liquid chromatography, and amino acid compositions were determined. Tryptic peptides were (a) nonglycosylated, (b) selectively enriched in serine, glutamic acid plus glutamine, and glycine, and (c) depeleted in threonine and proline compared with native gallbladder mutin. Bilirubin derivatized with Woodward’s reagent K covalently bound to purified mucin. Tryptic digestion of the mucin-bilirubin complex yielded lowmolecular-weight nonglycosylated peptides with covalently bound bilirubin. These data indicate that the mucin protein core contains at least two distinct domains. One domain is rich in threonine and proline and contains the majority of covalently bound carbohydrate. A second domain, possibly internally located, is nonglycosylated, enriched in serine, glutamic acid plus glutamine, and glycine, and binds hydrophobic ligands such as bilirubin and l-anilino8-naphthalene sulfonate. Hydrophobic domains on the mucin protein core may contribute to the pathogenesis of cholesterol cholelithiasis.
M
ucin is the major secretory product of the gallbladder epithelium and is the principal organic constituent of mucous gel. Recent evidence from this laboratory (l-31 and others (4-6) indicates that gallbladder mucin is a critical element in the pathogenesis of cholesterol cholelithiasis. Mucin hypersecretion precedes gallstone formation in both
of
experimental animals (4-6) and humans (7,8) and results in the accumulation of a thick mucous gel adherent to the gallbladder epithelium. Nucleation of cholesterol monohydrate crystals, the initial event in cholesterol stone formation, occurs within the mucous gel (1,8,9). Moreover, inhibition of mucin secretion with aspirin prevents gallstone formation in experimental animals (5). It has been shown that gallbladder mucin can bind biliary lipids and accelerate the nucleation of cholesterol monohydrate crystals in supersaturated model bile (2,3). The structural integrity of the protease-sensitive areas on the mucin peptide core is essential for the acceleration of cholesterol crystal nucleation by mucin in model bile (2). Mucin is a densely glycosylated macromolecule that consists of a central peptide core accounting for 16% of its weight and a covalently bound carbohydrate accounting for approximately 76% of its weight (10). Extensive posttranslational glycosylation of mucin results in molecular heterogeneity with respect to size and polydispersity with respect to charge. Mucin monomers aggregate in aqueous solution and polymerize via both electrostatic and hydrophobic interactions 111). Mucin viscosity increases above a concentration of 2.5 mg/ml, and gel formation occurs at concentrations of 20-30 mg/ml (11). In this study, the structure of the protein core of bovine gallbladder mucin has been examined. The
Abbreviations used in this paper: ANS, I-anilino-g-naphthalene sulfonate; BW, bilirubin-Woodward’s reagent K; cDNA, complementary deoxyribonucleic acid; HPLC, high-performance liquid chromatography; RP-HPLC, reverse-phase high-performance liquid chromatography; SDS-PAGE, sodium dodecyl sulfate-polyacrylamide gel electrophoresis; TFA, trifluoroacetic acid. 0 1990 by the American Gastroenterological Association 0016-5065/90/$3.00
GASTROENTEROLOGY Vol. 98,No. 6
1634 AFDHAL ET AL.
results indicate that nonglycosylated areas hydrophobically bind bilirubin and the fluourescent ligand l-anilino-8-naphthalene sulfonic acid (ANS) (10). These areas are internally located on the mucin peptide core and have an amino acid composition that is distinct from the glycosylated segments of the mucin peptide core. Materials and Methods Purification
of Gallbladder
Mucin
Mucin was purified from bovine gallbladders obtained fresh at a local abbatoir (A. Arena and Sons, Hopkinton, Mass.]. Gallbladder mucus was scraped from the epithelial surface with a glass slide and homogenized for 3 min in a Waring blender in 0.2 M NaCl. Following centrifugation at 50,000 xg for 30 min, soluble mucin was purified as previously described (3,10,11] by Sepharose CL-4B (Sigma Chemica! Co., St. Louis, MO.) exclusion gel chromatography and two sequential equilibrium-density gradient ultracentrifugations in 60% cesium chloride. Purified mucin is free of low-molecular-weight protein, glycoprotein, and lipid contaminants (lo], but remains polydisperse with respect to size and charge.
Proteolytic
Digestion
For trypsin and chymotrypsin digestion, lyophilized mucin (10mg] was dissolved in 2 ml of 0.05 mM ammonium bicarbonate, 0.01 M CaCI,, and 0.04% sodium azide (pH 8.0) containing 0.1 ml of penicillin-streptomycin (1000 U + 1000 for 12 h at 37°C and wg-' * ml-‘). Mucin was incubated vortexed periodically to solubilize the mucin beffre digestion. Trypsin [type II, treated with phenylmethyl-sulfonylfluoride to inactivate chymotrypsin; Sigma Chemical Co., St. Louis, MO.) was added to mucin in a ratio of 1:lOO (enzymemucin, wt/wt] and incubated for either 24, 48, or 72 h at 37°C. Trypsin activity was terminated by adding 0.1% trifluoroacetic acid (TFA] to obtain pH 1.0 and freezing at -20°C. Chymotrypsin [type VII, treated with Na-p-tosyl+lysine chloromethyl ketone to inactivate trypsin; Sigma] was added to mucin (10 mg) in 0.08 M Tris, 0.01 M CaCl, (pH 7.8) at a ratio of 1:lOO (enzyme-mucin, wt/wt), and digestion was performed under conditions identical to those described for trypsin. For digestion with pepsin [Sigma], lyophilized mucin (10 mg) was reconstituted in 2 ml of a buffer of 0.08 M Tris HCl, 0.01 M CaCl, (pH 1.0) and incubated with pepsin (pepsin A, Sigma) at an enzyme-mucin ratio of 1:lOO (wt/wt) for either 24, 48, or 72 h at 37°C. Pepsin activity was terminated by the addition of 0.1 M sodium hydroxide to pH 8.0 and freezing at -20°C. Leucine aminopeptidase (5 mg; Worthington Biochemical, Freehold, N.J.) was dissolved in 1 ml of 0.14 M trimethylamine, 0.002 M MgCl, (pH 8.5) and incubated for 12 h at 4°C with diisopropyl-fluorophosphate to remove contaminating tryptic or chymotryptic activity. Enzyme activity was confirmed by the spectrophotometric method of Mitz and
Schlueter (12). Lyophilized mucin (10 mg] was reconstituted in 5 ml of the same buffer and incubated at an enzymemucin ratio of 1:20 (wt/wt] for either 24, 48, or 72 h at 37°C. Leucine aminopeptidase activity was terminated by acidification to pH 1.5 and freezing at -20°C. Carboxypeptidase A [Worthington) was prepared as a 1 x lo-‘-g/ml stock solution, and enzyme activity was confirmed by the reaction-velocity method of Folk and Schirmer (13) using hippuryl-L-phenylalanine as substrate. Lyophilized mucin (10 mg) was reconstituted in 10 mM Tris (pH 8.0) and incubated as previously described. Carboxypeptidase Y (Worthington] was prepared as a 2 x IO-‘-g/ml stock solution, and activity was confirmed using the enzymatic hydrolysis method of Kuhn (14). Lyophilized mucin (10 mg] was dissolved in 10 mM Tris (pH 6.0) and incubated as previously described.
High-Performance
Liquid Chromatography
Gallbladder mucin-derived peptides were analyzed by high-performance liquid chromatography (HPLC) using a Waters system with monitoring of column eluates at 229 nm (for peptides) an in some experiments at 436 nm [for bilirubin). Analytic separation of mucin peptides by molecular sieve was performed either on Bio-Gel TSK-60 and Bio-Sil TSK-400 columns connected in series or on a Bio-Sil TSK-125 column (Bio-Rad, Richmond, Calif.). Following preparative molecular-sieve HPLC, mucin tryptic peptides were dialyzed against deionized water at 4’C for 48 h, and anion exchange chromatography was performed on a Protein Pak DEAE 5PW anion-exchange column (Waters, Milford, Mass.]. Selected fractions were collected and pooled for subsequent amino acid analysis and sodium dodecyl sulfate-polyacrylamide gel electrophoresis [SDS-PAGE]. Mucin tryptic peptides were also analyzed directly without prior dialysis by reverse-phase HPLC (RP-HPLC) using a Bio-Rad Hi-Pore C-18 column. Selected peptides were further purified by RP-HPLC run isocratically at a concentration of the eluting solvent (0.01% TFA in 80% acetonitrile and 20% water) that was 16% less than the concentration at which the peptide originally eluted from the reverse-phase column.
l-Aniline-8-naphthalene Studies
Sulfonate-Binding
The hydrophobic binding properties of mucin were studied by fluorescence spectroscopy, which examined the binding of the hydrophobic fluorescent probe ANS to native and protease-digested mucin as previously described (10). After protease digestion, mucin glycopeptides were purified by gel-filtration chromatography using a Sepharose 2B column. Glycoprotein in the column effluent was detected by the periodic acid-Schiff assay (15], and glycopeptide fractions in the included volume were collected, pooled, dialyzed against deionized water for 48 h at 4”C, and lyophilized. Fluorescence spectoscopy was performed on a Perkin-Elmer Model LS-3B fluorimeter by mixing mucin glycopeptides (400 wg) with increasing concentrations of ANS (2-40 wM) in a final volume of 1 ml in 0.01 M Tris (pH
GALLBLADDER
June 1990
7.0). Relative fluorescence was measured wavelength of 365 nm and an emission nm.
PoJyacryJamide
using an excitation wavelength of 475
Gel Electrophoresis
Peptides separated by anion-exchange chromatography were analyzed by SDS-PAGE according to the method of Laemmli (IS] using a Bio-Rad Protean II slab-gel system with a acrylamide stacking gel and a 5%-30% acrylamide separating gel. Protein was detected by silver staining. 4%
Amino
Acid Analysis
Peptides were hydrolyzed in 6 N HCl in vacua at for 22 hours and analysed on a Beckman System 6300 amino acid analyzer. 110°C
Covalent
Binding
of Bilirubin
to Mucin
Bilirubin (Porphyrin Products, Logan, Utah] was purified to isolate the IXa isomer (17) and then derivatized with N-ethyl-5-phenylisoxazlium-3’-sulfonate (Woodward’s reagent K] according to the method of Kuenzle et al. (18). Bilirubin (116.8 mg; 0.2 mM] was incubated with Woodward’s reagent K (151.8 mg; 0.6 mM) and stirred at 20°C for 45 min in 20 ml of acetonitrile containing triethylamine (0.3 ml; 2.0 mM]. The mixture was evaporated to dryness at 3O”C, resuspended in 5 ml water, and chromatographed on a Sephadex G-25 column (50 cm x 1.0 cm] eluted with water at 40 ml/min. The bilirubin-Woodward’s reagent K complex (BW) was recovered in the third peak to elute from the column that was orange. The BW was lyophilized and stored in the dark at -20°C until further use. The BW was covalently bound to purified mucin by a modification of the method described by Kuenzle et al. for (18) and by Boyer for glutathione serum albumin S-tran:sferase (19). Lyophilized mucin (90 mg) was reconstituted in 23 ml of 0.01 M sodium phosphate and 0.145 M NaCl (pH 7.4). The BW was suspended in 5 ml water, and 1.6 ml was mixed with the mucin solution and incubated at 20°C for 2 h under N, in the dark. Ethylenediaminetetraacetic acid was added to obtain a final concentration of 1 mM, and the pH was brought to 9.4 by the addition of 700 mg of imidazole. After an additional 8 h of incubation in the dark at 20”C, the solution was chromatographed on a Sepharose CL-2B column (86.0 cm x 2.5 cm) and eluted with 0.1 M sodium phosphate with 0.145 M NaCl (pH 7.4) at a flow rate of 42 ml/h. Fractions (12 ml] were collected and monitored at A,,,, for protein and A,,, for bilirubin. Void volume fractions containing mucin and covalently bound BW were dialyzed against water in the dark at 4°C for 48 h and lyophilized. The mucin-BW complex (10 mg] was digested with trypsin for 24 h, and mucin peptides were separated by molecular sieve and RP-HPLC as previously described. Selected peptides were further fractionated by molecular-sieve HPLC using a Bio-Rad TSK-125 column.
MUtXN
PEPTIDES
1635
Results Protease Digestion Chromatography
and Molecular-Sieve
Endopeptidase digestion of purified bovine gallbladder mucin with trypsin, chymotrypsin, and pepsin yielded similar elution profiles through molecularsieve HPLC (Figure 1). Proteolysis resulted in the appearance of a second, well-defined, large-molecularweight peak that eluted just after the void volume peak. In addition, small poorly defined peaks were present in the intermediateand low-molecularweight portions of the included volume. Column fractions of the trypsin digest [Figure 1B) were pooled as indicated, and the peptides in these fractions were examined by anion-exchange HPLC and RP-HPLC. The large peak at the bed volume represents absorbance at 229 nm by sodium azide, which was added to all digests at a concentration of 0.04%. The time course of enzymatic digestion was examined for trypsin at 2, 24, and 48 h (not shown]. Digestion of mucin was apparent by molecular-sieve HPLC as early as 2 h and was essentially complete by 48 h.
l-Aniline-&naphthaJene
Protease-Digested
Sulfonate
Binding
to
Mucin
Digestion of mucin with trypsin, chymotrypsin, and pepsin resulted in a shift of the elution of glycoprotein from the void volume to the included volume of the Sepharose CL-2B column following proteolysis (not shown). After exopeptidic digestion with leucine amino-peptidase and carboxypeptidases A and Y, no alteration in the elution profile of glycoprotein from the Sepharose CL-2B column was noted (not shown). Digestion of bovine gallbladder mucin with trypsin, chymotrypsin, and pepsin resulted in a >7O% decrease in the binding of ANS to mucin as gauged by the decrease in relative fluorescence of ANS compared with the native mucin [Figure 2). Carboxypeptidases A and Y caused no change in ANS-mucin binding as gauged by ANS fluorescence. Leucine aminopeptidase digestion, on the other hand, caused an increase in ANS-mucin binding compared with native mucin (Figure 2).
Anion-Exchange
Chromatography
The elution of mucin tryptic peptides contained in peak III (Figure 1B) from the Protein Pak DEAE5PW is shown in Figure 3. Fractions A-G were collected and pooled as indicated. Peak II (Figure 1B) gave an identical elution profile but yielded a considerably lower quantity of peptides as gauged by absorbance at 229 nm.
1636 AFDHAL
GASTROENTEROLOGYVol.98.No.6
ET AL.
A
0.1~
0.05
-
I 0
/
I
I
I
I
I
I
I
I
B
O.l-
0.05
-
0 -C
ure 4). Each fraction from the anion-exchange column contained large-molecular-weight aggregates, a predominant low-molecular-weight peptide, and multiple intermediate-sized peptides. The electrophoretic pattern of the peptides from each anion-exchange fraction was nearly identical. The similarity of the peptides in anion-exchange fractions A-G was also reflected in the amino acid composition of these peptides [Table 1). Fractions A and B contained amino sugars, whereas the remainder did not. The absence of amino sugars in fractions C-F suggests that trypsin cleaved these peptides from nonglycosylated segments of the mucin peptide core. Compared with the amino acid composition of native mucin, the nonglycosylated peptides were significantly enriched in serine, glutamic acid plus glutamine, and glycine, and significantly depleted in threonine, proline, valine, leucine, and arginine. Reverse-Phase High-Performance Chromatography
Liquid
Given the apparent difficulty in peptide separation using anion exchange chromatography, peptide separation by RP-HPLC using a C-18 stationary
0.1
phase was attempted and Figure 5 shows the chromatogram obtained for fractions II, III, and IV (Figure 1B)
0.05 :
L
nl
I
I
I
I
I
I
I
I
D
O.l-
P
Aminopeptadase
0.05Carboxypeptldase
O-
I 10
I 20
A
Carboxypeptfdase
30
40
50
60
70
80
Time (minutes)
Figure 1. Molecular-sieve HPLC of protease-digested bovine gallbladder mucin. Purified bovine gallbladder mucin was digested with either trypsin, chymotrypsin, or pepsin for 72 h as described. After proteolytic digestion, mucin structure was analyzed by molecular-sieve HPLC using Bio-Gel TSK-60 (7.5 mm x 300 mm) and Bio-Sil TSK-400 (7.5 mm x 600 mm) columns connected in series. Chromatography was performed in 0.01 M Tris HCI with 0.05 M NaCl (pH 6.6) at a flow rate of 1 ml/min. Absorbance of the column effluent was monitored at 226 nm, and l-ml fractions were collected. A, native mucin; B, trypsin-digested mucin; C, chymotrypsin-digested mucin; D, pepsin-digested mucin. Column fractions of trypsin-digested mucin were pooled as indicated in B (fractions I-IV) and stored at -20% for further analysis.
Sodium DodecyJ Sulfate-PoJyacryJamide Electrophoresis
Gel
The tryptic peptides in fractions A-G (Figure 3) showed anomalous electrophoretic behavior through SDS-PAGE, each fraction showing multiple peptides on silver staining that appeared as a stepladder (Fig-
10
20
WW.
30
40
PM
Figure 2. Binding of ANS to protease-digested mucin. After protease digestion, mucin glycopeptides were repurified by gelfiltration chromatography using a Sepharose 2B column (80 cm x 1.5 cm] eluted with 0.2 M NaCl at a flow rate of 2 ml/min. Glycoprotein in the column effluent was detected by the periodic acid-Schiff assay (15), and glycopeptide fractions were collected, pooled, dialyzed against deionized water for 48 h at 4%, and lyophilized. Fluorescence spectoscopy was performed by mixing mucin glycopeptides (400 ag] with increasing concentrations of ANS (2-40 PM) in a final volume of 1 ml 0.01 M Tris buffer (pH 7.0). Relative fluorescence was measured at an excitation wavelength of 365 nm and an emission wavelength of 475 nm.
GALLBLADDER
Tune 19!)0
MUCIN
PEPTIDES
1637
Covalent Binding of Bilirubin to Mucin I
i
fi a
0.04
I
oL-=-7
10
I
Bilirubin was covalently bound to purified mutin after derivitization with Woodward’s reagent K. Sepharose CL-2B chromatography separated the mutin-bilirubin complex from unbound bilirubin (Figure 7). The mucin-bilirubin complex was recovered in the void volume of the Sepharose CL-2B column with an apparent mol wt >2 x 106. Tryptic digestion of the mucin-bilirubin complex was performed exactly as described for native gallbladder mucin. The elution profile of the trypsin-digested mucin-bilirubin complex after molecular-sieve HPLC was identical to that shown in Figure 1B. Mucin-bilirubin peptides that
/’ ,’
-
I----_ 20
/’
, 30
’
,
I
40
50
’
Mr
A
B
C
D
E
F
Time (minutes) Figure 3. Anion-exchange chromatography of tryptic mucin peptides. Mucin tryptic peptides in peak III of Figure 1B were dialyzed against deionized water for 48 h at 4OC. Anion-exchange HPLC was performed with a Protein Pak DEAE 5PW anion exchange column (7.5 mm x 75 mm]. Samples were loaded in 30 mM KH,PO, (pH 7.0) buffer and eluted with a curvilinear gradient over 1 h at a flow rate of 1 ml/min. The elution gradient was from 100% 30 mM KH,PO, (pH 7.0) to 100% 30 mM KH,PO, and 1 M NaCl (pH 3.0). Absorbance was monitored at 229 nm, and fractions (1 ml) were collected as indicated (fractions A-G) and pooled for subsequent amino acid analysis and SDS-PAGE.
from t‘he C-18 column. As with anion exchange chromatography, very similar elution profiles of these fractions were obtained with RP-HPLC. The major peaks designated as A, B, and c in Figure 5B were collected, pooled, and subjected to amino acid analysis (Table 2). Amino sugars were not present in the peptides purified by RP-HPLC, and these peptides were significantly enriched in serine, glutamic acid plus glutamine, and glycine, and depleted in threonine, proline, valine, and arginine. Attempts to further purify these peptides to homogeneity by molecularsieve IHPLC or by repeated RP-HPLC under isocratic conditions were unsuccessful. Figure 6 shows the molecular-sieve HPLC elution profile of fraction c from F:igure 5B after this fraction had further purified by RP-HPLC using isocratic elution. The major peptide elutes with an apparent molecular weight of approximately 19,000, but several smaller peptides were resolved as well. Because of the persistent aggregation of these peptides in a variety of solvents and the small quantities of peptide available, no tryptic peptide was purified to homogeneity in sufficient quantities to obtain an amino acid sequence from the nonglycosylated portion of the mucin protein core.
92,500
66,200
45,000
31,000
i
Figure 4. Polyacrylamide-gel electrophoresis of tryptic mucin peptides purified by anion-exchange chromatography. Fractions A-F from the anion exchange column (Figure 3) were diluted 1:4 in a sample buffer containing 0.125 M Tris-HCl (pH 6.8),10% glycerol (wt/vol), 2% SDS (wt/vol], 0.1 M 2-mercaptoethanol, and 0.004% bromophenol blue (wt/vol) and analyzed by SDSPAGE according to the method of Laemmli (16). Electrophoresis was performed using a stacking gel of 4% acrylamide and 1% SDS in 0.125 M Tris (pH 6.8) and a separating gradient gel that was 5%-30% acrylamide and 1% SDS in 0.375 M Tris (pH 8.8). Peptides were detected by silver staining. The positions of molecular weight standards are shown at left.
1638
AFDHAL
GASTROENTEROLOGY
ET AL.
Table 1. Amino Acid Composition of Mucin Tryptic Peptides Purified by Anion-Exchange Chromatography Residues Amino Asx Thr Ser Glx Pro GIY Ala CYS Val Met Be Leu Tyr Phe His LYS Arg Peptides Peptides
acid
per 1000 amino
acids
A
B
C
D
E
F
73 45 200 158 30 200 77 6 32 4 21 36 21 17 30 28 20
75 49 197 156 29 195 75 7 34 5 22 38 27 17 29 29 20
78 45 211 167 25 203 68 0 31 5 21 35 23 17 25 26 16
78 44 201 165 25 210 70 0 30 4 22 37 23 18 25 27 19
95 53 260 203 27 219 84 0 40 4 22 34 25 18 28 26 14
80 43 206 179 24 217 78 0 30 4 16 32 10 16 21 23 22
isolated by anion exchange chromatography are identified by same letters as in Figure 3.
as described.
eluted in peak III were further examined by RPHPLC under operating conditions identical to those shown in Figure 5. The major bilirubin-binding peptide eluted with a similar retention time to peak C in Figure 5B. Figure 8 shows the elution profile of this peptide from a Bio-Sil TSK 125 column. Identical aliquots were chromatographed, and column eluates were monitored either at A,,, for the detection of protein [Figure 8A) or at A,,, for the detection of bilirubin (Figure 8B). The elution of the mucinbilirubin peptide was retarded from the Bio-Sil TSK 125 column compared with the elution of similar peptides in Figure 6. Nonspecific interaction of the bilirubin with the column may account for the difference in apparent molecular weight as determined by chromatography. The amino acid composition of the peptides that bind bilirubin are shown in Table 2 and are similar to that of the nonglycosylated tryptic peptides that were purified by anion-exchange chromatography (Table 1) and RP-HPLC (Table 2) after trypsin digestion of native mucin. Discussion
Vol. 98, No. 6
mucus is believed to provide a barrier that retards the diffusion of detergents such as bile salts to the mucosal surface of the gallbladder. Current knowledge of gallbladder mucin structure is limited. In this study, gallbladder mucin structure was examined by the isolation and characterization of tryptic peptides from the protein core. Endopeptidases with different peptide-bond specificities disaggregated the mucin polymer (Figure 1) into large-molecular-weight glycopeptides and lowmolecular-weight nonglycosylated peptides. Furthermore, endopeptidase digestion of mucin decreased the hydrophobic binding of the fluorescent ligand ANS to mucin (Figure 2), indicating that the hydrophobic domains that bind ANS are internally located on the mucin peptide core. Digestion of mucin with carboxypeptidases A and Y did not alter the mucin polymer structure or decrease the binding of ANS. Leucine aminopeptidase, an amino-terminal-specific peptidase, caused an increase in the binding of ANS to mucin. A definitive interpretation of the effect of exopeptidase digestion on ANS binding cannot be made based on the current data, but the findings are consistent with the location of hydrophobic domains on an interior segment of the mucin peptide core. The increase in ANS-mucin binding observed after leutine aminopeptidase digestion suggests that aminoterminal digestion of mucin facilitates ANS binding by removal of the N-terminus of the molecule. Previous attempts to identify the N-terminal amino acid in Table 2. Amino Acid Composition
of Native Mucin, Tryptic Peptides Purified by Reverse-Phase High-Performance Liquid Chromatography, and the Mucin-Bilirubin Peptide Residues
Amino Asx Thr Ser Glx Pro GIY Ala Val CYS Met Ile Leu
The structure of gallbladder mucin is of interest because of the physiologic functions of mucus in health and the pathophysiologic role of mucus in
‘W
gallstone disease. The gallbladder epithelium is exposed to potentially cytotoxic detergents during the interprandial state, and the mechanisms responsible for mucosal protection are unknown. Gallbladder
A%
Phe LYS His
Peptides described in Figure
acid
per 1000 amino
acids
NM
A
B
C
MB
97 147 101 126 76 127 76
73 45 155 138
82 45 183 149
73 38 176 146
68 42 205 134
220 71 96
214 73 42
146 68 27
206 86 27
40 25 51
47 25 59
85
51 28
28 20 41 22 22 38 28 28
21 24 55 19 23 46 24 38
33 18 31 18 ‘18 57 18
isolated by RP-HPLC are identified as A, B, and C as in Figure 5, and MB is the mucin-bilirubin peptide shown 6. NM, native mucin.
]une
1990
GALLBLADDER MUCIN PEPTIDES
1639
-A
0.04
_
0.03, a
Figure 5. Tryptic-mucin peptides after RPHPLC. After preparative molecular-sieve HPLC, mucin tryptic peptides in fractions II (A), III (B), and IV(C) from the chromatograph shown in Figure 1B were analyzed by RPHPLC using a Bio-Rad Hi-Pore C-18 column (7.5 mm x 150 mm). Mucin tryptic peptides were not dialysed before RP-HPLC. Samples were loaded in 0.01% TFA in water and eluted with a linear gradient over 90 min at a flow rate of 1 ml/min. The elution gradient went from 100% 0.01% TFA in water to 100% 0.01% TFA in 80% acetonitrile and 20% water (vol/ vol). Absorbance was monitored at 229 nm, and fractions (1 ml) A, B, and c were collected and pooled for further analysis.
w 0.02
-
0
bovine gallbladder mucin by dansylation have been unsuccessful, suggesting that the amino terminus of gallbladder mucin is blocked (Yee and Smith, unpublished observations]. The low-molecular-weight tryptic peptides were predominantly nonglycosylated, and apparent aggregation prevented characterization of their size and primary structure. The similarity of the gel-electrophoretic pattern and amino acid composition of the peptides fractionated by anion-exchange chromatography suggests that they aggregate under both native and denaturing conditions. The aggregation of these peptides was also evident after RP-HPLC (Figure 5) and analytic molecular-sieve HPLC (Figure 6). Compositional analysis of the tryptic peptides fractionated by both anion-exchange chromatography and RP-HPLC showed an enrichment of serine, glutamate plus glutamine, and glycine, and a relative depletion in threonine and proline compared with native mucin (10). The relative depletion of the tryptic peptides of gall-
!I I,10
20
30
40 lime
I 50
I
60
i--L 70
80
.ti
90
100
(minutes)
bladder mucin in threonine and proline suggests that these amino acids reside primarily on nonglycosylated segments of the protein core. A recently published partial sequence of a human intestinal mucin complementary deoxyribonucleic acid (cDNA) showed tandem repeating sequences of 23 amino acids on the glycosylated portion of the molecule containing 14 threonine and 5 proline residues in each repeat sequence (20). In addition, the 3’terminus of intestinal mucin contained a nonrepeating sequence that had a significantly different amino acid composition than the tandem repeats on the glycosylated segment of the molecule. Although the full primary structure of intestinal and gallbladder mucin have not been determined, there seems to be significant heterogeneity in the structure and amino acid composition of their protein cores. It is attractive to speculate that nonglycosylated peptides such as those resulting from trypsin digestion separate the repetitive glycosylated regions of the mucin molecule. Based on
1640
AFDHAL
GASTROENTEROLOGY
ET AL.
0-J
10
20
30
40
50
Time (minutes) Figure 6. Tryptic peptide C (Figure 5B) was further purified by isocratic RP-HPLC and eluted as a single peak from the C-16 column. This peptide was then analyzed by molecular-sieve HPLC using a Bio-Sil TSK-125 column (7.5 mm x 900 mm) eluted with 0.01M Tris HCl, 0.05 M NaCl (pH 6.5) at a flow rate of 1 ml/min. Absorbance was monitored at 229 nm. Arrows indicate elution of molecular weight standards.
quantitative amino acid analysis, it can be estimated that protease-sensitive, nonglycosylated segments account for approximately 10% of the gallbladder mucin protein core.
An additional aim of the present study was to identify the areas on the mucin molecule that bind the hydrophobic fluor ANS (lo), bilirubin (Zl), and biliary lipids (23). Previous reports from this laboratory had indicated that these ligands bound to proteasesensitive areas of the mucin core because the large glycopeptides resulting from proteolytic digestion of mucin were devoid of the hydrophobic-binding properties of the native molecule. Bilirubin was derivatized with Woodward’s reagent K and covalently bound to mucin at its presumed native binding domain in a manner similar to that used to study bilirubin binding to albumin (18) and glutathione-S-transferase (19). After partial purification by preparative molecular-sieve HPLC and RP-HPLC, the major tryptic peptide that had covalently bound bilirubin eluted from an analytical molecular-sieve column as a lowmolecular-weight peptide (Figure 81. The amino acid composition (Table 2) and the lack of amino sugars in this peptide indicate that it was derived from the nonglycosylated portion of the mucin protein core. The hydrophilic mucous gel acts as a diffusion barrier by creating an unstirred water layer at the epithelial surface. The present data suggest that nonglycosylated portions of the mucin protein core may nonspecifically absorb hydrophobic substances that are potentially toxic to the underlying epithelial cell
Vol. 98. No. 6
and, thereby, provide an additional protective function for mucous gel. The hydrophobic-binding properties of gastric mucin have also been described recently, and they were linked to the protective function of gastric mucus (22,231. Further elucidation of gallbladder mucin structure and function requires detailed structural knowledge of the mucin momomer. Attempts to obtain primary structure data on the mucin core have been unsuccessful because of the small quantities of nonglycosylated peptide that could be recovered and the tendency of these peptides to aggregate during both chromatography and gel electrophoresis. Detailed study of mucin structure by the standard techniques of protein chemistry is hampered by the unique structural and physical properties of mucin. An alternate strategy will be the use of nonglycosylated tryptic peptides to raise antibodies to the mucin protein core. The predominant antigenicity of native mucin resides in oligosaccarhide side chains that constitute the bulk of the molecule. Antibodies raised against native mucin have not identified the mucin protein core in in vitro translation studies using gallbladder epithelial cell messenger RNA (Turner and LaMont, personal communication, July 1989). This finding indicates that polyclonal antibodies raised against the densely glycosylated native mucin may lack reactivity toward the mucin protein core. Antibodies raised against nonglycosylated tryptic peptides should be directed against epitopes present on the
0.2
I aP
0.2
%
t
4
0.1
0.1
10
20
30
d
40
Fractions
Figure 7. Covalent binding of bilirubin to mucin. Bilirubin was derivatized with N-ethyl&phenylisoxazlium-3’-sulfonate (Woodward’s reagent K) as described. Lyophilized mucin (60 mg] was reconstituted in 23 ml of 0.01 M sodium phosphate, 0.145 M NaCl (pH 7.4). The BW complex was suspended in 5 ml water, and 1.6 ml was mixed with the mucin solution and incubated at 20% for 2 h under N, in the dark. Ethylenediaminetetraacetic acid was added to obtain a final concentration of I mM, and the pH was brought to 6.4 by the addition of 700 mg of imidazole. After an additional 6 h of incubation in the dark at 2o°C, the solution was chromatographed on a Sepharose 2B-CL column (66.0 cm x 2.5 cm) eluted with 0.1 M sodium phosphate, 0.145 M NaCl (pH 7.4) at a flow rate of 42 ml/h. Fractions (12 ml) were collected and monitored at A,,, for protein and at A,,, for bilirubin.
June 1990
oi
GALLBLADDER MLJCIN PEPTIDES
I
I
’
10
20
I
30
40
50
IL60
Time (minutes) Figure 5. Isolation of a bilirubin-binding tryptic mucin peptide. The mucin-BW complex that eluted in the void volume of a Sepbarose ZB-CL column (Figure 7) was digested with trypsin for 24 b as described and subjected to molecular-sieve HPLC as described in legend to Figure 1. Tryptic peptides eluting in the same position as those indicated as fraction III in Figure 1B were collected and analyzed by RPNPLC as described in Figure 5 legend. A peptide eluting with a retention time identical to that of peak c in Figure 58 was collected and analyzed by molecular-sieve HPLC using a Bio-Rad TSK-125 column (7.5 mm x 300 mm) eluted with 0.01 M Tris Hcl and 0.05 M NaCl (pH 6.5) at a flow rate of 1 ml/min. Fractions were monitored at A,,, (A] for the detection of protein and at A,,,(B) for detection of bilirubin.
mucin
protein
cDNA library
core
and
may
of the gallbladder
be useful
to screen
a
epithelium.
References Levy PF, Smith BF, LaMont JT. Human gallbladder mucin accelerates in vitro nucleation of cholesterol in artificial bile. Gastroenterology 1984;87:270-275. Smith BF. Human gallbladder mucin binds biliary lipids and promotes cholesterol crystal nucleation in model bile. J Lipid Res 1987;28:1088-1097. Lee TJ. Smith BF. Bovine gallbladder mucin accelerates cholesterol crystal nucleation from cholesterol enriched vesicles in supersaturated model bile. J Lipid Res 1989;30:491-498.
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4. Lee SP, LaMont JT, Carey MC. The role of gallbladder mucus hypersecretion in the evolution of cholesterol gallstones: studies in the prairie dog. J Clin Invest 1981;67:1712-1723. 5. Lee SP, Carey MC, LaMont JT. Aspirin prevention of cholesterol gallstone formation in prairie dogs. Science 1981;211:14291432. 6. MacPherson BR, Pemsingh RS, Scott GW. Experimental cholelithiasis in the ground squirrel. Lab Invest 1987;56:138-145, 7. Messing B, Boires C, Ku&linger F, Bernier JJ. Dooes total parenteral nutrition induce gallbladder sludge formation and lithiasis? Gastroenterology 1983;84:1012-1019. 8. Hulten 0. Formation of gallstones. II. Acta Chir Stand 1968;134: 557-560. 9. Lee SP, Nichols JF. The nature and composition of biliary sludge. Gastroenterology 1986;90:677-686. 10. Smith BF, LaMont JT. Hydrophobic binding properties of bovine gallbladder mucin. J Biol Chem 1984;259:12170-12177. 11. Smith BF, Peetermans JA, Tanaka T, LaMont JT. Subunit interactions and physical properties of bovine gallbladder mutin. Gastroenterology 1989;97:179-187. 12. Mitz MA, Schlueter RJ. Direct spectrophotometric measurement of the peptide bond: applications to the determination of acylase I. Biochem Biophys Acta 1958;27:168-177. 13. Folk JE, Schirmer EW. The porcine pancreatic carboxypeptidase A system. J Biol Chem 1963;238:3884-3889. 14. Kuhn RW. Reaction of yeast carboxypeptidase C with groupspecific reagents. Biochemistry 1976;15:4881. 15. Mantle M, Allen A. A colometeric assay for glycoproteins based on the periodic acid/Schiff stain. Biochem Sot Trans 1978:6:607609. 16. Laemmli UK. Cleavage of the structural proteins during assembly of the head of bacteriophage T,. Nature 1970;227:680-685. 17. McDonagh AF, Assisi F. The ready isomerization of bilirubin IXa in aqueous solution. Biochem J 1972;129:797-800, N, Wilson KJ. Affinity 18. Kuenzle CC, Gitzelmann-Cumarasamy labeling of the primary bilirubin binding site of human serum albumin. J Biol Chem 1976;251:801-807. ligand19. Boyer TD. Covalent labeling of the non-substrate binding site of glutathione-S-transferase with bilirubin-woodward’s reagent K. J Biol Chem 1986;261:5363-5367. 20. Gum JR, Byrd JC, Hicks JW, Toribara NW, Lamport DTA, Kim YS. Molecular cloning of human intestinal mucin cDNAs. Sequence analysis and evidence for genetic polymorphism. J Biol Chem 1989; 264:6480-6487. 21. Smith BF, LaMont JT. Bovine gallbladder mucin binds bilirubin in vitro. Gastroenterology 1983;85:707-712. 22. Sarosiek J, Piotrowski J, Gabryelewicz A, Slomiany A, Slomiant BL. Alterations in mucin hydrophobicity and molecular form distribution with peptic ulcer (abstr). Gastroenterology 1989;96: A441. 23. Slomiany BL, Nishikawa, Slomiany A. Gastric mucin hyrophobicity: effects of proteolysis, reduction and lipid removal (abstr]. Gastroenterology 1989;96:A478.
Received August 2,1989. Accepted November 30,1989. Address requests for reprints to: Bernard F. Smith, M.D., Section of Gastroenterology, Thorndike 503. Boston City Hospital, 818 Harrison Avenue, Boston, Massachusetts 02118. Dr. Smith was supported by National Institutes of Health research grant DK39017, and Dr. Afdal was supported by NIH BRSQ RR 0556924.