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Interaction of human gallbladder mucin with calcium hydroxyapatite: Binding studies and the effect on hydroxyapatite formation
Interaction of Human Gallbladder Mucin With Calcium Hydroxyapatite: Binding Studies and the Effect on Hydroxyapatite Formation SuI-MIN
QIu, 1 G A R Y
W E N , 1 J U L I E W E N , 2 R O G E R D . SOLOWAY, 1 AND R O G E R S. C R O W T H E R 1
Calcium h y d r o x y a p a t i t e (HAP) crystals f o r m e d in vitro in t h e p r e s e n c e o f p o l y m e r i c h u m a n gallbladder m u c i n (1.0 mg/mL) w e r e s m a l l e r (0.75 _+ 0.39/xm) t h a n c o n t r o l crystals (7.86 + 2.76 ttm), but t h e m u c i n did n o t affect t h e k i n e t i c s o f crystal f o r m a t i o n or alter t h e a m o u n t of m i n e r a l p h a s e p r e s e n t at equilibrium. In contrast, g l y c o p e p t i d e s u b u n i t s p r o d u c e d by p r o t e o l y s i s o f the n a t i v e m u c i n h a d n o effect o n HAP crystal size. B o t h native m u c i n a n d g l y c o p e p t i d e s b o u n d to m a t u r e HAP crystals, but t h e g l y c o p e p t i d e s w e r e m u c h m o r e readily displaced by p h o s p h a t e ions. Therefore, in e x p e r i m e n t s w h e r e HAP w a s b e i n g formed, t h e p h o s p h a t e ions inhibited t h e i n t e r a c t i o n o f g l y c o p e p t i d e s w i t h t h e n a s c e n t HAP. T h e s e results i n d i c a t e that gallbladder m u c i n m a y m o d u l a t e HAP f o r m a t i o n in vivo, a n d that this ability m a y be altered d u r i n g p a t h o l o g i c a l states, s u c h as neutrophil infiltration or bacterial c o l o n i z a t i o n , that m a y c a u s e t h e r e l e a s e of p r o t e i n a s e s capable o f digesting mucin. (HEPATOLOGY1995;21:1618-1624.)
The inclusion of mucin in the organic matrix of cholesterol and pigment gallstones TM indicates a potential role for these epithelial glycoproteins in the pathogenesis of cholelithiasis. H u m a n gallbladder mucin promotes cholesterol nucleation in vitro in model biles, 5 as well as in prairie dog hepatic bile 6 and h u m a n gallbladder bile. 7 Much less obvious, however, is whether mucins are qualitatively or quantitatively different in normal subjects and those with gallstones. Lee and Nicholls s measured higher gallbladder mucin concentrations in gallstone patients compared with control patients, but this result was not supported by Harvey et al. 9 Compositional analysis showed t h a t mucins from patients with cholesterol gallstones were more exten-
Abbreviation: HAP, hydroxyapatite. From the 1Division of Gastroenterology, Department of Internal Medicine, and the 2Department of Pathology, University of Texas Medical Branch, Galveston, TX. Received July 12, 1994; accepted December 29, 1994. Supported by grant AM-16549 from the National Institutes of Health and by grants from the Scaly and Smith and M. D. Anderson Foundations. Address reprint requests to: Roger S. Crowther, PhD, Division of Gastroenterology, 301 University Blvd, University of Texas Medical Branch, Galveston, TX 77555-0764. Copyright © 1995 by the American Association for the Study of Liver Diseases. 0270-9139/95/2106-002053.00/0
sively sulfated t h a n were mucins from control subjects, s but there was no difference in the efficiency with which the mucins promoted cholesterol nucleation, v Less attention has been paid to the effect of mucin on the formation of the calcium salts found in gallstones. Yamasaki et al 1° showed t h a t mucin isolated from the hepatic bile of a patient without gallstones inhibited calcium carbonate precipitation in vitro. Mucin purified from T-tube bile of a patient with intrahepatic stones was more highly sulfated t h a n was control mucin obtained from a patient with no biliary tract disease, and it also had a significantly greater ability to cause aggregation of finely divided calcium carbonate. 11 Bovine gallbladder mucin alone inhibited the precipitation of CaHPO4, but when the mucin was complexed with an acidic, amphiphilic protein isolated from h u m a n gallstones, precipitation of the mineral phase was enhanced. 12 The effect of gallbladder mucin on the formation of calcium hydroxyapatite, Ca5OH(PO4)3 (HAP), has not been studied. However, salivary mucins bind to HAP of tooth enamel ~3'14 and influence the demineralization and remineralization of teeth. ~5 Because salivary and gallbladder mucins have compositional similarities, we hypothesized t h a t gallbladder mucin m a y significantly affect the formation of HAP. Here we report on the binding of h u m a n gallbladder mucin and its constituent glycopeptides to HAP and the consequent effect on HAP formation. MATERIALS A N D M E T H O D S
Calcium chloride, dibasic sodium phosphate, Trizma buffer, sodium chloride, and calcium hydroxyapatite (type I) were obtained from Sigma Chemical Co., St Louis, MO. HAP was analyzed for calcium and phosphate content, and the Ca/ P ratio was 1.69 +_0.01, which is consistent with HAP (1.67). Pronase was purchased from Boehringer-Mannheim Corp., Indianapolis, IN. Mucin Purification. Gallbladders were obtained from patients with cholesterol or pigment gallstones undergoing cholecystectomy. Bile was removed by aspiration with a syringe and 16-gauge needle and was pooled and stored at -20°C. Mucin was isolated as described by Pearson et al, 1~with modifications. After thawing, bile was diluted 1:1 with KSCN buffer (0.22 mol/L KSCN, 10 mmol/L Na2HPO4, and 0.01% sodium azide, pH 7.5) and centrifuged at 2,000g for 20 minutes to remove the sludge and other insoluble debris. The
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HEPATOLOGY
Vo]. 21, No. 6, 1995
supernatant was chromatographed in 20-mL batches on a gel filtration column of Sepharose CL-2B (110 × 3 cm) eluted with KSCN buffer at 0.6 mL/min. Eluted protein was monitored by fluorescence at excitation and emission wavelengths of 280 and 340 nm, respectively (Perkin-Elmer Corp., Norwalk, CT). The excluded protein peak, which contained the high-molecular-weight mucin, was dialyzed against distilled water at 4°C to remove the bulk of the KSCN. Sufficient CsC1 was added to bring the density to 1.45 g/mL, and the samples were centrifuged at 105,000g for at least 48 hours at 4°C (L870M ultracentrifuge, Beckman Instruments, Palo Alto, CA). The resulting gradients were fractionated by hand, and the density of each fraction was determined gravimetrically. The protein profile was monitored by fluorescence, and proteincontaining fractions with densities between 1.40 and 1.50, which is typical of mucin, were pooled. The density of the pooled fraction was readjusted to 1.45 g/mL, and the sample was subjected to an additional two cycles of CsC1 density gradient ultracentrifugation to remove residual noncovalently bound protein. The purified mucin was exhaustively dialyzed against distilled/deionized water at 4°C and was concentrated by ultrafiltration through a 30,000 molecular weight cut-off membrane (YM30, Amicon Corp., Danvers, MA). An eightfold concentrated solution of Tris/NaC1 was added to the concentrated mucin to bring the final Tris and saline concentrations to 50 and 100 mmol/L, respectively. The resulting mucin concentration was determined fluorimetrically by assaying for O-linked oligosaccharides with 2-cyanoacetamide, 17 using a gravimetrically standardized sample of canine tracheal mucin as the standard. The mucin (2.4 rag/ mL) was stored in l-mL aliquots at -20°C. To establish that mucin solubility was not adversely affected by storage at -20°C, a sample of the frozen mucin was thawed and diluted to 1.0 mg/mL with Tris/NaC1. The diluted mucin was incubated at 37°C for 30 minutes, and mucin aggregates were dispersed by very gentle passage through a 26-gauge needle. A portion of the solution was centrifuged at 11,000g for 5 minutes to pellet undispersed mucin, and the mucin concentration in the centrifuged and uncentrifuged aliquots was determined fluorimetrically with 2-cyanoacetamide. There was no difference in the mucin concentration of the centrifuged (1.09 _+ 0.04 mg/mL) and uncentrifuged (1.10 _+ 0.11 mg/mL) samples, which demonstrated that mucin did not irreversibly aggregate during storage at -20°C. In all further experiments, whenever a mucin sample was thawed, the mucin was dispersed by incubation at 37°C and passage through a 26-gauge needle as described above. Chemical Analysis. Mucin amino acid content was measured with an automated analyzer (121MB Analyzer, Beckman Instruments). For neutral and amino sugar analysis, mucin was hydrolyzed at 125°C for I hour in 4 N trifluoroacetic acid, 17 freeze-dried, and reconstituted in water. For sialic acid analysis, mucin was hydrolyzed in 0.1 N HC1 for i hour at 80°C, 18 freeze-dried, and redissolved in water. The carbohydrate composition of the hydrolyzed mucin was then analyzed by anion exchange high-pressure liquid chromatography with pulsed amperometric detection (BioLC, Dionex Corp., Sunnyvale, CA). The amino acid and carbohydrate composition of the purified mucin was very similar to that given by Pearson et al TM for human gallbladder mucin (Table 1). Generation of Mucin Glycopeptides. Mucin was degraded to its constituent glycopeptides by digestion with pronase. Mucin (2 mg/mL in Tris/saline buffer) was incubated for 16 hours at 37°C with 0.2 mg/mL pronase. A control incubation was also performed in which mucin was incubated with pro-
QIU ET AL
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TABLE 1. C o m p o s i t i o n a l A n a l y s i s o f P u r i f i e d Gallbladder Mucin Amino Acid
Asp Tbr Ser Glu Pro Gly Ala Cys Val Met Ile Leu Tyr Pbe His Lys Arg
Residues/100 Residues
5.60* 19.24 10.88 7.66 9.00 7.87 9.01 ND 5.58 0.90 2.72 8.60 1.48 2.73 2.66 2.82 3.33
Carbohydrate
6.13t 19.75 12.83 7.95 12.83 8.74 8.51 ND 5.90 0.91 2.95 5.68 1.70 2.50 2.84 3.41 3.29
Fuc GalNAc GlcNAc Gal NANA
Molar Ratio
2.88* 1.00 4.38 4.98 1.29
2.67~ 1.00 4.03 3.72 ND
Abbreviation: ND, not determined. * This study. Data from Pearson et al. TM
nase that had been inactivated by boiling for 10 minutes. Comparison of the proteolytic activity of the native and heatinactivated pronase by measuring the solubilization of hide powder azure 19 showed that more than 90% of the enzyme activity was abolished by boiling. Effect of Mucin on Hydroxyapatite Formation. HAP was formed from solutions containing 4 mmol/L CaCI2, 4 mmol/ L Na2HPO4, 50 mmol/L Tris, and 100 mmol/L NaC1 exactly as previously described. 2° Briefly, twofold concentrated solutions of calcium or phosphate in Tris/saline buffer were equilibrated to 37°C, mixed, and then placed in quartz cuvettes maintained at 37°C. Mineral phase formation was monitored by measuring turbidity at 220 nm (model 4053 spectrophotometer, Biochrom, Cambridge, England). 2° The effect of mucin was studied over the concentration range of 0.1 to 1.0 mg/ mL. In some experiments, mucin glycopeptides were used instead of native mucin. At the end of the experiment (usually 250 minutes), the solutions were removed from the cuvettes and centrifuged at 11,000g for 5 minutes. The phosphate concentration in the supernatant was determined spectrophotometrically by the molybdenum blue method, 21 and the amount of precipitated HAP was calculated. Transmission Electron Microscopy. Hydr0xyapatite was allowed to form as described above for 250 minutes in the presence of increasing concentrations of mucin or glycopeptides. At the end of the incubation, the samples were gently agitated to resuspend any sedimented particles, and 2 #L of the suspension was removed and transferred to a formvarcoated, carbon-stabilized copper grid (Ernest F. Fullam Inc., Latham, NY). The suspension was allowed to remain in contact with the grid for 5 minutes, and the drop was then removed by carefully touching it with tissue paper. Samples were examined at 60 kV by transmission electron microscopy. The particle size distribution was estimated by measuring with a ruler the largest dimension of 10 individual particles in each of at least three separate photographic enlargements for every sample. Particles that appeared to be aggregates of smaller particles were treated as a single particle.
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QIU ET AL
HEPATOLOGYJune 1995
Mucin Binding to Hydroxyapatite. Ten milligrams of HAP were suspended in 0.5 mL of Tris/sa]ine buffer containing up to 1.0 mg/mL of native mucin or mucin glycopeptides in a polypropylene microtube. Control incubations without HAP were also performed. The suspension was incubated for 2 hours at room temperature with intermittent vortexing, and then centrifuged at ll,000g for 5 minutes to pellet HAP and bound mucin. Mucin remaining in the supernatant was measured fluorimetrically with 2-cyanoacetamide, and the amount bound was calculated by difference. Preliminary experiments showed that equilibrium was reached in approximately 60 minutes, To determine the relative binding to HAP of glycopeptides of different sizes, 200 #L of the supernatant was removed and subjected to gel filtration chromatography on Sepharose CL-2B (30 × 1.5 cm). The column was eluted with 0.15 mol/ L NaC] at 0.4 mL/min, and fractions of 1.0 mL were collected and analyzed for O-linked oligosaccharides with 2-cyanoacetamide. ~2 The effect of phosphate ions on mucin binding to HAP was investigated at a fixed mucin or glycopeptide concentration of 0.2 mg/mL by adding increasing concentrations of Na2HPO4, and the ionic strength was kept constant by reciprocally reducing the NaC1 concentration. All other conditions were as described above. RESULTS
Calcium Phosphate Precipitation. In the absence of mucin, H A P began to precipitate at ~ 5 0 minutes. The time at which H A P formation commences is referred to as the induction time. 2° We have previously confirmed by chemical analysis and infrared spectroscopy t h a t this mineral phase is HAP. 2° The optical density of the solution rose for a f u r t h e r 10 minutes, b u t t h e n began to decline as the HAP particles became sufficiently large to sediment (Fig. 1). Native mucin at a concentration of 0.5 mg/mL h a d no effect on the induction time, b u t p r e v e n t e d the decline in optical density t h a t was associated with sedimentation (Fig. 1). Similar results were observed with native mucin concentrations of 0.1 and 1.0 mg/mL (not shown). W h e n mucin glycopeptides (0.5 mg/mL) were substituted for native mucin, the inhibition of HAP sedim e n t a t i o n was abolished (Fig. 1). The a m o u n t of HAP precipitated at 250 minutes was not affected by either native mucin or glycopeptides (Table 2). W h e n HAP precipitation was allowed to proceed for 24 hours, there was no change in the a m o u n t of HAP formed (not shown), which indicated t h a t equilibrium was attained within 250 minutes. The p r o n a s e p r e p a r a t i o n u s e d to g e n e r a t e glycopeptides c o n t a i n e d Ca 2+, b u t this r e s u l t e d in a less t h a n 1% increase in Ca 2+ c o n c e n t r a t i o n in p r e c i p i t a t i o n exp e r i m e n t s u s i n g glycopeptides. This small i n c r e a s e would not be expected to a l t e r the kinetics of H A P formation, a n d in a control e x p e r i m e n t with p r o n a s e alone (final c o n c e n t r a t i o n , 50 #g/mL), t h e induction time was a l m o s t identical to t h e i n d u c t i o n t i m e w i t h o u t pronase, 50 a n d 54 m i n u t e s , respectively. U s i n g t r a n s m i s s i o n electron microscopy, H A P was observed to form i r r e g u l a r l y s h a p e d particles a n d agg r e g a t e s w i t h " f e a t h e r e d " edges (Fig. 2A). S i m i l a r particles w e r e observed for H A P f o r m e d in the p r e s e n c e of
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FIG. 1. Formation of calcium hydroxyapatite monitored by optical density. Ca2÷ and phosphate (final concentration of each, 4 mmol/ L) in 50 mmol/L Tris/100 mmol/L NaC1 buffer, pH 7.3, were mixed and incubated at 37°C with or without native mucin or glycopeptides. Mineral phase separation was followed by monitoring optical density at 220 nm. 0.5 m g / m L glycopeptides (Fig. 2B), b u t the particles were s m a l l e r w h e n 0.5 m g / m L n a t i v e m u c i n was present (Fig. 2C). The effect of n a t i v e m u c i n a n d glycopeptides on t h e m e a n H A P particle size is given in Table 2. Binding of Mucin and Glyeopeptides to HAP. B o t h n a t i v e m u c i n a n d glycopeptides were b o u n d to HAP, b u t H A P a p p e a r e d to h a v e a h i g h e r capacity for t h e glycopeptides (Fig. 3). In control incubations, o m i t t i n g HAP, all the m u c i n was r e c o v e r e d in t h e s u p e r n a t a n t a f t e r centrifugation, which d e m o n s t r a t e d t h a t t h e r e was no nonspecific b i n d i n g to t h e t u b e s a n d t h a t the m u c i n was sufficiently dispersed to r e m a i n in solution. W h e n m u c i n was i n c u b a t e d for 16 h o u r s at 37°C with h e a t - i n a c t i v a t e d pronase, t h e r e was no effect on the a m o u n t of m u c i n s u b s e q u e n t l y b o u n d to H A P (Fig. 3). To e s t i m a t e t h e size distribution of the glycopeptides p r o d u c e d by p r o n a s e digestion, t h e y were chromatog r a p h e d on a c o l u m n of S e p h a r o s e CL-2B. N a t i v e mucin e l u t e d as a single p e a k in t h e c o l u m n void volume, w h e r e a s the p r o n a s e - t r e a t e d m u c i n was d e g r a d e d into glycopeptides t h a t showed considerable size heterogen e i t y (Fig. 4A). This h e t e r o g e n e i t y is in a g r e e m e n t w i t h the observation of P e a r s o n et al, 1~ who e s t i m a t e d t h e m o l e c u l a r size of p r o t e i n a s e - d i g e s t e d h u m a n gallbladder m u c i n to be on t h e o r d e r of m a g n i t u d e of 5 × 105. To d e t e r m i n e if glycopeptide size influenced binding to HAP, glycopeptides a n d H A P w e r e i n c u b a t e d together, a n d the s u p e r n a t a n t a f t e r c e n t r i f u g a t i o n was chrom a t o g r a p h e d on S e p h a r o s e CL-2B (Fig. 4B). Midsize
QIU E T A L
HEPATOLOGY Vol. 21, No. 6, 1995 TABLE 2. E f f e c t o f N a t i v e M u c i n a n d G l y c o p e p t i d e s
on HAP Formation
Control
0.1
0.5
1.0
Glycopeptides (mg/mL) 0.5
50 0.38 _+ 0.02 7.86 _+ 2.76
50 0.39 _+ 0.01 2.79 _+ 1.76"
50 0.38 _+ 0.01 1.16 _+ 0.73*
45 0.37 _+ 0.01 0.75 _+ 0.39*
54 0.39 _+ 0.01 8.24 _+ 4.53
Native Mucin (mg/mL)
Induction time, min HAP formed, #mol/mL Particle size, pm
1621
* Significantly different from control (P < .9001, t-test).
glycopeptides were bound to HAP to a greater extent than were either residual intact mucin or very small glycopeptides, and the maximum binding efficiency occurred in fractions 17 and 18 (Fig. 4B). Because glycopeptides had no effect on HAP formation, but were bound more extensively to HAP than was the native mucin, we hypothesized that specific components that were present only in the precipitation experiments preferentially inhibited glycopeptide binding to HAP. We have previously shown that bile salt binding to HAP is competitively inhibited by phosphate ions, 23 and we therefore chose to investigate the effect of phosphate on mucin binding. Low concentrations of phosphate had no effect on either mucin or glycopeptide binding to HAP, but above 1 mmol/L phosphate, there was a progressive decline in binding, and this effect
was more pronounced for the glycopeptides than for the native mucin (Fig. 5). DISCUSSION
The current study has shown that native h u m a n gallbladder mucin binds to HAP crystals in vitro and reduces the particle size of nascent HAP, but it does not alter the kinetics of HAP formation or reduce the amount of HAP precipitated at equilibrium. Proteolytically produced mucin glycopeptides also bound to HAP, but were without effect on HAP particle size. The effect of native mucin on HAP particle size was probably underestimated in these experiments because HAP was placed on the electron microscopy grid by allowing it to settle under gravity, which would favor the sampling of the largest particles. In addition, our measurement
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FIG. 2. Transmission electron micrographs of HAP crystals. HAP was allowed to form for 250 minutes under the conditions described in the legend for Fig. 1. either without mucin (A~, or with 0.5 rag/ mL of mucin glycopeptides (B) or native mucin (C). In each case the original magnification is x17,500, and the bar represents i #m.
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1622
QIU E T A L
HEPATOLOGY J u n e 1995
increase the stability of the HAP-mucin complex. Alternatively, the hydrophobicity of the nonglycosylated regions may be less important than their function of covalently linking the glycosy]ated segments together, giving these segments the ability to bind to HAP cooperatively rather than as individual units. The easier displacement of glycopeptides by phosphate was somewhat surprising because analysis of the effect ofglycopeptide size on bind-
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of particle size was relatively imprecise, and did not account for the three-dimensional nature of the particles. Despite these technical limitations, the reduction of particle size by mucin was unambiguous, and was consistent with the sustained increase in optical density seen in the precipitation experiments. Although native mucins have a much greater effect on solution viscosity than do glycopeptides,24 this effect probably did not cause the reduction of particle size because Blumenthal et a125 showed that viscosity per se does not affect HAP formation. Therefore, the effect of mucin on HAP precipitation probably resulted from the interaction with the crystal surface. Although glycopeptides also bound to HAP, they were much more readily displaced by phosphate ions than was the native mucin, and therefore, in precipitation experiments containing initial phosphate concentrations of 4 mmol/L, there was probably minimal interaction of the glycopeptides with the nascent HAP. The greater ease of displacement of glycopeptides compared with native mucin indicates the importance of the nonglycosylated segments of the mucin molecule for binding to HAP, because these regions are largely removed by digestion with pronase. In gallbladder mucin these nonglycosylated segments are relatively hydrophobic2~ and have been shown to be responsible for the interaction of mucin with phosphatidylcholine and cholesterol27 and bilirubin 2s'29 and are postulated to have a role in the self-association of bovine gallbladder mucin. 29 It is unlikely that mucin interacts with HAP hydrophobically, but adjacent mucin molecules on the HAP surface may form hydrophobic bonds with each other that
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Elution volume, ml FIG. 4. Effect of glycopeptide size on binding to HAP. (A) Two hundred microliters native mucin or pronase-produced g]ycopeptides, each 1.0 mg/mL, was chromatographed on Sepharose CL-2B, 1-mL fractions were collected and analyzed for O-linked oligosaccharides with 2-cyanoacetamide. (B) Mucin glycopeptides (1.0 mg/mL) were incubated with HAP for 2 hours, the unbound fraction was collected by centrifugation, and 200 #L was chromatographed on Sepharose CL-2B. The percentage of glycopeptides bound to HAP (bars) was calculated from the difference between the fluorescence of the fractions obtained from the total glycopeptide mixture before the addition of HAP (from Fig. 4A) and the fluorescence of the fractions obtained from the unbound glycopeptides.
HEPATOLOGY Vol. 21, No. 6,1995
QIU E T A L
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P h o s p h a t e c o n c e n t r a t i on, mM FIG. 5. Effect of phosphate on native mucin and glycopeptide binding to HAP. Native mucin or glycopeptides (0.2 mg/mL) were incubated with HAP in buffer with increasing concentrations of Na2HPO4, and the ionic s t r e n g t h was kept constant by reciprocally reducing the NaC1 concentration. All other conditions were as described in the legend to Fig. 3.
1623
but that exposed parts of the protein core were also involved. 35 Using different techniques, the binding of glycoprotein from rat minor salivary gland was also concluded to be mediated mainly by ester sulfate groups. 3~ The possible importance of sulfated carbohydrates is interesting, because it has been reported that the sulfation of biliary mucins is increased in lithogenic bile. s'11 Both sialic acid and ester sulfate groups are negatively charged under physiologic conditions, which indicates that electrostatic interactions with surface calcium ions may be important for mucin binding to HAP. This interpretation is consistent with the reduction in binding caused by phosphate ions, which may be viewed as competition between the phosphate and the polyanion for a common binding site on the HAP surface. Other high-molecular-weight glycoconjugates also affect HAP formation. Proteoglycan aggregates and subunits had different effects than did mucin, because these macromolecules increased the time required for HAP precipitation to commenceY Hyaluron, the polysaccharide backbone required for proteoglycan aggregation, was bound to HAP but had no effect on HAP precipitation. 37 Mucins from various sources have also been shown to regulate the precipitation of other calcium salts. Mucin from h u m a n hepatic bile inhibited calcium carbonate precipitation, 1° but the effect was small compared with that of a low-molecular-weight acidic protein that was isolated from cholesterol 3s or black pigment gallstones. 39 Calcium carbonate precipitation is also regulated by the intestinal mucus of the silver eel, b u t in this case the mucous gel acts as a matrix for biomineratization that results in the production of calcite crystals. 4° Bovine submaxillary mucin altered the size and crystal habit of calcium oxalate crystals by inhibiting the growth of specific crystal
ing to HAP indicated that midsize glycopeptides were adsorbed in preference to residual intact mucin. However, this apparent preference for midsize glycopeptides may indicate faster binding kinetics for these smaller, more diffusible species, rather than a higher binding affinity. The greater binding capacity of HAP for glycopep- f a c e s . 41 tides versus native mucin may reflect the collapse of the An important observation about gallstone pathogenexpanded mucin conformation caused by proteolysis, 24 esis that has resulted from work with animal models which would allow more efficient packing of the glycosyl- is that cholesteroP 2'43 and pigment 43 gallstones are freated subunits on the HAP surface. The reduced effect of quently nidated in the mucous gel layer adherent to the glycopeptides in comparison with native mucin on HAP gallbladder mucosa. A limitation of the current study is formation may have pathologic significance because, al- that it was performed at mucin concentrations that though neither normal bile nor gallbladder mucosa con- were too low to permit gel formation, b u t extrapolation tains proteolytic enzymes that digest mucin, 3° many bac- from the current results suggests that significant efterial proteases are capable of degrading mucins, 3133 and fects on HAP formation would be likely to occur in a bacteria have been assigned a potential role in gallstone mucin gel. Gels frequently act as nucleation sites for pathogenesis. In addition, the recruitment and activation crystals, by mechanisms that are imperfectly underof neutrophils may also lead to mucin degradation be- stood, 44 and it is possible that the smaller crystals cause human neutrophil elastase has been shown to di- formed in the presence of these relatively dilute mucin gest mucin. 32 solutions resulted from the nucleation of a greater The interaction of salivary mucins with HAP has been number of embryos than was formed in the control exextensively investigated, but no common structural motif periments. The ability of mucin to bind to HAP crystals responsible for binding has been identified. For example, m a y enable it to cement crystals together and thus removal of sialic acid from the oligosaccharides of ovine cause crystal agglomeration that may be an essential submandibular mucin prevented its binding to HAP, but precursor to gallstone formation. The effect of mucins similar treatment of human submandibular mucins had may also be modified by other biliary proteins, and it only a small effect on binding to HAP. ~4Analysis by infra- has been shown that the calcium binding protein that red spectroscopy of the binding of a sulfated mucin from was isolated from h u m a n gallstones 3s'39 alters the abilhuman whole saliva suggested that the sulfated carbohy- ity of mucin to inhibit CaHPO4 precipitation. ~2 The imdrates were particularly important for binding to HAP, portance of the chemical composition of gallbladder
1624
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HEPATOLOGYJune 1995
mucin and in particular the effect of sulfation on the formation of HAP also remain to be investigated. REFERENCES
1. Womack NA, Zeppa R, Irvin GL. The anatomy of gallstones. Ann Surg 1963; 157:670-686. 2. LaMent JT, Ventola AS, Trotman BW, Soloway RD. Mucin glycoprotein content of human pigment gallstones. HEPATOLOGY 1983;3:377-382. 3. Pearson JP, Foster SNE. Mucous glycoprotein content of human cholesterol gallstones. Digestion 1987;36:132-140. 4. Smith BF, LaMent JT. Identification of gallbladder mucin-bilirubin complex in human cholesterol gallstone matrix: effects of reducing agents on in vitro dissolution of matrix and intact gallstones. J Clin Invest 1985; 76:439-445. 5. Levy PF, Smith BF, LaMent JT. Human gallbladder mucin accelerates nucleation of cholesterol in artificial bile. Gastroenterology 1984;87:270-275. 6. Lee SP, LaMent JT, Carey MC. Role of gallbladder mucus hypersecretion in the evolution of cholesterol gallstones. J Clin Invest 1981;67:1712-1723. 7. Gallinger S, Taylor RD, Harvey PCR, Petrunka CN, Strasberg SM. Effect of mucous glycoprotein on nucleation time of human bile. Gastroenterology 1985;89:648-658. 8. Lee SP, Nicholls JF. Nature and composition of biliary sludge. Gastroenterology 1986;90:677-686. 9. Harvey PRC, Rupar CA, Gallinger S, Petrunka CN, Strasberg SM. Quantitative and qualitative comparison of gallbladder mucus glycoprotein from patients with and without gallstones. Gut 1986;27:374-381. 10. Yamasaki T, Chijiwa K, Endo M. Isolation of mucin from human hepatic bile and its effects on precipitation of cholesterol and calcium carbonate in vitro. Dig Dis Sci 1993;38:909-915. 11. Nagashima H, MasabuchiM, Yosizawa Z. Sulfated glycoproteins capable of coagulating calcium carbonate isolated from pathological human bile. J Biochem (Tokyo) 1974; 75:779-786. 12. Afdahl NH, Niu N, Offner GD, Ostrow JD, Koehler R, Veis A, Sabsay B. Gallbladder mucin and the calcium-binding protein from gallstones, bind hydrophobically and regulate CaHPO4 precipitation [Abstract]. HEPATOLOGY1993; 18:144A. 13. Tabak LA, Levine MJ, Jain NK, Bryan AR, Cohen RE, Monte LD, Zawacki S, et al. Adsorption of human salivary mucins to hydroxyapatite. Arch Oral Biol 1985;30:423-427. 14. Nieuw Amerongen AV, Oderkerk CH, Veerman ECI. Interaction of human salivary mucins with hydroxyapatite. J Biol Buccale 1989; 17:85-92. 15. Zahradnik RT. Modification by salivary pellicles of in vitro tooth remineralization. J Dent Res 1979;58:2066-2073. 16. Pearson JP, Kaura R, Taylor W, Allen A. The composition and polymeric structure of mucus glycoprotein from human gallbladder bile. Biochim Biophys Acta 1982; 706:221-228. 17. Neeser J-R, Schweizer TF. A quantitative determination by capillary gas-liquid chromatography of neutral and amino sugars (as O-methyloxime acetates), and a study on hydrolytic conditions for glycoproteins and polysaccharides in order to increase sugar recoveries. Anal Biochem 1984; 142:58-67. 18. Honda S. High-performance liquid chromatography ofmono- and oligosaccharides. Anal Biochem 1984; 140:1-47. 19. Rinderknecht H, Geokas MC, Silverman P, Haverback BJ. A new ultrasensitive method for the determination of proteolytic activity. Clin Chim Acta 1968;21:197-203. 20. Qiu S-M, Wen G, Hirakawa N, Soloway RD, Hang N-K, Crowther RS. Glycochenodeoxycholic acid inhibits calcium phosphate formation in vitro by preventing the transformation of amorphous calcium phosphate to calcium hydroxyapatite. J Clin Invest 1991;88:1265-1271. 21. Crouch SR, Malmstadt HV. A mechanistic investigation of molybdenum blue method for determination of phosphate. Anal Chem 1967;39:1084-1089.
22. Crowther RS, Wetmore RF. Fluorometric assay of O-linked glycoproteins by reaction with 2-cyanoacetamide. Anal Biochem 1987; 163:170-174. 23. Qiu S-M, Soloway RD, Crowther RS. Interaction of bile salts with calcium hydroxyapatite: inhabiters of apatite formation exhibit high-affinity premicellar binding. HEPATOLOGY1992;16:12801289. 24. Pain RH. The viscoelasticity of mucus: a molecular model. Symp Soc Exp Biol 1981;24:359-376. 25. Blumenthal NC, Posner AS, Silverman LD, Rosenberg LC. Effect of proteoglycans on in vitro hydroxyapatite formation. Calcif Tissue Int 1979;27:75-82. 26. Smith BF, LaMent JT. Hydrophobic binding properties of bovine gallbladder mucin. J Biol Chem 1984;259:12170-12177. 27. Smith BF. Human gallbladder mucin binds biliary lipids and promotes cholesterol nucleation in model bile. J Lipid Res 1987; 28:1088-1097. 28. Smith BF, LaMent JT. Bovine gallbladder mucin binds bilirubin in vitro. Gastroenterology 1983;85:414-428. 29. Smith BF, Peetermans JA, Tanaka T, LaMent JT. Subunit interactions and physical properties of bovine gallbladder mucin. Gastroenterology 1989;97:179-187. 30. Reuben A. Biliary proteins. HEPATOLOGY1984;4:46S-50S. 31. Slomiany BL, Bliski J, Sarosiek J, Murty VLN, Dworkin B, VanHorn K, Zielenski J, et al. Campylobacter pyloridis degrades mucin and undermines gastric mucosal integrity. Biochem Biophys Res Commun 1987;144:307-314. 32. Poncz L, Jentoft N, He M-CD, Dearborn DG. Kinetics of proteolysis of hog gastric mucin by human neutrophil elastase and by Pseudomonas aeruginosa elastase. Infect Immun 1988;56:703704. 33. Crowther RS, Roomi NW, Fahim REF, Forstner JF. Vibrio cholerae metalloproteinase degrades intestinal mucin and facilitates enterotoxin-induced secretion from rat intestine. Biochim Biophys Acta 1987;924:393-402. 34. Roukema PA, Nieuw Amerongen AV. Sulphated glycoproteins in human saliva. In: Kleinberg I, Ellison SA, Mandel ID, eds. Workshop conference on saliva and dental caries. New York: Retrieval Inc., 1979:67-80. 35. Embery G, Green DRJ, Rolla G. Structural probe analysis on the attachment of salivary glycoproteins to hydroxyapatite using Fourier-transform infrared spectroscopy. Caries Res 1989;23: 247-251. 36. Embery G, Green DJR. The hydroxyapatite-bindingregions of a rat salivary glycoprotein. J Biol Buccale 1989;17:193-198. 37. Boskey AL, Dick BL. Hyaluronan interactions with hydroxyaparite do not alter in vitro hydroxyapatite crystal proliferation and growth. Matrix 1991; 11:442-446. 38. Shimizu S, Sabsay B, Veis A, Ostrow JD, Rege RV, Dawes LG. Isolation of an acidic protein from cholesterol gallstones, which inhibits the precipitation of calcium carbonate in vitro. J Clin Invest 1989;84:1990-1996. 39. Okido M, Shimizu S, Ostrow JD, Nakayama F. Isolation of a calcium-regulatory protein from black pigment gallstones: similarity with a protein from cholesterol gallstones. HEPATOLOGY 1992; 15:1079-1085. 40. Humbert W, Voegel JC, Kirsch R, Simonneaux V. Role of intestinal mucus in crystal biogenesis: an electron-microscopical, diffraction and X-ray microanalytical study. Cell Tissue Res 1989;255:575-583. 41. Akbarieh M, Tawashi R. Calcium oxalate crystal growth in the presence of mucin. Scanning Microsc 1991;5:1019-1027. 42. CareyMC, Calahane MJ. Whither biliary sludge? Gastroenterology 1988; 95:508-523. 43. Malet PF, Deng S-Q, Soloway RD. Gallbladder mucin and cholesterol and pigment gallstone formation in hamsters. Scand J Gastroenterol 1989;24:1055-1060. 44. Henisch HK. Crystal growth in gels. 1970. Pennsylvania State University Press, University Park, PA.
QIu, 1 G A R Y
W E N , 1 J U L I E W E N , 2 R O G E R D . SOLOWAY, 1 AND R O G E R S. C R O W T H E R 1
Calcium h y d r o x y a p a t i t e (HAP) crystals f o r m e d in vitro in t h e p r e s e n c e o f p o l y m e r i c h u m a n gallbladder m u c i n (1.0 mg/mL) w e r e s m a l l e r (0.75 _+ 0.39/xm) t h a n c o n t r o l crystals (7.86 + 2.76 ttm), but t h e m u c i n did n o t affect t h e k i n e t i c s o f crystal f o r m a t i o n or alter t h e a m o u n t of m i n e r a l p h a s e p r e s e n t at equilibrium. In contrast, g l y c o p e p t i d e s u b u n i t s p r o d u c e d by p r o t e o l y s i s o f the n a t i v e m u c i n h a d n o effect o n HAP crystal size. B o t h native m u c i n a n d g l y c o p e p t i d e s b o u n d to m a t u r e HAP crystals, but t h e g l y c o p e p t i d e s w e r e m u c h m o r e readily displaced by p h o s p h a t e ions. Therefore, in e x p e r i m e n t s w h e r e HAP w a s b e i n g formed, t h e p h o s p h a t e ions inhibited t h e i n t e r a c t i o n o f g l y c o p e p t i d e s w i t h t h e n a s c e n t HAP. T h e s e results i n d i c a t e that gallbladder m u c i n m a y m o d u l a t e HAP f o r m a t i o n in vivo, a n d that this ability m a y be altered d u r i n g p a t h o l o g i c a l states, s u c h as neutrophil infiltration or bacterial c o l o n i z a t i o n , that m a y c a u s e t h e r e l e a s e of p r o t e i n a s e s capable o f digesting mucin. (HEPATOLOGY1995;21:1618-1624.)
The inclusion of mucin in the organic matrix of cholesterol and pigment gallstones TM indicates a potential role for these epithelial glycoproteins in the pathogenesis of cholelithiasis. H u m a n gallbladder mucin promotes cholesterol nucleation in vitro in model biles, 5 as well as in prairie dog hepatic bile 6 and h u m a n gallbladder bile. 7 Much less obvious, however, is whether mucins are qualitatively or quantitatively different in normal subjects and those with gallstones. Lee and Nicholls s measured higher gallbladder mucin concentrations in gallstone patients compared with control patients, but this result was not supported by Harvey et al. 9 Compositional analysis showed t h a t mucins from patients with cholesterol gallstones were more exten-
Abbreviation: HAP, hydroxyapatite. From the 1Division of Gastroenterology, Department of Internal Medicine, and the 2Department of Pathology, University of Texas Medical Branch, Galveston, TX. Received July 12, 1994; accepted December 29, 1994. Supported by grant AM-16549 from the National Institutes of Health and by grants from the Scaly and Smith and M. D. Anderson Foundations. Address reprint requests to: Roger S. Crowther, PhD, Division of Gastroenterology, 301 University Blvd, University of Texas Medical Branch, Galveston, TX 77555-0764. Copyright © 1995 by the American Association for the Study of Liver Diseases. 0270-9139/95/2106-002053.00/0
sively sulfated t h a n were mucins from control subjects, s but there was no difference in the efficiency with which the mucins promoted cholesterol nucleation, v Less attention has been paid to the effect of mucin on the formation of the calcium salts found in gallstones. Yamasaki et al 1° showed t h a t mucin isolated from the hepatic bile of a patient without gallstones inhibited calcium carbonate precipitation in vitro. Mucin purified from T-tube bile of a patient with intrahepatic stones was more highly sulfated t h a n was control mucin obtained from a patient with no biliary tract disease, and it also had a significantly greater ability to cause aggregation of finely divided calcium carbonate. 11 Bovine gallbladder mucin alone inhibited the precipitation of CaHPO4, but when the mucin was complexed with an acidic, amphiphilic protein isolated from h u m a n gallstones, precipitation of the mineral phase was enhanced. 12 The effect of gallbladder mucin on the formation of calcium hydroxyapatite, Ca5OH(PO4)3 (HAP), has not been studied. However, salivary mucins bind to HAP of tooth enamel ~3'14 and influence the demineralization and remineralization of teeth. ~5 Because salivary and gallbladder mucins have compositional similarities, we hypothesized t h a t gallbladder mucin m a y significantly affect the formation of HAP. Here we report on the binding of h u m a n gallbladder mucin and its constituent glycopeptides to HAP and the consequent effect on HAP formation. MATERIALS A N D M E T H O D S
Calcium chloride, dibasic sodium phosphate, Trizma buffer, sodium chloride, and calcium hydroxyapatite (type I) were obtained from Sigma Chemical Co., St Louis, MO. HAP was analyzed for calcium and phosphate content, and the Ca/ P ratio was 1.69 +_0.01, which is consistent with HAP (1.67). Pronase was purchased from Boehringer-Mannheim Corp., Indianapolis, IN. Mucin Purification. Gallbladders were obtained from patients with cholesterol or pigment gallstones undergoing cholecystectomy. Bile was removed by aspiration with a syringe and 16-gauge needle and was pooled and stored at -20°C. Mucin was isolated as described by Pearson et al, 1~with modifications. After thawing, bile was diluted 1:1 with KSCN buffer (0.22 mol/L KSCN, 10 mmol/L Na2HPO4, and 0.01% sodium azide, pH 7.5) and centrifuged at 2,000g for 20 minutes to remove the sludge and other insoluble debris. The
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Vo]. 21, No. 6, 1995
supernatant was chromatographed in 20-mL batches on a gel filtration column of Sepharose CL-2B (110 × 3 cm) eluted with KSCN buffer at 0.6 mL/min. Eluted protein was monitored by fluorescence at excitation and emission wavelengths of 280 and 340 nm, respectively (Perkin-Elmer Corp., Norwalk, CT). The excluded protein peak, which contained the high-molecular-weight mucin, was dialyzed against distilled water at 4°C to remove the bulk of the KSCN. Sufficient CsC1 was added to bring the density to 1.45 g/mL, and the samples were centrifuged at 105,000g for at least 48 hours at 4°C (L870M ultracentrifuge, Beckman Instruments, Palo Alto, CA). The resulting gradients were fractionated by hand, and the density of each fraction was determined gravimetrically. The protein profile was monitored by fluorescence, and proteincontaining fractions with densities between 1.40 and 1.50, which is typical of mucin, were pooled. The density of the pooled fraction was readjusted to 1.45 g/mL, and the sample was subjected to an additional two cycles of CsC1 density gradient ultracentrifugation to remove residual noncovalently bound protein. The purified mucin was exhaustively dialyzed against distilled/deionized water at 4°C and was concentrated by ultrafiltration through a 30,000 molecular weight cut-off membrane (YM30, Amicon Corp., Danvers, MA). An eightfold concentrated solution of Tris/NaC1 was added to the concentrated mucin to bring the final Tris and saline concentrations to 50 and 100 mmol/L, respectively. The resulting mucin concentration was determined fluorimetrically by assaying for O-linked oligosaccharides with 2-cyanoacetamide, 17 using a gravimetrically standardized sample of canine tracheal mucin as the standard. The mucin (2.4 rag/ mL) was stored in l-mL aliquots at -20°C. To establish that mucin solubility was not adversely affected by storage at -20°C, a sample of the frozen mucin was thawed and diluted to 1.0 mg/mL with Tris/NaC1. The diluted mucin was incubated at 37°C for 30 minutes, and mucin aggregates were dispersed by very gentle passage through a 26-gauge needle. A portion of the solution was centrifuged at 11,000g for 5 minutes to pellet undispersed mucin, and the mucin concentration in the centrifuged and uncentrifuged aliquots was determined fluorimetrically with 2-cyanoacetamide. There was no difference in the mucin concentration of the centrifuged (1.09 _+ 0.04 mg/mL) and uncentrifuged (1.10 _+ 0.11 mg/mL) samples, which demonstrated that mucin did not irreversibly aggregate during storage at -20°C. In all further experiments, whenever a mucin sample was thawed, the mucin was dispersed by incubation at 37°C and passage through a 26-gauge needle as described above. Chemical Analysis. Mucin amino acid content was measured with an automated analyzer (121MB Analyzer, Beckman Instruments). For neutral and amino sugar analysis, mucin was hydrolyzed at 125°C for I hour in 4 N trifluoroacetic acid, 17 freeze-dried, and reconstituted in water. For sialic acid analysis, mucin was hydrolyzed in 0.1 N HC1 for i hour at 80°C, 18 freeze-dried, and redissolved in water. The carbohydrate composition of the hydrolyzed mucin was then analyzed by anion exchange high-pressure liquid chromatography with pulsed amperometric detection (BioLC, Dionex Corp., Sunnyvale, CA). The amino acid and carbohydrate composition of the purified mucin was very similar to that given by Pearson et al TM for human gallbladder mucin (Table 1). Generation of Mucin Glycopeptides. Mucin was degraded to its constituent glycopeptides by digestion with pronase. Mucin (2 mg/mL in Tris/saline buffer) was incubated for 16 hours at 37°C with 0.2 mg/mL pronase. A control incubation was also performed in which mucin was incubated with pro-
QIU ET AL
1619
TABLE 1. C o m p o s i t i o n a l A n a l y s i s o f P u r i f i e d Gallbladder Mucin Amino Acid
Asp Tbr Ser Glu Pro Gly Ala Cys Val Met Ile Leu Tyr Pbe His Lys Arg
Residues/100 Residues
5.60* 19.24 10.88 7.66 9.00 7.87 9.01 ND 5.58 0.90 2.72 8.60 1.48 2.73 2.66 2.82 3.33
Carbohydrate
6.13t 19.75 12.83 7.95 12.83 8.74 8.51 ND 5.90 0.91 2.95 5.68 1.70 2.50 2.84 3.41 3.29
Fuc GalNAc GlcNAc Gal NANA
Molar Ratio
2.88* 1.00 4.38 4.98 1.29
2.67~ 1.00 4.03 3.72 ND
Abbreviation: ND, not determined. * This study. Data from Pearson et al. TM
nase that had been inactivated by boiling for 10 minutes. Comparison of the proteolytic activity of the native and heatinactivated pronase by measuring the solubilization of hide powder azure 19 showed that more than 90% of the enzyme activity was abolished by boiling. Effect of Mucin on Hydroxyapatite Formation. HAP was formed from solutions containing 4 mmol/L CaCI2, 4 mmol/ L Na2HPO4, 50 mmol/L Tris, and 100 mmol/L NaC1 exactly as previously described. 2° Briefly, twofold concentrated solutions of calcium or phosphate in Tris/saline buffer were equilibrated to 37°C, mixed, and then placed in quartz cuvettes maintained at 37°C. Mineral phase formation was monitored by measuring turbidity at 220 nm (model 4053 spectrophotometer, Biochrom, Cambridge, England). 2° The effect of mucin was studied over the concentration range of 0.1 to 1.0 mg/ mL. In some experiments, mucin glycopeptides were used instead of native mucin. At the end of the experiment (usually 250 minutes), the solutions were removed from the cuvettes and centrifuged at 11,000g for 5 minutes. The phosphate concentration in the supernatant was determined spectrophotometrically by the molybdenum blue method, 21 and the amount of precipitated HAP was calculated. Transmission Electron Microscopy. Hydr0xyapatite was allowed to form as described above for 250 minutes in the presence of increasing concentrations of mucin or glycopeptides. At the end of the incubation, the samples were gently agitated to resuspend any sedimented particles, and 2 #L of the suspension was removed and transferred to a formvarcoated, carbon-stabilized copper grid (Ernest F. Fullam Inc., Latham, NY). The suspension was allowed to remain in contact with the grid for 5 minutes, and the drop was then removed by carefully touching it with tissue paper. Samples were examined at 60 kV by transmission electron microscopy. The particle size distribution was estimated by measuring with a ruler the largest dimension of 10 individual particles in each of at least three separate photographic enlargements for every sample. Particles that appeared to be aggregates of smaller particles were treated as a single particle.
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HEPATOLOGYJune 1995
Mucin Binding to Hydroxyapatite. Ten milligrams of HAP were suspended in 0.5 mL of Tris/sa]ine buffer containing up to 1.0 mg/mL of native mucin or mucin glycopeptides in a polypropylene microtube. Control incubations without HAP were also performed. The suspension was incubated for 2 hours at room temperature with intermittent vortexing, and then centrifuged at ll,000g for 5 minutes to pellet HAP and bound mucin. Mucin remaining in the supernatant was measured fluorimetrically with 2-cyanoacetamide, and the amount bound was calculated by difference. Preliminary experiments showed that equilibrium was reached in approximately 60 minutes, To determine the relative binding to HAP of glycopeptides of different sizes, 200 #L of the supernatant was removed and subjected to gel filtration chromatography on Sepharose CL-2B (30 × 1.5 cm). The column was eluted with 0.15 mol/ L NaC] at 0.4 mL/min, and fractions of 1.0 mL were collected and analyzed for O-linked oligosaccharides with 2-cyanoacetamide. ~2 The effect of phosphate ions on mucin binding to HAP was investigated at a fixed mucin or glycopeptide concentration of 0.2 mg/mL by adding increasing concentrations of Na2HPO4, and the ionic strength was kept constant by reciprocally reducing the NaC1 concentration. All other conditions were as described above. RESULTS
Calcium Phosphate Precipitation. In the absence of mucin, H A P began to precipitate at ~ 5 0 minutes. The time at which H A P formation commences is referred to as the induction time. 2° We have previously confirmed by chemical analysis and infrared spectroscopy t h a t this mineral phase is HAP. 2° The optical density of the solution rose for a f u r t h e r 10 minutes, b u t t h e n began to decline as the HAP particles became sufficiently large to sediment (Fig. 1). Native mucin at a concentration of 0.5 mg/mL h a d no effect on the induction time, b u t p r e v e n t e d the decline in optical density t h a t was associated with sedimentation (Fig. 1). Similar results were observed with native mucin concentrations of 0.1 and 1.0 mg/mL (not shown). W h e n mucin glycopeptides (0.5 mg/mL) were substituted for native mucin, the inhibition of HAP sedim e n t a t i o n was abolished (Fig. 1). The a m o u n t of HAP precipitated at 250 minutes was not affected by either native mucin or glycopeptides (Table 2). W h e n HAP precipitation was allowed to proceed for 24 hours, there was no change in the a m o u n t of HAP formed (not shown), which indicated t h a t equilibrium was attained within 250 minutes. The p r o n a s e p r e p a r a t i o n u s e d to g e n e r a t e glycopeptides c o n t a i n e d Ca 2+, b u t this r e s u l t e d in a less t h a n 1% increase in Ca 2+ c o n c e n t r a t i o n in p r e c i p i t a t i o n exp e r i m e n t s u s i n g glycopeptides. This small i n c r e a s e would not be expected to a l t e r the kinetics of H A P formation, a n d in a control e x p e r i m e n t with p r o n a s e alone (final c o n c e n t r a t i o n , 50 #g/mL), t h e induction time was a l m o s t identical to t h e i n d u c t i o n t i m e w i t h o u t pronase, 50 a n d 54 m i n u t e s , respectively. U s i n g t r a n s m i s s i o n electron microscopy, H A P was observed to form i r r e g u l a r l y s h a p e d particles a n d agg r e g a t e s w i t h " f e a t h e r e d " edges (Fig. 2A). S i m i l a r particles w e r e observed for H A P f o r m e d in the p r e s e n c e of
1.5 I:p----~EI
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0.5 mg/ml mucin 0.5 mg/ml gl ycopepti de
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0.0 0
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90
120
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240
T i m e , mJ n
FIG. 1. Formation of calcium hydroxyapatite monitored by optical density. Ca2÷ and phosphate (final concentration of each, 4 mmol/ L) in 50 mmol/L Tris/100 mmol/L NaC1 buffer, pH 7.3, were mixed and incubated at 37°C with or without native mucin or glycopeptides. Mineral phase separation was followed by monitoring optical density at 220 nm. 0.5 m g / m L glycopeptides (Fig. 2B), b u t the particles were s m a l l e r w h e n 0.5 m g / m L n a t i v e m u c i n was present (Fig. 2C). The effect of n a t i v e m u c i n a n d glycopeptides on t h e m e a n H A P particle size is given in Table 2. Binding of Mucin and Glyeopeptides to HAP. B o t h n a t i v e m u c i n a n d glycopeptides were b o u n d to HAP, b u t H A P a p p e a r e d to h a v e a h i g h e r capacity for t h e glycopeptides (Fig. 3). In control incubations, o m i t t i n g HAP, all the m u c i n was r e c o v e r e d in t h e s u p e r n a t a n t a f t e r centrifugation, which d e m o n s t r a t e d t h a t t h e r e was no nonspecific b i n d i n g to t h e t u b e s a n d t h a t the m u c i n was sufficiently dispersed to r e m a i n in solution. W h e n m u c i n was i n c u b a t e d for 16 h o u r s at 37°C with h e a t - i n a c t i v a t e d pronase, t h e r e was no effect on the a m o u n t of m u c i n s u b s e q u e n t l y b o u n d to H A P (Fig. 3). To e s t i m a t e t h e size distribution of the glycopeptides p r o d u c e d by p r o n a s e digestion, t h e y were chromatog r a p h e d on a c o l u m n of S e p h a r o s e CL-2B. N a t i v e mucin e l u t e d as a single p e a k in t h e c o l u m n void volume, w h e r e a s the p r o n a s e - t r e a t e d m u c i n was d e g r a d e d into glycopeptides t h a t showed considerable size heterogen e i t y (Fig. 4A). This h e t e r o g e n e i t y is in a g r e e m e n t w i t h the observation of P e a r s o n et al, 1~ who e s t i m a t e d t h e m o l e c u l a r size of p r o t e i n a s e - d i g e s t e d h u m a n gallbladder m u c i n to be on t h e o r d e r of m a g n i t u d e of 5 × 105. To d e t e r m i n e if glycopeptide size influenced binding to HAP, glycopeptides a n d H A P w e r e i n c u b a t e d together, a n d the s u p e r n a t a n t a f t e r c e n t r i f u g a t i o n was chrom a t o g r a p h e d on S e p h a r o s e CL-2B (Fig. 4B). Midsize
QIU E T A L
HEPATOLOGY Vol. 21, No. 6, 1995 TABLE 2. E f f e c t o f N a t i v e M u c i n a n d G l y c o p e p t i d e s
on HAP Formation
Control
0.1
0.5
1.0
Glycopeptides (mg/mL) 0.5
50 0.38 _+ 0.02 7.86 _+ 2.76
50 0.39 _+ 0.01 2.79 _+ 1.76"
50 0.38 _+ 0.01 1.16 _+ 0.73*
45 0.37 _+ 0.01 0.75 _+ 0.39*
54 0.39 _+ 0.01 8.24 _+ 4.53
Native Mucin (mg/mL)
Induction time, min HAP formed, #mol/mL Particle size, pm
1621
* Significantly different from control (P < .9001, t-test).
glycopeptides were bound to HAP to a greater extent than were either residual intact mucin or very small glycopeptides, and the maximum binding efficiency occurred in fractions 17 and 18 (Fig. 4B). Because glycopeptides had no effect on HAP formation, but were bound more extensively to HAP than was the native mucin, we hypothesized that specific components that were present only in the precipitation experiments preferentially inhibited glycopeptide binding to HAP. We have previously shown that bile salt binding to HAP is competitively inhibited by phosphate ions, 23 and we therefore chose to investigate the effect of phosphate on mucin binding. Low concentrations of phosphate had no effect on either mucin or glycopeptide binding to HAP, but above 1 mmol/L phosphate, there was a progressive decline in binding, and this effect
was more pronounced for the glycopeptides than for the native mucin (Fig. 5). DISCUSSION
The current study has shown that native h u m a n gallbladder mucin binds to HAP crystals in vitro and reduces the particle size of nascent HAP, but it does not alter the kinetics of HAP formation or reduce the amount of HAP precipitated at equilibrium. Proteolytically produced mucin glycopeptides also bound to HAP, but were without effect on HAP particle size. The effect of native mucin on HAP particle size was probably underestimated in these experiments because HAP was placed on the electron microscopy grid by allowing it to settle under gravity, which would favor the sampling of the largest particles. In addition, our measurement
B
c
FIG. 2. Transmission electron micrographs of HAP crystals. HAP was allowed to form for 250 minutes under the conditions described in the legend for Fig. 1. either without mucin (A~, or with 0.5 rag/ mL of mucin glycopeptides (B) or native mucin (C). In each case the original magnification is x17,500, and the bar represents i #m.
Q
1622
QIU E T A L
HEPATOLOGY J u n e 1995
increase the stability of the HAP-mucin complex. Alternatively, the hydrophobicity of the nonglycosylated regions may be less important than their function of covalently linking the glycosy]ated segments together, giving these segments the ability to bind to HAP cooperatively rather than as individual units. The easier displacement of glycopeptides by phosphate was somewhat surprising because analysis of the effect ofglycopeptide size on bind-
350
300
2S0 I
..d r." :::I
200
o
.Q ¢..
150
140 C
1 30 °
"
100
~"
0
//
50
•
0.0
N
,ve m c,n/he
i nacti vated pronase
y
Ol
Glycopepti des
0.2 Initial
0.4
0.6
0.8
mucin concentration,
1.0
,
20
•
s
o
1.z
mg/ml
FIG. 3. Mucin binding to HAP. Increasing concentrations of native mucin or glycopeptides were incubated for 2 hours at room temperature with 10 mg HAP suspended in 0.5 mL Tris/NaC1 buffer. After centrifuging at ll,000g for 5 minutes, the mucin remaining in the s u p e r n a t a n t was measured, and the amount bound was calculated by difference.
E 2o
,',
z
0000000
0 0
of particle size was relatively imprecise, and did not account for the three-dimensional nature of the particles. Despite these technical limitations, the reduction of particle size by mucin was unambiguous, and was consistent with the sustained increase in optical density seen in the precipitation experiments. Although native mucins have a much greater effect on solution viscosity than do glycopeptides,24 this effect probably did not cause the reduction of particle size because Blumenthal et a125 showed that viscosity per se does not affect HAP formation. Therefore, the effect of mucin on HAP precipitation probably resulted from the interaction with the crystal surface. Although glycopeptides also bound to HAP, they were much more readily displaced by phosphate ions than was the native mucin, and therefore, in precipitation experiments containing initial phosphate concentrations of 4 mmol/L, there was probably minimal interaction of the glycopeptides with the nascent HAP. The greater ease of displacement of glycopeptides compared with native mucin indicates the importance of the nonglycosylated segments of the mucin molecule for binding to HAP, because these regions are largely removed by digestion with pronase. In gallbladder mucin these nonglycosylated segments are relatively hydrophobic2~ and have been shown to be responsible for the interaction of mucin with phosphatidylcholine and cholesterol27 and bilirubin 2s'29 and are postulated to have a role in the self-association of bovine gallbladder mucin. 29 It is unlikely that mucin interacts with HAP hydrophobically, but adjacent mucin molecules on the HAP surface may form hydrophobic bonds with each other that
S
10
15
0
20
•
25
30
II
25
v
II
0 30
100
~--
8O
~, <
20
60 ,,,,i
"~
o 4-J "O e" O
40
~
20
e~
10
e~
o U
5
O
0 0
S
10
15
20
25
0 30
Elution volume, ml FIG. 4. Effect of glycopeptide size on binding to HAP. (A) Two hundred microliters native mucin or pronase-produced g]ycopeptides, each 1.0 mg/mL, was chromatographed on Sepharose CL-2B, 1-mL fractions were collected and analyzed for O-linked oligosaccharides with 2-cyanoacetamide. (B) Mucin glycopeptides (1.0 mg/mL) were incubated with HAP for 2 hours, the unbound fraction was collected by centrifugation, and 200 #L was chromatographed on Sepharose CL-2B. The percentage of glycopeptides bound to HAP (bars) was calculated from the difference between the fluorescence of the fractions obtained from the total glycopeptide mixture before the addition of HAP (from Fig. 4A) and the fluorescence of the fractions obtained from the unbound glycopeptides.
HEPATOLOGY Vol. 21, No. 6,1995
QIU E T A L
120
I
1O0 -
=L
~
Native mucin
~
Glycopepti des
80
eo
60
~
40
i
20
~
0
1
2
1,1,[ 3
4
5
P h o s p h a t e c o n c e n t r a t i on, mM FIG. 5. Effect of phosphate on native mucin and glycopeptide binding to HAP. Native mucin or glycopeptides (0.2 mg/mL) were incubated with HAP in buffer with increasing concentrations of Na2HPO4, and the ionic s t r e n g t h was kept constant by reciprocally reducing the NaC1 concentration. All other conditions were as described in the legend to Fig. 3.
1623
but that exposed parts of the protein core were also involved. 35 Using different techniques, the binding of glycoprotein from rat minor salivary gland was also concluded to be mediated mainly by ester sulfate groups. 3~ The possible importance of sulfated carbohydrates is interesting, because it has been reported that the sulfation of biliary mucins is increased in lithogenic bile. s'11 Both sialic acid and ester sulfate groups are negatively charged under physiologic conditions, which indicates that electrostatic interactions with surface calcium ions may be important for mucin binding to HAP. This interpretation is consistent with the reduction in binding caused by phosphate ions, which may be viewed as competition between the phosphate and the polyanion for a common binding site on the HAP surface. Other high-molecular-weight glycoconjugates also affect HAP formation. Proteoglycan aggregates and subunits had different effects than did mucin, because these macromolecules increased the time required for HAP precipitation to commenceY Hyaluron, the polysaccharide backbone required for proteoglycan aggregation, was bound to HAP but had no effect on HAP precipitation. 37 Mucins from various sources have also been shown to regulate the precipitation of other calcium salts. Mucin from h u m a n hepatic bile inhibited calcium carbonate precipitation, 1° but the effect was small compared with that of a low-molecular-weight acidic protein that was isolated from cholesterol 3s or black pigment gallstones. 39 Calcium carbonate precipitation is also regulated by the intestinal mucus of the silver eel, b u t in this case the mucous gel acts as a matrix for biomineratization that results in the production of calcite crystals. 4° Bovine submaxillary mucin altered the size and crystal habit of calcium oxalate crystals by inhibiting the growth of specific crystal
ing to HAP indicated that midsize glycopeptides were adsorbed in preference to residual intact mucin. However, this apparent preference for midsize glycopeptides may indicate faster binding kinetics for these smaller, more diffusible species, rather than a higher binding affinity. The greater binding capacity of HAP for glycopep- f a c e s . 41 tides versus native mucin may reflect the collapse of the An important observation about gallstone pathogenexpanded mucin conformation caused by proteolysis, 24 esis that has resulted from work with animal models which would allow more efficient packing of the glycosyl- is that cholesteroP 2'43 and pigment 43 gallstones are freated subunits on the HAP surface. The reduced effect of quently nidated in the mucous gel layer adherent to the glycopeptides in comparison with native mucin on HAP gallbladder mucosa. A limitation of the current study is formation may have pathologic significance because, al- that it was performed at mucin concentrations that though neither normal bile nor gallbladder mucosa con- were too low to permit gel formation, b u t extrapolation tains proteolytic enzymes that digest mucin, 3° many bac- from the current results suggests that significant efterial proteases are capable of degrading mucins, 3133 and fects on HAP formation would be likely to occur in a bacteria have been assigned a potential role in gallstone mucin gel. Gels frequently act as nucleation sites for pathogenesis. In addition, the recruitment and activation crystals, by mechanisms that are imperfectly underof neutrophils may also lead to mucin degradation be- stood, 44 and it is possible that the smaller crystals cause human neutrophil elastase has been shown to di- formed in the presence of these relatively dilute mucin gest mucin. 32 solutions resulted from the nucleation of a greater The interaction of salivary mucins with HAP has been number of embryos than was formed in the control exextensively investigated, but no common structural motif periments. The ability of mucin to bind to HAP crystals responsible for binding has been identified. For example, m a y enable it to cement crystals together and thus removal of sialic acid from the oligosaccharides of ovine cause crystal agglomeration that may be an essential submandibular mucin prevented its binding to HAP, but precursor to gallstone formation. The effect of mucins similar treatment of human submandibular mucins had may also be modified by other biliary proteins, and it only a small effect on binding to HAP. ~4Analysis by infra- has been shown that the calcium binding protein that red spectroscopy of the binding of a sulfated mucin from was isolated from h u m a n gallstones 3s'39 alters the abilhuman whole saliva suggested that the sulfated carbohy- ity of mucin to inhibit CaHPO4 precipitation. ~2 The imdrates were particularly important for binding to HAP, portance of the chemical composition of gallbladder
1624
QIU ET AL
HEPATOLOGYJune 1995
mucin and in particular the effect of sulfation on the formation of HAP also remain to be investigated. REFERENCES
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