Biomacromolecules 2004, 5, 269-275
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Articles Interaction of Mucin with Cholesterol Enriched Vesicles: Role of Mucin Structural Domains Nezam H. Afdhal,*,† Xingxiang Cao,‡,§ Rama Bansil,‡ Zhenning Hong,‡ Christine Thompson,| Beth Brown,| and David Wolf|,# Liver Center, Beth Israel Deaconess Medical Center, 110 Francis Street, Boston, Massachusetts 02215, Physics Department, Boston University, 590 Commonwealth Avenue, Boston Massachusetts 02215, and Worcester Foundation for Biomedical Research, Worcester, Massachusetts 01545 Received June 4, 2003; Revised Manuscript Received November 26, 2003
We utilized fluorescence recovery after photobleaching (FRAP) and fluorescence correlation spectroscopy (FCS) to examine the role of gallbladder mucin (GBM) in promoting the aggregation and/or fusion of cholesterol enriched vesicles. By fluorescent labeling either the vesicle or the mucin, we could examine the change in vesicle size as well as changes in mucin’s diffusion constant. Both FRAP and FCS show that GBM has a profound effect in inducing vesicles to aggregate/fuse, particularly after overnight incubation. GBM mucin domains (either protease digested or reduced GBM) are not as effective as native GBM. Intact GBM alone was able to shorten crystal appearance time and increase the number of crystals nucleated by polarized optical microscopy. In summary, our findings would suggest that both glycosylated and nonglycosylated domains of GBM are involved in early aggregation of cholesterol enriched vesicles but that this effect is reversible in the absence of nonglycosylated domains. Introduction The pathogenesis of cholesterol gallstones is multifactorial and involves secretion of cholesterol supersaturated bile, nucleation of cholesterol monohydrate crystals, and impaired gallbladder contraction resulting in growth of crystals.1 Nucleation of cholesterol (Ch) monohydrate crystals from Ch supersaturated gallbladder bile is a heterogeneous process and is controlled by the interplay of factors that promote (pronucleating) and inhibit (antinucleating) nucleation in bile.1 Pronucleating factors include potentially biliary proteins, apolipoproteins, and biliary lipid particles. A major pro-nucleating glycoprotein is gallbladder mucin which is also a criticial component for the formation of the biliary gel or sludge in which gallstones form and grow. Cholesterol is transported in bile complexed with bile salts and lecithin either as micelles or as simple unilamellar vesicles. Supersaturation of gallbladder bile with Ch is almost universally present in individuals who develop cholesterol gallstones. The common occurrence of Ch supersaturation in the gallbladder bile of normal individuals, however, indicates that additional factors, such as pronucleating agents, * To whom correspondence should be addressed. † Beth Isarel Deaconess Medical Center. ‡ Boston University. § Current address: Wyatt Technology Corporation, 30 South La Patera Lane, B-7, Santa Barbara, California 93117. | Worcester Foundation for Biomedical Research. # Current address: Sensor Technologies, 910 Boston Turnpike, Shrewsbury, Massachusetts 01545.
are needed to promote the initiation and growth of cholesterol gallstones. In the presence of increasing cholesterol supersaturation of bile, simple unilamellar vesicles are enriched with cholesterol and can aggregate and fuse to become multilamellar vesicles (liquid crystals) and nucleate into solid cholesterol monohydrate crystals.2,3 In cholesterol supersaturated gallbladder bile (total lipid content of ∼10 g/dL), cholesterol exists in the form of simple small unilamellar vesicles (SUV): these are a single lipid bilayer with a diameter 40-80 nm, composed of about 1:1 Ch and lecithin (PC) (phosphatidylcholine, the most predominant phospholipid in bile). Nucleation studies of Ch crystals suggest that SUVs fuse to form large multilamellar vesicles of about 500 nm in diameter that represent liquid crystals of Ch within the nucleation pathway.2,3 Multiple biliary glycoproteins have been shown to accelerate the Ch crystallization in model bile systems.4-8 The molecular mechanisms by which these proteins promote Ch crystallization in bile have not been identified, although gallbladder mucin (GBM) has been shown to preferentially bind to vesicles in cholesterol supersaturated bile.9 GBM, a major secretory product of the gallbladder epithelium, has a typical structure that is common to members in the mucin family. GBM is a macromolecular glycoprotein consisting of hydrophilic regions which are heavily glycosylated with oligosaccharide side chains 8-14 sugar residues in length and hydrophobic, non - glycosylated regions that bind biliary lipids.10-14 The effects of GBM on
10.1021/bm0341733 CCC: $27.50 © 2004 American Chemical Society Published on Web 01/07/2004
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vesicle aggregation and fusion have been studied by light scattering and fluorescence resonance energy transfer assays.9 These studies suggested that native GBM was able to initiate and accelerate vesicle fusion. However, these studies were not able to identify the mechanism of interaction of mucin or its different structural domains with the vesicles because they only examined changes in vesicle size and fluorescence without selective labeling of mucin. The present work is intended to demonstrate conclusively that GBM is directly involved in promoting the aggregation and/or fusion of Ch-enriched vesicles and that the unique architecture of the GBM molecule plays an important role in this promotion mechanism. Fluorescence recovery after photobleaching (FRAP) and the related technique of fluorescence correlation spectroscopy (FCS) are sensitive techniques for measuring the lateral diffusion of macromolecules in membranes and aqueous solutions. By selective fluorescent labeling of one component in a multicomponent system, it is possible to measure the diffusion of the labeled target. Because the diffusion constant is inversely proportional to the size of the diffusing species, one can follow the changes in the aggregation state of the selected species. Thus, by labeling either the vesicles or the mucin macromolecules, we were able to monitor the effects of mucin on vesicle fusion, as well as address the question of whether mucin is in any way altered or incorporated into the fusion product. Our results show that specific domains of GBM are involved in aggregation and binding of biliary lipids and may lead to strategies to prevent cholesterol nucleation and growth. Experimental Methods Model Bile and Vesicle Preparation. In brief, cholesterol supersaturated model biles with a cholesterol saturation index (CSI, defined as equal to 1 at saturation) of 1.4 were prepared by coprecipitation of the lipids from organic solvents as previously described.9 Cholesterol (Ch) was purified by double recrystallization from hot 95% ethanol, and sodium taurocholate (NaTC) was recrystallized twice by the method of Pope.13 Phosphatidylcholine (PC) was used as supplied in chloroform:methanol 9:1 (vol:vol). The total lipid concentration of model bile was 10 g/dL, reflecting the average concentration of lipids in lithogenic human gallbladder bile. The molar ratio of PC/(NaTC + PC) in model bile is 0.2. Following coprecipitation of lipids in a rotary evaporator, model biles were suspended in 0.01 M Tris, pH 7.0 containing 0.145 M NaCl, heated to 80 °C for 1 h to obtain an isotropic phase, and then filtered through a sterile 0.22 µm filter. Model biles were equilibrated at 37 °C for 4 h. 0.5 mL aliquots of the model bile were applied to a Pharmacia FPLC system equipped with a Superose 6 gel filtration column equilibrated with 7.5 mM NaTC at 1 mL/ min.9 Separation of bile into vesicular and micellar fractions was confirmed by measuring cholesterol, bile salts, and phospholipid. This procedure results in effective separation of vesicles within 1 h. Fluorescent labeling of vesicles was achieved by the addition of fluorescein-DHPE (N-(5-fluoresceinthiocarbamoyl)-1,2-dihexadecanoyl-sn-glycero-3-phos-
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phoethanolamine, triethylammonium salt). The DHPE labeled vesicles were also separated on the Superose 6 gel filtration column. Bovine Gallbladder Mucin (GBM) Preparation. Mucin was purified from bovine gallbladders by scraping the epithelial surface with a glass slide, homogenizing the mucus in 0.2 M NaCl with 0.04% sodium azide, and centrifuging for 30 min at 50 000 g in a Beckman J2-21 centrifuge. Soluble mucin was further purified by size exclusion chromatography on Sepharose CL-4B and subsequent equilibrium density gradient ultracentrifugation in 60% cesium chloride. Glycoprotein at a buoyant density of 1.48 g/mL was repurified through a second 60% cesium chloride gradient. Purified mucin was shown to be free of low molecular weight protein and glycoprotein contaminants by SDS-PAGE (sodium dodecyl sulfate polyacrylamide gel electrophoresis) gel stained with both silver and PAS.15 Thinlayer chromatography of mucin associated lipids showed no contamination of purified mucin with either neutral lipids or phospholipids. Mucin was labeled with fluorescein isothiocyanante (FITC-GBM) for studies of changes in mucin diffusion by FRAP. Nonglycosylated free amine groups on the mucin protein core were fluorescently labeled by conjugation with carboxytetramethylrhodamine succinimidyl ester (CTR) as per manufacturers protocol (Molecular Probes Inc, Eugene, OR) for binding studies described below. Reduced GBM (r-GBM) Preparation. The reduction of disulfide bonds of GBM was performed according to a modified method of Snary.15 In brief, GBM was incubated in 6.0 M guanidinium chloride/5 mM EDTA/10 mM dithiothreitol/0.10 M Tris/HCl buffer, pH 8.0, for 5 h at 37 °C. Free thiol groups were subsequently alkylated by the addition of iodoacetamide. After dialysis against sodium phosphate buffer of pH 6.5, the fragments were subjected to densitygradient centrifugation and collected, lyophilized. Protease Digested GBM (p-GBM) Preparation. The digestion of hydrophobic domains of GBM was performed according to a modification of the method of Scawen and Allen.16 In brief, GBM was incubated in 0.1 M sodium phosphate buffer (0.04% sodium azide, pH 6.8) with nonspecific protease (Sigma Chemical Co. Type XIV: from Streptomyces griseus) at a ratio of 1:20 (wt/wt) at 37 °C for 48 h. After digestion, the mixture was dialyzed for 72 h against water and lyophilized. Confirmation of digestion of mucin was performed by running the digested mucin on SDS-PAGE and staining with both silver and PAS as described above. Digested mucin appeared as a smear of peptides of varying molecular weight, whereas native mucin was present at the interface of the stacking and running gels. Immunoglobulins. Immunoglobulins, which are potent pro-nucleating agents, were utilized as control proteins, for the vesicle fusion assays by FRS. Immunoglobins IgG and IgA were purchased from Sigma Chemicals, St. Louis, MO. In addition, IgG and IgA were purified from bile of gallstones patients using affinity chromatography as previously described5 (gift of R. Harvey, St. Louis, MO). Vesicle-Protein Interactions. GBM, r-GBM, and pGBM (1 and 4 mg/mL, dissolved in model bile buffer) were
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mixed with labeled vesicles at a total lipid concentration of 800 mM which we had previously used for vesicle fusion studies.9 The vesicle-protein interaction was then studied by monitoring the diffusion behavior selectively of either the protein or the vesicle, as described below. Fluorescence Recovery after Photobleaching (FRAP). In FRAP measurements, the molecules whose diffusion is to be measured must be labeled with a fluorescent chromophore directly in a non-cross-linking manner. A laser beam is focused using a fluorescence microscope to a small area (∼µm) on the sample. The light from this area is monitored, and the fluorescence intensity is found to be essentially constant with time. The intensity of the incident light is then momentarily raised approximately 104-fold so as to irreversibly photobleach a significant fraction of the fluorescence within the area, thereby significantly reducing the fluorescence intensity. The rate at which the bleached area recovers its fluorescence intensity, as monitored by the fluorescence microscope, indicates the rate at which unbleached and bleached fluorescence labeled molecules diffuse in to and out of the bleached area, respectively. The diffusion coefficient is obtained by fitting the recovery data to diffusion theory.17,18 In addition to being a conventional technique for determining the lateral diffuison of lipids and membrane proteins in the membrane cells, FRAP can also be used as a powerful tool to study the diffusion of fluorescence labeled macromolecules and particles in solutions, especially in a multicomponent system. FRAP can selectively measure the diffusion of a particular target of interest in a complex solution where more than one species coexist as solute, if the target species is fluorescence labeled. In the study of mucin-induced vesicle aggregation and fusion, FRAP is used to selectively monitor the vesicle fusion process with vesicles labeled with fluorescein-DHPE. All FRAP measurements for our vesicle + protein study were performed at room temperature, the bleaching time was set at 200 ms, with the bleaching laser power ∼50 mW using the λ ) 514.5 nm line of an argon laser. Monitoring intensity was about 5 µW. The data were analyzed according to the theories described by Barisas and Leuther and Wolf and Edidin.19,20 Vesicle + protein mixtures for FRAP measurements were prepared as described in the previous section. Samples were then taken up in 100-µm path-length flat capillary tubes (Vitro Dynamics, Rockaway, NJ) for photobleaching measurements. Twenty or more measurements were taken for each sample at different spots, and the average value was presented as the final result. The FRAP system was calibrated by measuring the diffusion of Fluorescein-labeled bovine serum albumin (BSA) in solution, which has a known diffusion constant of 3.25 × 10-7 cm2/s, to obtain the absolute value of diffusion constants of samples measured. When necessary, the diffusion coefficient D can be used to obtain the hydrodynamic diameter dH or hydrodyanmic radius RH of the particle via the Stokes-Einstein equation, dH ≡ 2RH ) kBT/3πηD, where kB is the usual Boltzmann constant, T is the temperature in degrees Kelvin, and η is the solvent viscosity. In a multicomponent system, a change
in the diffusion constant D can reflect either the change in its size dH or the change in the viscosity of the medium due to the presence of the other components. For the system of SUV’s in mucin solutions at 1-4 mg/mL, we have shown that the vesicles sense the microviscosity of the solution, rather than the bulk macroscopic viscosity of the polymer solution.21 This viscosity effect corresponds to less than 20% change in the diffusion constant, and the change in D values is largely attributed to change in the size of the vesicles. Fluorescence Correlation Spectroscopy (FCS). Fluorescence correlation spectroscopy (FCS) has been widely applied to the study of molecular diffusion and molecular complexing in solution,17,18,22,23 in membranes,24 and on surfaces when coupled with total internal reflection.25 As described below, this technique extracts diffusion rates and complex size from the temporal fluctuations of a system about equilibrium. In FCS, one measures fluctuations in fluorescence intensity which result from diffusion of fluorescent molecules in and out of the spot, as well as other phenomena which alter fluorescence. These latter include binding and in the case of polarized fluorescence, polarization.18 The basic theory involves the calculation of the correlation between one point in time and later points in time by dividing the data stream into a series of counting intervals of duration t. Then the autocorrelation function G(m) is given by17 G(m) )
1 (N - 1)
∑n (I(n∆t) - 〈I〉)(I(n∆t + m∆t) - 〈I〉)
Using ∆I(τ) ) I(τ) - 〈I〉 and going to integral form this becomes G(t) )
∫0∞ ∆I(τ)∆I(τ + t) dτ
In the case of multiple component diffusion in three dimensions, the autocorrelation function takes the form G(t) )
∑n
An (1 + t/τn)(1 + K2t/τn)1/2
where An and the τn represent the fractions and diffusion times for the different components. For instance, if we have two species of diffusants, monomers and dimers G(t) ) (1/N)({(1 - fD)}/{(1 + {t}/{τM})(1 + {K2t}/ {τM})1/2} + {fD}/{(1 + {t}/{τD})(1 + {K2t}/{τD})1/2}) where fD is the fraction of molecules which are dimers, τM and τD are monomer and dimer diffusion correlation times, and N is the total number of monomers and dimers.17 These correlation times may be related to their respective diffusion coefficients by τ ) ω2/4D and K ) ω/δ, where ω is a measure of the beam radius and δ is the beam length. For the FCS measurements we used a Zeiss-Evotec Confocor Spectrofluorimeter. This microscope-based system focuses a laser beam to an ω of approximately 0.25 µm and a δ of approximately 0.6 µm. The illumination source is an
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Ar laser (488 and 514 nm) coupled with a fiber optic to a Zeiss Axiovert microscope. This microscope has an electronic xyz stage and an electronically adjustable and positionable aperture at the image plane. The detector is a fast quenching avalanche photodiode with at least 106 photons per second resolution. In addition, the system has a CCD camera useful for adjustment, calibration, and location of the appropriate regions of the sample. Data were processed by a 288 channel logarithmic autocorrelator with adjustable sampling times from 200 ns to 3438.8 s. The Confocor fits data by nonlinear least squares to one, two, or three diffusion components according to the equations above. Additionally, the instrument calculates and corrects for occupancy of the triplet state, which causes additional fast (µs) fluctuations in the autocorrelation function. The calibration of the instrument was checked by using monodisperse fluorescently labeled latex particles (Molecular Probes) of known sizes. All of our measurements were performed at room temperature. At least 15 measurements were made for each sample, and the results were presented as average value ( std. dev. Lipid Binding Studies. Fluorescently labeled GBM-CTR was incubated with C14-cholesterol labeled vesicles at 37 °C for 6 h. The vesicle fraction was separated from free GBMCTR by sucrose gradient ultracentrifugation with vesicles appearing as low-density particles at the top of the gradient. The vesicle fraction was then examined on a BioRad MRC 1000 Confocal microscope equipped with a Krypton-Argon laser with emission and detection at 480 nm for lissamine -rhodamine. Crystal Appearance (Nucleation) Time Studies. GBM and r-GBM at a concentration of 1 mg/mL were mixed with vesicle fractions and model bile with CSI 1.4 and a total lipid concentration of 10 g/dL. As a control bovine serum albumin at a concentration of 1 mg/mL was used. The mixtures of protein and model bile were incubated in the dark under N2 at 37 °C and aliquots examined daily using polarizing light microscopy (Nikon Optiphot Pol) at a magnification of 40X. The appearance of solid notched rhomboidal cholesterol monohydrate crystals was noted, and the day of the appearance of the crystals was recorded as the nucleation time. Studies were performed in triplicate and data presented is mean of three experiments.
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Results
Figure 1. FRAP measurements of DHPE-FPLC vesicles + proteins. Panel A: There is a significant increase in the relative diameter (d/ d0) of the vesicle when incubated with GBM as compared to vesicle alone (black bars): a >3-fold increase when concentration of GBM ) 1.0 mg/mL (white bars) and a >5-fold increase when GBM ) 4.0 mg/mL (grey bars). After overnight incubation, the increase in vesicles’ diameter was even more profound: a >5-fold increase at 1.0 mg/mL and a >7-fold increase at 4.0 mg/mL. Panel B. Both p-GBM and r-GBM induced quite significant increase in vesicles’ diameter at mixing (p-GBM: ∼3-fold at 1.0 mg/mL, >4-fold at 4.0 mg/mL; r-GBM: >3-fold at 1.0 mg/mL, >7-fold at 4.0 mg/mL). The effect of p-GBM and r-GBM on the increase of vesicles’ diameter was greatly reduced after overnight incubation (p-GBM: 2-fold at 4.0 mg/mL; r-GBM: ∼2-fold at 1.0 mg/mL, ∼5-fold at 4.0 mg/mL). The bars are color coded as in panel A: vesicle alone (black bars), with GBM at 1 mg/ml (white bars), and with GBM at 4 mg/ml (grey bars). The bars for r-GBM are solid, whereas those for p-GBM are crosshatched.
Effect of GBM on Vesicles by FRAP. The FRAP measurements give us the changes in the diffusion coefficients D of the target component in a mixture of vesicle suspension and mucin molecules compared to D0, the diffusion coefficient of the target component without mixing with the other interaction component. From prior studies, we know that the viscosity change due to the presence of GBM at these concentrations has little effect on the diffusion of unilamellar FPLC vesicles.9,21 Therefore, the diffusion coefficient can be used to calculate the diameter of the vesicles via the Stokes-Einstein equation by keeping the relative viscosity constant.The results are presented in the format of relative diameter d/d0 ) D0/D, where d and d0
denote the hydrodynamic diameter of the labeled vesicle with and without mixing with mucins, respectively. This normalization eliminates the potential errors for comparing results from different measurements without calibrating the system per usage. GBM induced a 3-5-fold increase in d (proportional to a similar decrease in diffusion coefficient, D) suggesting that in the presence of GBM, the FPLC vesicles aggregate and/ or fuse (Figure 1A). The increase in d is larger with increasing concentration. The diffusion constant decreased almost immediately after mixing and did not decrease further over the first 2 h of measurement. However, at 24 h, GBM produced a slower decrease in D resulting in an overall 5-7-
Role of Mucin Structural Domains
Figure 2. FCS measurements of DHPE-FPLC vesicles + protein, FCS of mucins and immunoglobulins incubated with fluorescently labeled simple unilamellar vesicles. All mucins are at a concentration of 4 mg/mL and immunoglobulins are at 1 mg/mL. All of our measurements were performed at room temperature. At least 15 measurements were made for each sample and the results are presented as average value. GBM increases vesicle size immediately and further at 24 h. r-GBM is a potent immediate aggregator of vesicles whose effect is lost at 24 h. No major effect is seen with the Ig species.
fold increase in diameter d of the vesicles. This immediate change in D followed by the slower decline in D over 24 h suggests a bi-phasic kinetic process. No changes in diffusion constants were observed in the control experiment where bovine serum albumin was added to the vesicles (data not shown). Effect of r-GBM and p-GBM on Vesicles. There was a 3-7-fold decrease in D values (3-7-fold increase in size d) of FPLC vesicles upon addition of either r-GBM or p-GBM (Figure 1B). This change occurred immediately upon mixing as for GBM. However, at 24 h, the decrease in D was not sustained and D/D0 increased toward the baseline seen for vesicles alone. FRAP of Labelled Mucin. FITC labeled GBM (1 and 4 mg/mL) was mixed with unlabeled FPLC vesicles, and the D values of GBM were measured by FRAP to evaluate changes in GBM diffusion. Within a time period of 2 h, no systematic change of D for GBM was apparent suggesting that aggregation of mucin was not occurring and confirming our DLS findings that GBM interacts with vesicles irrespective of viscosity change.9,21 However, there was a decrease in the D values (from normalized 1.0 to 0.77) of GBM after 24 h incubation with vesicles. This increase in GBM size cannot be attributed to polymer aggregation and we believe that it represents binding of vesicles to GBM with a resulting increase in size. Mucin-Lipid Binding Studies. To confirm the FRAP findings of vesicles binding to mucin and increasing mucin size, we examined binding of unlabeled vesicles to GBMCTR (0.5 mg/mL). Examination by Confocal microscopy of the vesicle fraction demonstrated an aggregate of vesicles within a fluorescent mucin background and confirmed that mucin is tightly associated with the cholesterol enriched vesicles.
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Figure 3. Crystal appearance time in model bile, CSI 1.4 after incubation alone (white bars) and with GBM (solid bars), r-GBM (grey bars), and p-GBM (crosshatched bars), all at 1 mg/ mL. The crystal appearance time was accelerated by GBM at day 2 but not by r-GBM or p-GBM.
Fluorescence Correlation Spectroscopy of MucinVesicle Mixtures. To confirm that the effects seen with mucins on FRAP were valid, we also studied pro-nucleating immunoglobulins IgG and IgA purified from bile of gallstone patients (gift of Dr. Harvey, St. Louis, MO). The results are shown in Figure 2 which again demonstrates the ability of mucin to increase vesicle size on immediate mixing. The maximal effect was seen with r-GBM but there was a large standard deviation in this group of experiments. Similar to the FRAP studies a reduction in vesicle size was seen at 24 h for r-GBM and p-GBM. Immunoglobulins A and G, although potent pronucleators, had no effect on vesicle size by FRS. FCS of mucins and immunoglobulins were incubated with fluorescently labeled simple unilamellar vesicles. All mucins are at a concentration of 4 mg/mL, and Immunoglobulins are at 1 mg/mL. All of our measurements were performed at room temperature. At least 15 measurements were made for each sample, and the results are presented as an average value. GBM increases vesicle size immediately and further at 24 h. r-GBM is a potent immediate aggregator of vesicles whose effect is lost at 24 h. No major effect is seen with the Ig species. Crystal Appearance Time. The appearance of cholesterol crystals from model bile and their morphology has been investigated extensively by microscopic techniques.26 These studies reveal a complex pathway leading to platelike monohydrate cholesterol crystals. We did not observe any crystals with mixing of proteins with vesicles due to the low overall concentration of lipid in the vesicle preparations. Platelike crystals were seen in model bile mixed with GBM or its derivatives after some incubation time. The number of crystals for model bile with GBM, r-GBM and p-GBM versus incubation time is shown in Figure 3 and compared to the control model bile to which albumin is added. Only GBM reduced the crystal appearance time and increased crystal number compared to model bile with albumin as control.
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Discussion Both FRAP and FCS methods show that GBM has a profound effect in inducing FPLC vesicles to aggregate/fuse. This effect is more pronounced after overnight incubation. The altered GBM (either p-GBM and r-GBM) is not as effective as native GBM, particularly after overnight incubation. IgA and IgG proteins showed little effect. Similarly, synthetic water soluble polymer polyacrylamide was not effective in causing fusion of the vesicles used in this study (data not shown). We have previously shown that reduction of GBM alters the conformation of the protein core in such a way as to increase the number of exposed hydrophobic binding sites.14 Therefore, the increase in vesicle size seen with r-GBM could be explained by increased binding to mucin with promotion of aggregation. Because r-GBM is unable to polymerize, the immediate decrease in D cannot be explained by alteration of mucin viscosity. However, the immediate effect of p-GBM on D was unexpected because p-GBM does not promote nucleation in this model. The rapid initial reduction in D was not sustained because after 24 h there was an increase in D unlike the continued decrease in D seen with native GBM. We have speculated that this may indicate a two phase kinetic reaction with initial rapid aggregation and then fusion as suggested by our prior TEM studies.9 Could the aggregation be caused by purely entropic interactions? It has been shown that when a small amount of linear polymer is added to a colloidal suspension the loss of entropy of the polymer chain lying between two or more spherical particles causes an unfavorable increase in the free energy; and the system segregates into a particle rich region depleted of polymer and a polymer rich region depleted of particles.27 However, this purely physical mechanism occurs at much higher concentrations of colloid than those used in the experiments with vesicles (
