Atomic Force Microscopy Reveals Aggregation of Gastric Mucin at

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Atomic Force Microscopy Reveals Aggregation of Gastric Mucin at Low pH Zhenning Hong,† Bernard Chasan,*,† Rama Bansil,*,† Bradley S. Turner,‡ K. Ramakrishnan Bhaskar,‡ and Nezam H. Afdhal‡ Department of Physics, Boston University, Boston, Massachusetts 02215, and Division of Gastroenterology, Beth Israel Deaconess Medical Center, Harvard Medical School, Boston, Massachusetts 02115 Received August 16, 2005; Revised Manuscript Received October 5, 2005

Mammalian gastric mucin, at high concentration, is known to form a gel at low pH, behavior essential to the protection of the stomach from auto-digestion. Atomic force microscopy (AFM) measurements of dilute solutions of porcine gastric mucin in an aqueous environment in the pH range 6-2 provide a direct visualization of extended fiberlike molecules at pH 6 that aggregate at pH 4 and below forming welldefined clusters at pH 2. The clusters consist of 10 or less molecules. AFM images of mucin at high concentration at pH 2 reveal clusters similar to those seen in the dilute solutions at low pH. We also imaged human gastric mucus revealing a network having a “pearl necklace” structure. The “pearls” are similar in size to the clusters found in the purified porcine gastric mucin gels. AFM images of deglycosylated mucin reveal that the deglycosylated portions of the molecule re-fold into compact, globular structures suggesting that the oligosaccharide chains are important in maintaining the extended conformation of mucin. However, the oligosaccharides do not appear to be directly involved in the aggregation at low pH, as clusters of similar size are observed at pH 2 in both native and deglycosylated mucin. Introduction Mucin glycoproteins constitute a family of macromolecules known to be responsible for the protective and lubricative properties of mucus secretions that adhere to epithelial cell surfaces.1,2 Although mucins from different organs exhibit chemical differences, presumably related to their different functions, recent advances using recombinant techniques have shown a striking similarity in the basic structure of all of the mucin genes.3 The important viscoelastic properties of mucins depend on their ability to polymerize to molecular weights of over 106 Da. 4 These polymers further aggregate and, in the case of the gastric mucin, gel under acidic pH, at the typical concentrations found in mucus secretions in mammalian stomachs.5,6 A fundamental understanding of mucin structure and its interactions is essential to gaining an insight into the gelation characteristics of gastric mucin. Identification of the structural features of mucins has been difficult because of their large size, polydisperse nature and high degree of glycosylation, approximately 80% carbohydrate by weight. Further complications arise due to their propensity to aggregate and form mucoadhesive interactions with other molecules. In the last several years, however, molecular biological techniques have provided information on the primary structures of these molecules. All of the mucin genes share several common features.7 Each contains a domain comprised of tandem repeating sequences that encode * To whom correspondence should [email protected] (B.C.); [email protected] (R.B.). † Boston University. ‡ Harvard Medical School.

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from 6 to 169 amino acids, repeated up to 100 times, to produce large protein subunits. The tandem repeated sequences code for domains rich in serine (Ser), threonine (Thr), and proline (Pro). The oligosaccharides are attached to the protein core by O-glycosidic bonds to the hydroxyl side chains of Ser/Thr and arranged in a bottle-brush configuration about the protein core. The oligosaccharide side chains consist of 5-15 monomers, exhibit moderate branching, and contain primarily N-acetyl galactosamine, N-acetyl glucosamine, fucose, galactose, N-acetyl neuraminic (sialic) acid, and traces of mannose and sulfate. Recently Escande et al.8 have described the occurrence of super repeats consisting of cysteine rich regions followed by Pro/Ser/Thr rich repeating regions in human MUC5AC which is homologous to porcine gastric mucin (PGM).9 In all mucins for which the structural organization has been elucidated, these tandem repeat domains occupy the central region of the polypeptide backbone and are flanked by nontandem repeating sequences at the amino- and carboxyl-termini, which have significantly less serines and threonines and are rich in cysteines. These cysteine-rich regions have very few O-linked sugars but may contain some N-linked oligosaccharides (we refer to these as “nonglycosylated” regions). The nonglycosylated regions resemble typical globular proteins as regards amino acid composition and folding and are involved in the further polymerization of the primary bottle-brush like glycosylated subunit via disulfide linkages to produce a very high molecular weight polymer typically in the several million Dalton range. Thus, mucin can be considered as an alternating multiblock copolymer of gly-

10.1021/bm0505843 CCC: $30.25 © 2005 American Chemical Society Published on Web 10/26/2005

Aggregation of Gastric Mucin at Low pH

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Figure 1. (a) Schematic drawing of the PGM monomer consisting of glycosylated regions flanked by regions with little glycosylation. (b) The symbols indicate the different domains in the sketch in (a). (This representation is based in part on Figures 1 and 2 of Dekker et al.10). At the bottom of the figure we show (c) a dimer formed by two monomeric subunits linked via disulfide bonds in the nonglycosylated regions and in (d) The dimers are further disulfide linked to form higher multimers which give rise to the polydispersity and high molecular weight of secretory mucins. Polymers >16-mers have been described in MUC5AC from human airway secretions by Sheehan et al.11 (The bottom part of the Figure is adapted from Figure 8 of ref 11.)

cosylated and nonglycosylated domains. A sketch of PGM showing super repeats and oligosaccharide side chains is displayed in Figure 1. The picture also shows the nonglycosylated regions with cystine knots and von Willebrand factor (vWF) C and D domains, which are likely to be involved in the polymerization of mucin. Electron microscopy12 and dynamic light scattering (DLS) studies suggest that most mucins are highly elongated rodlike or wormlike polymers, ranging in size from 50 to 400 nm.4 The aggregation/gelation of such complex macromolecules is a fascinating problem in biopolymer physics. Of all of the organs, it is in the stomach that mucus faces its severest challenges from secreted HCl, bile salts, alcohol, and drugs. We have previously demonstrated6 that gastric mucin has the unique ability to gel at pH 2, the ambient pH of the mammalian stomach during digestion, a feature crucial to the protective function of gastric mucus. Using depolarized DLS to investigate porcine gastric mucin,13 we showed that a conformational transition occurs at pH < 4, with the molecule becoming elongated to around 400 nm in length. At higher concentrations, DLS studies showed clearly that the aggregation of the rodlike molecules leads to gelation.13 Fluorescent dye-binding studies suggest that the association arises from the hydrophobic interactions of the nonglycosylated regions of the mucin polymer.13 Atomic force microscopy (AFM) offers a unique tool to examine in an aqueous environment the molecular and structural mechanisms involved in this still poorly understood process. AFM has been used extensively to visualize the conformation of single molecules,14 and previous studies of mucins in dilute solutions15,16,17 have shown its extended conformation. Recent AFM measurements18 have observed in situ depolymerization of mucin by chemically reducing the S-S bonds. However, AFM has not been used to observe their aggregation states. Unlike DLS measurements, which are routinely used to determine the average hydrodynamic size of molecules and aggregates from their diffusional dynamics in the bulk solution state, AFM measurements provide a direct visual observation of the conformation and aggregation state, albeit on a substrate, with which it must interact. Our goal in this work is to elucidate and visualize

changes in the molecular conformation and consequent aggregation state of porcine gastric mucin in the pH range 6-2. To the best of our knowledge, the study of pH dependent aggregation reported here is a unique application of AFM. Results on both dilute mucin solutions and mucin gels are presented. During active acid secretion, the pH of the mucus layer drops to around 2, so the changes observed are expected to represent the actual physiological processes that must take place. To this end, we also present AFM images of human mucus gels obtained from endoscopy. Materials and Methods Atomic Force Microscopy. A Dimension 3000 AFM (Digital Instruments, Santa Barbara, CA) attached to a Nanoscope IIIa controller with an electronic extender box was used for the present studies. Tapping mode in aqueous solution was used as the least perturbative approach. The tapping frequency employed was in the range 6-8 kHz. We used a set-point in the vicinity of 200 mV with the amplitude voltage set slightly higher than the set point. To improve the image quality and keep compression forces to a minimum, we fine-tuned the set-point by raising it as high as we could while still getting a good image. For viscous samples, amplitude voltages and set-points both had to be increased while keeping the difference to a minimum. Images were obtained with oxide-sharpened silicon nitride tips (DNP-S Digital Instruments) and were flattened with a zero order or first-order polynomial fit, but no further filtering or data modification was employed. Preparation of Samples for AFM. For all scans, the substrate was untreated freshly cleaved mica with the exception of a pH 6 measurement where APTES [(3aminopropyl) triethoxysilane; Sigma Chemicals] treatment was used to increase the binding of the negatively charged protein to the mica substrate. The APTES coated mica disks were made using protocols from Lyubchenko et al.19 Briefly, freshly cleaved mica was left in the APTES vapor created by letting a small drop of APTES solution (∼10 mL) evaporate in a covered Petri dish for at least 30 min. Some silane groups of the APTES bind to the mica surface, while

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amino groups bind to the negatively charged mucin. At lower values of pH, as both mucin and mica are in highly protonated states, treatment of the substrate proved to be not necessary. Samples for AFM imaging were prepared by pipetting 25-50 µL of mucin solution in a buffer at the appropriate pH (see below for details) typically at a concentration of 1-5 µg/mL onto the mica, waiting for 3 min, washing with the same buffer that was used in preparing PGM solution, and than adding sufficient buffer to carry out the scans. Gel samples were prepared by dropping 10 µL of mucin gel (14 mg/mL) onto the mica, letting it dry for 5 min, then depositing 100 µL of 1-butanol on top of the sample. Butanol functions by displacing the water,20 leading to dehydration thus stiffening the sample and making it easier to image with AFM. Data Analysis. All of the image data were acquired using the Nanoscope software provided with the AFM. The 3-D plots and height profile analysis were also done using this software. We used approximately the same imaging conditions for all our studies involving comparison of heights and estimate that the average height is known to an accuracy of about 15%. For measurements of the volume of mucin aggregates, the ad hoc approach of Berge et al.21 was used in which the aggregate on the mica is modeled as a the cap of a sphere, V ) (πh/6)(3r2 + h2), where r is the half width at half-maximum and h is the maximum (central) height. For asymmetric objects, we take an appropriate average r. Although this was developed to estimate the size of globular proteins, we assume its approximate applicability to aggregates as well. Image-Pro Plus software (Media Cybernetics) was used to analyze the distribution of areas of the clusters observed in the images. Objects of higher contrast above the background than an appropriately chosen threshold were outlined and the software provides a histogram of the area distribution of the outlined objects. Preparation of Purified Native Porcine Gastric Mucin. Mucin was isolated from mucosal scrapings of pig stomach epithelium obtained fresh from local slaughterhouses.13 Briefly, the scrapings were solubilized by stirring gently overnight in 0.2 M NaCl containing 0.04% sodium azide and protease inhibitors benzamidine HCl, phenyl methyl sulfonyl fluoride, dibromoacetophenone, and EDTA adjusted to pH 7 with 1 M NaOH. Coarse debris was removed by centrifugation at 50 000g for 1 h. The supernatant containing soluble mucin was fractionated by column chromatography on Sepharose CL-4B. Void volume fractions containing high molecular weight (greater than 2 million Da) periodic acidSchiff (PAS) positive glycoproteins were pooled and concentrated by ultrafiltration with an XM-300 (300 000 Dalton cutoff) filter. Purified mucin was prepared from the concentrate by density gradient ultracentrifugation in CsCl6 with an initial concentration of 42% (w/w) at 300 000g for 24 h. Eight 1 mL fractions were recovered from the top of the tubes. Densities of the fractions were determined by weighing aliquots and the glycoprotein content determined by PAS assay. Those fractions containing glycoprotein (average density 1.45 g/mL) were pooled, exhaustively dialyzed, and lyophilized for further study.

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Porcine gastric mucin prepared by this method was essentially free from contaminating proteins and lipids22,23 and its amino acid and carbohydrate composition9 was typical of mucins, with a predominance of serine, threonine, and proline (230, 163, and 135 residues per 1000, respectively). Molecular weights of mucins prepared by essentially similar methods have been reported to range from 21-9 M Da.24 The pH of the mucin solutions was adjusted using 10 mM phosphate-citrate or 10 mM phosphate-succinate buffers over the entire range of pH values from 2 to 7 thus maintaining the same buffer composition and ionic strength. Physical properties of PGM in these buffers are comparable to those found in solutions mimicking physiologic gastric juice solutions of the same pH and at constant ionic strength as previously reported by Bhaskar et al.6 Preparation of Deglycosylated PGM. Deglycoslyated PGM was prepared using the protocols, adapted from Current Protocols in Molecular Biology,25 commonly referred to as beta-elimination. This method cleaves oligosaccharides linked O-glycosidically to the hydroxy groups of the amino acids serine and threonine in the protein backbone. In brief, lyophilized PGM was dissolved in the minimum volume of alkaline borohydride (22.4 mg sodium borohydride, NaBH4, dissolved in 2 mL of 0.4 M NaOH) just before use and incubated at room temperature for 24 h. Hydrochloric acid (2 M) was added to convert excess borohydride to H2 gas. After the evolution of hydrogen had ceased, 1 M NaOH was added until a neutral pH (6-8) was reached. The deglycosylated PGM was dialyzed and lyophilized. Aliquots of solutions of native and deglycosylated PGM were subjected to the microplate assay for neutral sugars26 which indicated that deglycosylation had removed ∼50% of carbohydrate. Native and deglycosylated PGM solutions were also examined by SDS-PAGE followed by western blotting to a monoclonal anti-MUC5AC antibody (Gastric mucin Ab1, Labvision Corp., CA). Native PGM gave rise to an intense band in the stacking gel (molecular weight . 200 kD), whereas deglycosylated PGM showed no reactivity (results not shown). Since this antibody was raised to a portion of the peptide backbone,27 it appears that the strong alkali treatment is probably responsible for some destruction of the protein backbone also. Preliminary gel filtration studies indicate that deglycosylation results in a considerable reduction in molecular weight. Preparation of Human Mucus Gels. Human gastric mucus gels were obtained from gastric aspirates collected during endoscopy of healthy humans at Beth Israel Deaconess Medical Center. Gastric aspirates were first clarified by centrifugation at 5000g for 15 min at 25 °C to remove cellular and other debris. A small quantity of pH 2 buffer was added to the clear supernatant which was then subjected to centrifugation at low speed. A gel-like sediment resulted and a portion of this was deposited on untreated freshly cleaved mica for AFM measurements.


Figure 2. Mucin imaged in dilute solution at pH 6. (a) Mucin on untreated mica disk. (b) Mucin on mica disk treated with APTES. In both cases, a 2-D top-down image is shown together with a representative height profile along the line indicated by the cursors. The big white blobs seen in Figure 2b are polymerized APTES left in the preparation of the APTES coating process on mica as confirmed by a scan of an APTES treated disk without mucin (not shown).

Results and Discussion I. Observations of Mucin Structure in Dilute Solutions. a. Studies of PGM at Neutral pH. Figure 2a shows images


of fiberlike individual molecules obtained from a dilute solution of purified PGM at pH 6 on a mica substrate in aqueous buffer, using tapping mode AFM. Several regions of the sample and many different fields of view showed qualitatively similar results, although some images were clearer than others. Typical results are shown along with representative height measurements along a line indicated on the top-down view. We note that height measurements are not subject to the tip size related broadening which is an important limitation in interpreting lateral in-plane measurements. The individual fibers seen in Figure 2a appear to have been straightened out. This is caused by the poor adherence of the negatively charged mucin at neutral pH to the mica substrate making it difficult to get good scans. Therefore, as discussed in the Methods section, we fixed the protein molecules on the surface by treating the mica with APTES to produce a positively charged substrate without altering the pH. AFM images of APTES coated mica disks without mucin revealed some bright white blobs but were otherwise featureless. A typical image of mucin on an APTES treated mica surface is shown in Figure 2b. The mucin fibers on APTES are unperturbed by the tip, and their orientation remains unchanged after several scans. Otherwise both images of Figure 2, panels a and b, at pH 6 show fiberlike individual molecules, sometimes in loose association with each other. The fibers in the image of Figure 2a range from 350 to 850 nm in length, and the height ranges from a fraction of 1 nm to about 2 nm along the length of an individual fiber. The average length is about 560 ( 160 nm. Some of the fibers from the untreated mica substrate (Figure 2a) could be longer than 850 nm as they extend beyond the field of view in the images. The individual fibers seen in the present work at pH 6 are similar to those seen in a tapping mode AFM scan in buffer carried out on ocular mucin at neutral pH, by McMaster et al.,16 and the height measurements in the two experiments are comparable as well. Likewise the two experiments exhibit the same regular variations in height along the length of the fiber which McMaster et al.16 interpret as glycosylated regions of the mucin molecules. This is a reasonable interpretation for our results as well, particularly since the higher regions tend to be clustered together on stretches of length around 100 nm and alternate with lower height regions as expected from the structure of mucin, shown in Figure 1. We suggest that the sugar chains are too mobile in their solvated state to be seen fully extended in an AFM image, which requires a high degree of immobilization on the substrate. It is also important to recognize the “nonsolid” nature of the chains: a recent force measurement on a brush polymer surface suggests that the AFM tip penetrated the brush without sensing it.28 b. Effects of Deglycosylation. In the hope of examining the bare-protein core of mucin, we imaged a deglycosylated mucin solution at pH 5. A typical image is shown in Figure 3. The resulting conformations differ significantly from those seen in native PGM, revealing both compact spherical structures (which we refer to as “heads”) and extended fibers (referred to as “tails”). Sometimes the heads appear to be attached to a tail, or two heads appear to be bridged by a

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Figure 3. Deglycosylated PGM at pH 5. The circled objects show a typical “head” (top right), “head with 1 tail” (top left), “two heads bridged with one tail” (middle, enclosed within cursors) and extended fibers or “tail” (line at bottom). A representative height analysis is shown in the bottom from the object with two heads bridged by a tail.

tail. We attribute the collapsed structures to a decreased solubility which is a consequence of the removal of 50% of the sugars and which causes the deglycosylated portions of the mucin molecules to form globules, as they effectively re-fold. We outlined these objects by choosing an appropriate threshold value of the contrast to discriminate between objects and background in the Image Pro Plus program. Analysis of the resulting image shows that about 33% of the objects are fully collapsed heads, about 25% are fully extended tails with no heads, 30% have a head and a tail, and the remaining 12% are two heads bridged by a tail. The “no head” sample of nine includes two that may be assumed not to have been subject to the deglycosylation reaction, by virtue of their length. The other seven have an average length of 180 nm, significantly shorter than the native PGM with intact carbohydrate side chains. The volumes of the heads, calculated using the Berge et al.21 approach (see Methods section), varied uniformly over 200-600 nm3 with a few outliers. Assuming that the heads are tightly packed, we can find the length of chain coiled up in the heads, using the radius of the chain as r ) 0.75 nm, typical of the isolated mucin molecule. This length, LHEAD, varies uniformly from 100 to 300 nm. We take 200 nm as a reasonable average. Applying this result to the two-headed objects, we find an average observed length of 100 nm for the bridging tail, and an average total length 100 + 2LHEAD ) 500 nm. Within the considerable

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experimental uncertainties, we consider that this value is consistent with the length of the native fully glycosylated fibers. As mentioned in the Methods section, we estimated that about 50% of the sugars were removed and that some of the core protein may have been clipped in the deglycosylation process. It appears that the “no head” cases and the “two head” cases represent two extremes, maximum clipping and no clipping. Within rather difficult to estimate error, we have a consistent picture that deglycosylation causes the newly exposed portion of the mucin molecule to re-fold into a compact globule, and that the ends of the native mucin molecule are easier to deglycosylate than the middle, since we do not observe structures with a head in the middle and tails on both ends. Thus, the extended fiberlike structure of mucin is maintained by the sugar residues, in the absence of which the molecule re-folds significantly. c. Effects of Varying pH. As discussed in the Introduction, DLS studies of PGM solutions show that it forms a gel at low pH.13 To obtain visual evidence of this, we imaged PGM solutions prepared at different pH’s by tapping mode AFM in an aqueous environment of buffer of the same pH. Typical images from dilute solutions of PGM prepared in buffers at pH 5, 4, and 2 are shown in Figure 4a-c. This figure also shows an AFM image of deglycosylated PGM at pH 2. All of the data shown are imaged on untreated mica substrates. At pH 5 (Figure 4a) the more compact but still loose aggregates observed are of the same height as the monomeric fibers observed at pH 6 (cf. Figure 2) and exhibit considerable fluctuation in height suggesting that at pH 5 the mucin strands are not tightly aggregated. In contrast, at pH 4 and 2 (Figure 4, panels b and c) there is a qualitative change in the images which show well defined clusters within which individual strands are no longer identifiable as they still were at pH 5. They are also significantly higher, averaging around 5 nm at pH 4 and around 7 nm at pH 2. The individual aggregates are polydisperse, but the largest ones at pH 2, which are prolate in shape, have lateral dimensions of order 50 nm × 20 nm and heights of 6-7 nm. Similar structures, slightly lower in height and slightly larger in area, are seen in the deglycosylated PGM sample at pH 2 (Figure 4d), implying that removal of a large fraction of the sugar does not disrupt the aggregation process. The histogram in Figure 5 showing the area distribution of the images of clusters at pH 4 and 2 clearly reveals a change in the distribution of aggregates. The distribution at pH 4 with clusters ranging in size to 1150 nm2 is quite symmetric, whereas that at pH 2 is highly asymmetric with a tail extending to about 3400 nm2, indicating that larger clusters are formed at pH 2. The peak in the distribution also occurs at a larger value, 700 nm2 at pH 2 as compared to 500 nm2 at pH 4. The average size of about 600 ( 300 nm2 at pH 4 is considerably smaller than the average of about 1750 ( 1100 nm2 at pH 2. The higher polydispersity and asymmetry of the distribution at pH 2 as compared to pH 4 may reflect differences in the nature of the association of the mucin molecules as pH is changed. In a recent study, Lee et al. 29 have shown from circular dichroism measurements on commercially available PGM (Sigma) in dilute



Figure 4. Mucin imaged in dilute solution at different values of pH on untreated mica substrates. (a) pH 5, (b) pH 4, (c) pH 2, and (d) deglycosylated mucin at pH 2. Note that here we show 3-D perspective images together with height profiles along the line shown in the top-down 2-D view (bottom left of each image). The height scale is zoomed to a higher magnification for the pH 5 image (a) as compared to the lower pH images in panels b-d.

solutions that the tendency of certain nonpolar amino acids to remain folded is diminished with decreasing pH. This may lead to increased hydrophobic interactions at lower pH thus promoting larger aggregates at pH 2. To estimate the number of molecules in these aggregates, we estimate the monomer volume from the pH 6 data as around 500 nm3, considering it as a cylinder of length 350 nm and average diameter 1.5 nm. Comparing this with the volume of the aggregate seen at pH 2, and calculated using the Berge et al.21 approach (see Materials and Methods), we estimate that the largest clusters formed at pH 2 consist of about 10 monomers, whereas the pH 4 clusters are half as large. Although the error in the measurement of height (about 15%), the variability in length of the individual molecules, the high polydispersity, and the change in the shape of the cluster distribution affects our calculation of the number of fibers in a cluster, all of the parameters indicate that the clusters are smaller at pH 4 than at pH 2. We also note that these estimates of aggregate size are made under the assumption that the clusters are as tightly packed as protein molecules, and do not take into account the heavy glycosy-

lation of mucin. Consequently, they must be considered as an upper limit only. Although fiberlike structures have been reported in the literature, none of the previous AFM measurements on ocular mucin15,16 or PGM,17 all of which were made at fixed pH of 4.5, show clustering. The lack of clustering in the previous PGM experiment17 probably reflects the very low PGM concentration and a higher salt concentration (0.1 M), as it is known that PGM does not gel at high ionic strengths.6 Deacon et al.17 also observe much longer fibers: average length 2 µm, with a few fibers as small as 400 nm. At pH 6, we see some fibers longer than 800 nm, but predominantly smaller fibers. Although our measurements overlap in range with those in ref 17, we clearly have different averages. Round et al.15a obtain a wide range of contour lengths, from 100 nm to a few µm in ocular mucin, and they found that mucin fractions with differences in glycosylation exhibit differences in length distribution and conformation.15b The molecular weight distribution in a highly polydisperse polymer such as mucin may be sensitive to slight variations in the preparation method, e.g., lyophilization, the exact

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Figure 6. 10 µL of 14.5 mg/mL mucin prepared at pH 2 was deposited onto mica, air-dried, and then scanned under butanol. The mucin aggregates are similar in shape to the low concentration aqueous sample shown in Figure 4c.

Figure 5. Area distribution of the dilute solution mucin sample at (a) pH 2 and (b) pH 4.

selection of the void volume fraction, and differences in ionic strengths. An STM measurement30 on PGM at pH 4.5 also obtains long fibers and exhibits the loose aggregations seen in the present experiment along with a linear form. On the other hand, an earlier electron microscopy study of PGM12 also at pH 4.5 gives lengths in agreement with those reported here and some degree of entangling, similar to our pH 5 results. In view of the significant structural changes in the vicinity of pH 4, it is perhaps not surprising that mucin samples at pH 4.5 which are dried and imaged in air give different results than reported here in an aqueous buffer. II. AFM Observations of Gels. To obtain closer comparison with physiology, we imaged thick mucin films of concentration 14.5 mg/mL, large enough to produce a gel at pH 2 and roughly half the mucin concentration in naturally occurring porcine gastric mucus. Images of these samples shown in Figure 6 reveal an interface that is organized into clusters of similar shape to those seen in the lower concentration samples shown in Figure 4C. However, the average volume of the aggregates in the pH 2 gel samples is about 3 times larger than at low concentration suggesting that aggregate size depends on sample concentration. We also note that the gel sample, about 100 nm thick, was scanned under butanol rather than in aqueous buffer, a change necessitated by the soft, thick sample. In contrast, the aqueous pH 2 sample consists of a single layer of clusters, each in contact with the mica substrate. This

Figure 7. Area distribution of pH 2 high concentration gel sample.

difference in sample preparation and the dehydrating effects of butanol may well contribute to the difference in measured aggregate size. Figure 7 shows that the area distribution of the clusters of Figure 6 is also asymmetric, similar to that seen in the low concentration sample at pH 2 (Figure 5a). However, at higher concentration the total number of clusters is greater, they are bigger in size as revealed by the shift in the peak to 3000 nm2, and the tail of the distribution extends to 30 000 nm2. As mentioned above, the dehydrating effects of butanol may be a contributing factor to this change in cluster size distribution. III. AFM Studies of Human Mucus Gels. To further examine the physiological relevance of our observations, we examined human gastric mucus gels. In comparing the results on the human gastric mucus with the purified porcine gastric mucin gels, we assume that the interspecies mucin amino acid sequence similarity (80-90%) also reflect functional and structural similarities.31 The samples obtained from endoscopy were fragmented so we centrifuged the samples to remove the smaller fragments and to obtain the high concentration samples needed for formation of the gels. The



respectively) are involved in the interactions leading to aggregation. The central role of the block copolymer nature of mucin in determining mucus gel properties is supported by the striking similarity between Figure 8 and the picture obtained by Zhang et al.32 showing the so-called “pearl necklace” morphology obtained by acid induced gelation of micelles formed from polystyrene (PS)-poly(acrylic acid) (PAA) diblocks in aqueous solution. Theoretical calculations of hydrophobically modified polyelectrolytes33 show that the interplay between the surface energy of the hydrophobic blocks, which associate in an aqueous medium and the electrostatic repulsions and elastic energy of the polyelectrolytic blocks controls the pearl necklace morphology. We suggest that the “pearls” in the mucus gel and the clusters in purified PGM gels consist of micelle-like aggregates with hydrophobic regions sequestered in the interior and covered by sugars and hydrophilic amino acids, both of which contain charged groups. The similarity of the morphology to that obtained in PS-PAA diblocks suggest that although, hydrophobic interactions may lead to the formation of the micellar cores, ionic interactions of the negatively charged sugar and amino acid residues and the other ions present in mucus are contributing factors in determining the morphology of these gels. Conclusions

Figure 8. Human mucus gel at pH 2. (a) A 3 µm scan showing a clear network with well-defined pores. (b) A 1 µm × 1 µm region of the image shown in (a) is magnified revealing a “pearl necklace” morphology. The height scale in panel b has been adjusted to show the “pearls” clearly. We note that the distance between cursors spans four “pearls” of average diameter 100 nm. The diameter of the pores is around 200-400 nm as revealed by the higher magnification image.

mucus gel has a very clearly articulated porous structure as shown in Figure 8, in contrast to the ordered crowding together of the mucin gel shown in Figure 6. Nonetheless, there is an underlying similarity between the pH 2 human mucus gastric gel and the pH 2 PGM gel. In both cases, we observe the presence of clusters of similar size. The different solubilities of the alternating glycosylated and nonglycosylated domains (see Figure 1) in water could lead to solvophobic association, similar to that seen in block copolymers in selective solvents.23 Cao et al.13 have shown by DLS and hydrophobic dye binding studies that aggregation/gelation of mucin involves the interplay of hydrophobic and electrostatic interactions. Our observation that removal of the sugar does not disrupt aggregation points to the involvement of the portion of the protein with little or no glycosylation in the aggregation mechanism. The critical pH of 4 observed in both the AFM and DLS studies suggests that glutamic and aspartic acid residues (pK 4.1 and 3.9,

The AFM results presented here provide direct visual confirmation that gastric mucin aggregates at or below pH 4, in agreement with previous DLS findings that showed PGM forms a gel at pH 2. In dilute solution, mucin molecules are in an extended fiber like conformation at pH 5-7, with length of the order of 400 nm, whereas at pH 4 and below, they cluster together. The pH 2 results, both at relatively low concentrations and at gel concentrations, indicate that the aggregates consist of relatively few mucin molecules. Studies with deglycosylated mucin at pH 5 show the importance of the sugar side chains in maintaining the solubility and the extended structure of the mucin molecule, since the deglycosylated portions of the molecule re-folded into compact spherical structures. However, the deglycosylated sample also aggregates at pH 2, suggesting that the oligosaccharide side chains are not directly involved in aggregation. AFM images of mucus gels from endoscopy specimens reveal networks exhibiting the characteristic “pearl necklace” morphology observed in hydrophobic/hydrophilic block copolymers. Our results are consistent with the model suggested by Cao et al.13 that aggregation/gelation involves the interplay of hydrophobic and electrostatic interactions involving the portions of the mucin molecule with little or no glycosylation. In conclusion, we note that the aggregation of gastric mucin at pH 2 shows a clear correlation with the physiological function of gastric mucus, as a protective coating for the stomach at the low values of pH at which food must be digested. Our results also confirm that tapping mode AFM in an aqueous environment is a useful probe of glycoprotein structure and aggregation. Acknowledgment. We thank Anlee Krupp of the Boston University Photonics Center for her aid and advice in running

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Atomic Force Microscopy Reveals Aggregation of Gastric Mucin at

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