Glycobiology Advance Access originally published online on March 13, 2008
Glycobiology 2008 18(6):463-472; doi:10.1093/glycob/cwn024
| ||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
Analyzing the functions of large glycoconjugates through the dissipative properties of their absorbed layers using the gel-forming mucin MUC5B as an example
Department of Biochemistry and Biophysics, University of North Carolina, Chapel Hill, NC 27599, USA
1 To whom correspondence should be addressed: Tel: +1-9198432577; Fax: +1-9199667524; e-mail: kesimer{at}med.unc.edu
Received on January 9, 2008; revised on February 17, 2008; accepted on March 10, 2008
 |
Abstract |
|---|
 Top Abstract IntroductionResultsDiscussionConclusionsMaterials and methodsConflict of interest statementReferences |
|---|
Glyconjugates such as mucins, proteoglycans, and polysaccharides form the structural basis of protective cell-surface layers. In particular gel-forming mucins define a zone between the epithelial cell layer and the environment. Such molecules are of extreme molecular weight 5–100 x 106 and size (Rg 20–300 nm). On this account their biochemistry is inseparable from their physical biochemistry. Combining laser light scattering and quartz crystal mass balance with dissipation methods (QCM-D) we have investigated the properties of the MUC5B mucin and its cognate fragments when bound to a hydrophobic surface. MUC5B forms the basis of gels responsible for the protection of the oral cavity, lung, and cervical canal surfaces. Here we show, by analyzing dissipative interactions of hydrophobic, gold, and polystyrene surfaces, with the intact MUC5B molecule, its reduced subunits, and glycosylated tryptic fragments (obtained after reduction), the formation of 40- to 100-nm-thick highly structured, hydrated interfaces. These interfaces are dominated in their geometry and dissipative properties by the negatively charged carbohydrate-rich domains of the molecule, the naked protein domains being responsible for attachment. These carbohydrate-rich surfaces have well-defined absorptive properties and permit the entry and entrapment of albumin-coated micro-beads into the absorbed layer at and below a size of 60 nm. However beads larger than 100 nm are completely excluded from the surfaces. These absorptive phenomena correlate with large changes in film dissipation and thus may not only be important in biological functions, e.g. binding viruses, but could also be informative to the surfaces (often ciliated) onto which such mucus films are attached.
Key words: adhesion / MUC5B / mucin / mucus / QCM-D
|
Introduction |
|---|
|
TopAbstractIntroductionResultsDiscussionConclusionsMaterials and methodsConflict of interest statementReferences |
|---|
Major families of biological macromolecules such as polysaccharides, proteoglycans, mucins, and glycolipids play important roles in structuring and controlling the properties of the extracellular milieu. The polysaccharide, proteoglycan, and mucin families often comprise molecules of unusually large molecular weight, size, and complexity that form the basis of complex exoskeletons, supportive extracellular gels, or epithelial surface protection mechanisms. These biological functions relate to the rheological properties of their complexes and yet there are few approaches by which we may feel these dissipative properties while at the same time manipulating their biochemistry. Here we present a study combining light scattering and surface mass and dissipation measurements on a large gel-forming mucin (MUC5B) to demonstrate the value of these approaches in gaining insight into both its biochemistry and biological functions.
MUC5B is one of a small family of what are termed gel-forming mucins which provide the structural basis of protective mucus gels to be found on a number of human epithelial surfaces. These gels provide surface entrapment and protection from toxic particles/molecules and also from pathogenic, chemical, and physical erosion while at the same time maintaining appropriate properties for removal by flow. They underpin the basis of many innate immunity properties and their disregulation is involved with important hypersecretory diseases. Analogs of these molecules are to be found in a variety of animals stretching back over time. The other common members of this family are called MUC5AC, MUC2, and MUC6. MUC5B, in particular, is a major contributor to saliva, glandular secretions of the airway, and cervical secretions (Sheehan et al. 1986
; Thornton et al. 1994, 1999; Nielsen et al. 1997; Troxler et al. 1997). We are particularly interested in the properties of this molecule in the airway as being an important and perhaps under some circumstances dominant contributor to the viscoelastic properties of airway secretions moving over ciliated surfaces (Thornton et al. 1994, 1996, 2000; Kirkham et al. 2002; Holmen et al. 2004; Sheehan et al. 2006).
A schematic diagram outlining the major features of the structure of the MUC5B molecule is shown in Figure 1. This model is based upon a body of biochemical, biophysical, and gene sequence data (Gerken and Dearborn 1984; Desseyn, Guyonnet-Duperat, et al. 1997; Desseyn, Aubert, et al. 1997; Desseyn et al. 1998; Offner et al. 1998). These data indicate that the molecule as found in secretions is assembled from multiple large subunits. In some systems this assembly has been demonstrated to produce a long, linear polymeric thread whereas in other studied cases the multimerization may lead to complex crosslinked outcomes. Multimeric forms of MUC5B in excess of Mr 
100 x 106 have been documented in the mucus obtained from an individual patient who died in status asthmaticus (Sheehan et al. 1995, 1999).
|
As outlined in Figure 1 the structure of MUC5B is complex. Its large physical size and the presence of highly glycosylated domains of Mr
200–500,000 nullify many biochemical approaches that might be employed on normal proteins. Herein we seek to show that a relatively recently introduced method, i.e., quartz crystal mass measurement with dissipation (QCM-D) (Rodahl et al. 1995; Hook et al. 1998), can have a unique role to play in the analysis of these molecules both biochemically and functionally. This method exploits the fact that a vibrating quartz crystal will change both its frequencies of vibration and the damping of these frequencies depending on the amount of added matter accumulating on the crystal surface. The method is arranged so that the dynamics of the formation of the layers may be readily measured as well as interactions of these layers with interacting partners. In particular this is a valuable tool for those interested in the relationship of the mechanics of hydrated surface layers such as found in the extracellular matrix with the chemistry of the interactions happening within them. Thus the method yields intuition as to the emergent physiology and signaling potential for such interactions. We have exploited a property of the mucins discovered in previous electron microscopy studies (Sheehan and Carlstedt 1990), i.e., that colloidal gold binds exclusively to unglycosylated protein domains and is completely excluded from the carbohydrate-covered sequences of the protein as illustrated in Figure 1. We have thus employed gold-coated quartz crystals on which to study the absorbed mucin layers. |
Results |
|---|
|
TopAbstractIntroductionResultsDiscussionConclusionsMaterials and methodsConflict of interest statementReferences |
|---|
MUC5B prepared from human saliva yields a complex distribution of size, molecular weight, and glycoforms of this molecule (Thornton et al. 1999
) and no attempt has been made to dissect these contributing features; thus only average properties are being discussed here. It is a readily available source and also yields a simple model for assessing the contribution that the MUC5B makes to the dissipative properties of a real secreted physiological fluid, i.e., saliva as compared with the other contributory proteins. The MUC5B mucin was isolated by two approaches as described in Materials and methods. The molecular weight and radius of gyration of the intact mucin preparations as measured by multiangle laser light scattering (MALLS) (see Materials and methods) were similar and the data reported in Table I. To provide the basis for understanding a surface molecular layer of such a molecule the reduced tryptic domains that may be obtained from these molecules after reduction followed by trypsin digestion as described in Materials and methods were first studied as previous physical studies, briefly illustrated in Figure 1, suggest that the interpretation of the surface binding of this fragment to a gold surface should be unambiguous.
|
Reduced tryptic fragments
Samples were prepared for QCM-D and run as described in detail in Materials and methods. MALLS studies (Table I) indicate that these fragments have an average molecular weight of around 600 x 103, a radius of gyration of 35 nm, and a configurational length of 100–150 nm in agreement with previous studies (Carlstedt et al. 1983
; Thornton et al. 1999). Electron microscopy of reduced tryptic fragments marked with colloidal gold (Sheehan and Carlstedt 1990) indicates that the gold attaches to one end of the glycopeptide fragments and also that a number of these fragments may bind to a single gold nanoparticle. Examination of the trypsin cleavage sites in the small cysteine repeating domain which punctuate the large glycosylated domain of MUC5B indicates the presence of a residual 10-amino-acid sequence VFCCNYCHCP at the N-terminal end of the last four large glycosylated domains which is presumably responsible for this behavior (Figure 1 bottom right), all the other potential tryptic peptides in this region being accounted for in mass spectrometry of the purified MUC5B (data not shown). To provide an alternative view in solution of how these particles bind to such surfaces, gold particles (nominal diameter 30 nm) and 380 nm hydrophobic polystyrene beads were coated with reduced tryptic domains and their size in water was measured by quasi-elastic light scattering as described in Materials and methods. The size distribution as estimated from intensity is shown in Figure 2A and is remarkably tight and uniform, there being no evidence of open-ended aggregations of the beads. This method which essentially measures the average increase of the equivalent Stokes diameter of the particles due to the presence of the mucin fragments bound to them shows an increase in the Stokes diameter for the gold from 28 nm to 60 nm consistent with two or more reduced tryptic fragments binding to a single gold particle (Figure 1). For the larger polystyrene particles (nominal diameter 380 nm) the measured increase in the Stokes radius was from 388 nm for the beads alone to 520 nm for the bead–glycopeptide complexes as measured in water (Figure 2A). A typical data profile for QCM-D is shown for this fragment in Figure 2(B) in which both the frequency shift (left-hand axis) and dissipation shift (right-hand axis) are plotted versus time for the three overtones. The dissipation increases slowly the solution being refreshed in this experiment on six occasions. Analysis of these data using the proprietary software supplied from Q-Sense (Sweden) predicts a layer that thickens with time to around 40 nm. This would be consistent with the formation of a relaxed monomolecular layer as anticipated by the binding of this fragment to the gold surface at one end. A further simple prediction for such a polyelectrolyte layer is that it will thicken substantially if the supporting electrolyte is removed. In Figure 2(B) the effect on the dissipation (a doubling) by exchanging 0.2 M NaCl with water is demonstrated and the thickness of the layer is predicted by QCM-D measurements to be about 70 nm in reasonable agreement with the data obtained by the light scattering approaches discussed above.
|
Reduced subunit absorption
The reduced subunit is an important though structurally compromised fragment. It is compromised not only on account of the unfolding of previously folded strongly hydrophobic domains both at its C- and N-termini but also due to the small cysteine-rich domains repeated seven times through the highly glycosylated region of the protein. However depending on its mode of preparation it represents most closely the primary gene product providing that no proteolysis has compromised the integrity of the core protein as described in Figure 1. Figure 3A shows a typical F and D trace after the addition of a freshly prepared subunit sample made from a CsCl-purified mucin preparation (see Materials and methods) that was reduced and then dialyzed against an excess of saline as described in Materials and methods. The data are consistent with a slightly thicker absorbed layer (59 nm) than those achieved by the reduced glycopeptides. This is consistent with the presence of unfolded hydrophobic domains as evidenced by the ability of colloidal gold to bind to these regions only after reduction (Figure 1 bottom left). Thus, the binding configurations of the reduced subunit to the gold surface will be more complex than that of the reduced glycopeptide due to the diversity of hydrophobic patches exposed on the macromolecule (Hook et al. 1998
; Rawle et al. 2007). Evidence for a potential for uncontrolled hydrophobic interaction effects may be seen in a separate approach where the mucin was first reduced in GuHCl and subsequently rapidly chromatographed on a CL-2B column (see Materials and methods) to rapidly exchange the GuHCl for saline. A dramatic nonspecific association was observed during the chromatography step (data not shown), and the QCM-D data obtained on the mucin fraction collected through the chromatography (Figure 3B) yielded the highest dissipation levels (D7 = 30 x 10–6) and thickest layer (80 nm) obtained in these studies. Interestingly washing the chip with increasing concentrations of GuHCl decreased both dissipation and frequency levels back to those obtained for the subunits obtained via the dialysis method (Figure 3C and D).
|
The whole MUC5B mucin and its comparison with saliva
The intact (i.e., nonreduced) mucins as isolated chromatographically and by density gradient were high molecular weight mass (20–100 x 106) and size (100–300 nm radius of gyration) as measured by MALLS (average values are given in Table I). Similar QCM-D profiles for F and D were obtained for both preparations and the density gradient isolation case is shown in Figure 4A where a layer of thickness of 47–50 nm was obtained. These values are to be compared with those obtained directly from saliva that was simply centrifuged in a benchtop centrifuge to remove debris. This fluid applied directly to the QCM-D chip (Figure 4B) showed similar binding kinetics and yielded a layer of the same thickness (49 nm) as the pure MUC5B mucin. The similarity of these numbers confirms that the large MUC5B mucin which is the main macromolecule (sometimes called MG1) in saliva is creating a similar dissipating layer even in the presence of other protein components. More surprising to us was the similarity of the calculated thickness of the native MUC5B layer with values obtained for its reduced subunits and this will be discussed at length below.
|
These data open up an avenue for the study of the formation and physical properties of such layers, and two specific examples concerning the porosity of absorbed mucin layers and the effects of adhesions to such layers are given below. The porosity of absorbed protective mucin layers is a key consideration in thinking about how particles of different sizes will interact with such a surface. Is the interaction to be considered with respect to surface or volume properties and can biosurfaces detect the difference, i.e., what can enter or possibly be entrapped by them as compared to what adheres to them? The porosity of absorbed mucin layers was tested employing albumin-coated polystyrene beads between 200 nm and 20 nm. The data (Figure 5A) indicate that the 200 nm and 100 nm beads were not attracted or bound to the adsorbed mucin surface whilst below this size, i.e., 60, 40, and 20 nm, the beads were absorbed into the matrix and in particular changed its dissipation dramatically as well. Intriguingly the layer was nonsaturable with beads of different sizes, i.e., it was not possible to saturate the mucin layer by the initial addition of the smallest beads perhaps indicating that there are pores of different sizes in the absorbed layer.
|
A key issue in understanding the biology of mucus layers relates to what adheres to them and whether their dissipative properties are substantially changed through these adhesions. This might be particularly important in the lung where viscid mucus plaques attached to bronchial surfaces are a feature of a variety of airway diseases such as asthma, CF, and chronic bronchitis. These plaques may indeed be a primary causative agent in attracting, selecting, and settling certain bacterial strains that prompt further damaging inflammatory responses. Anticipating that the charged carbohydrate nature of the surfaces generated here will have strong interactions with polyanions we sought to examine the inside/outside nature of the absorbed layers employing polylysines of different sizes. The absorbed layers of whole mucins were challenged in a QCM-D experiment with a polylysine of Mr 150–300 kDa. These molecules clearly bound to the absorbed mucin layer and enhanced its dissipation (Figure 5B) as compared with smaller polylysine molecules that bound but decreased the dissipation slightly (data not shown). Challenging these two situations again with an intact MUC5B mucin yields a dramatic increase in dissipation in the case of the large polylysine layer (Figure 5B) as against almost no effect for the small polylysine (data not shown). Thus there was a dramatic difference in the surface exposure for these two different size polylysines.
Plot of df/dt versus dD/dt
There is a clear indication in these data that the intact mucin dissipation change is similar for the two whole mucin preparations and also to that for whole saliva. This may be highlighted by plotting df/dt against dD/dt for the data from the different preparations (Figure 6). This mode of data presentation is valuable in comparing the bound mass to dissipation ratios globally and in this case indicates that the viscoelastic properties of these layers must be very similar. Intriguingly the slope of the reduced tryptic sample is significantly higher indicating that the layer has a more elastic dissipative structure proportionate to the mass of the molecules bound. At first look this is very surprising because this fragment is some 100 times lower in molecular weight and 10 times smaller than the intact molecules used in this study. An explanatory hypothesis is explored below.
|
|
Discussion |
|---|
|
TopAbstractIntroductionResultsDiscussionConclusionsMaterials and methodsConflict of interest statementReferences |
|---|
There are few dissipative studies on these glycoproteins to which these data can be compared. A recent study (Feiler et al. 2007
) of a bovine submaxilliary gland mucin employing QCM-D concluded that the method was valuable for studying the absorption properties of such molecules and their possible interactions with specific proteins. The preparation employed in those experiments was commercially available and limited physical characterization was attempted. However the goal of the authors was targeted at the biomaterials research value of such an approach not at its contribution to the biological research on mucus/mucin layers. In this study we have employed QCM-D together with modern biophysical approaches to ask three questions: (1) Can a reasonable physical model for a mucus surface created upon a hydrophobic interface be built up from such data? (2) Do the mucins play the dominant role in such surfaces as anticipated? and (3) Do such artificial surfaces provide a robust model that might mimic biological function and activity?
The tryptic fragment obtained after reduction is probably the only material simple enough to sustain a detailed analysis on the basis of binding to the gold surface by a defined sequence at one end of the molecule. However it provides an important constraint for building a model for subsequent mucin/mucus layers that assemble on hydrophobic surfaces. The thickness of the reduced tryptic fragment layer in water as measured by the increased Stokes diameter of coated polystyrene beads is around 70 nm similar to that for QCM-D using the proprietory software supplied which analyses the frequency and dissipation of the crystal overtones. These values are to be compared with the free solution radii of gyration of 20–35 nm for these fragments. These data are consistent with the oligosaccharide-rich domain being largely excluded from the hydrophobic positively charged gold surface as cartooned in Figure 7(A). The glycopeptide layer thus approximates to a relaxed brush-like structure (Degennes 1980; Harden and Cates 1996). These oligosaccharide-rich tryptic fragments are negatively charged, many containing both terminal sialic acid and sulfate residues (Shori et al. 2001; Veerman et al. 2003). Thus the increasing dissipation and decreasing frequency of the layer in going from salt to pure aqueous (Figure 2B) is consistent with the decrease of Debye–Huckel screening of these polyelectrolytes and the increased thickness and rigidity of the layer. A cartoon indicating our view of these fragments on the surface is shown in Figure 7A.
|
The avoidance of an oligosaccharide interaction with the chip surface is a powerful constraint and strong structuring influence. It provides a possible mechanism to rationalize the subsequent experiments on the reduced subunits which can be isolated in a highly pure form as can be shown by mass spectrometry (data not shown). Thus the interpretation of these data cannot be attributed to the presence of extraneous proteins. The surface interactions of these fragments, though not so relevant from a physiological point of view, are important in gaining confidence in our analysis of these layers and the kinds of artifacts that might interfere with data interpretation. High concentrations of GuHCl (2–6 M) are classically employed in protein denaturation studies to unfold the polypeptide chain into a random coil structure and similar effects are achieved by urea. This is in distinction to high concentrations of NaCl which is not a denaturant and will often stabilize a protein tertiary structure. However the rapid removal of GuHCl or urea from a denatured protein solution will often tend to leave the proteins highly crosslinked due to uncontrolled nonspecific hydrophobic interactions and this appears to be the case for our reduced subunits after the rapid removal of the GuHCl. These subunits appear to be extensively aggregated for this case and yield highly dissipating layers that get thicker with time. Washing such layers with increasing GuHCl concentrations removes the subunits that are nonspecifically crosslinked to each other but not directly bound to the chip. Gently renatured dilute subunit preparations do not show such phenomena and yield only slightly thicker dissipative surfaces than the glycopeptides, of which they are mainly composed. We envisage that such subunits have the possibility of making more than one point of contact with the gold surface employing both N- and C-terminal ends of the protein core (see Figure 1).
The whole mucins present a different situation again, in that their hydrophobic domains are still folded and globular and the small internal Cys-rich domains are now fully folded and buried under a carapace of oligosaccharides (Figure 1). However their large size would lead to random interactions of their multiple protein-rich termini with the gold surface flattening their profile with respect to the chip. We suggest that such a pattern of interactions would populate the chip with a loose mucin network not much thicker than the glycopeptides or the subunits (Figure 7B).
Finally our preliminary attempts to use the surfaces as substrates for binding, adhesion, and penetration studies are intriguing and promising. The absorbed layers formed by the intact mucins resist penetration of particles at and beyond 100 nm but readily absorb particles at 60 nm and below and subsequently modify the dissipative properties of the layer. Such properties may be vital in differentiating between and binding to different pathogens such as viruses and bacteria. Using polylysine as a rather crude mimic of an adhesive protein yields different behaviors depending on the size of the molecule. The small molecules show little effect on the dissipation of the layer and do not act as a focus for further mucin binding to it. This is in contrast to the high molecular weight polylysine which appears to be entrapped in the surface of the layer and permit the strong adhesion of a further layer of the mucin. Such adhesions may be an important analog of what is found on mucus-rich surfaces such as the lung during infection and subsequent inflammatory processes.
|
Conclusions |
|---|
|
TopAbstractIntroductionResultsDiscussionConclusionsMaterials and methodsConflict of interest statementReferences |
|---|
Binding macromolecules to surfaces, e.g., the Biacore or more typically membranes or ELISA plates, is a classic strategy both for their detection and analysis of their interactions with other molecules. For many globular protein studies there may be little to gain by employing QCM-D approaches as against other available methodologies. However for large space filling molecules such as mucins there is much insight to be gained from studies of their dissipative behavior. The MUC5B mucin forms the structural basis of the protective coats in the mouth, the cervix, and the lung. In controlling access to the surface these coats form the basis of many innate immune mechanisms. Here we show that the interaction of the fully assembled MUC5B mucin with a generic hydrophobic surface such as might be encountered on a biosurface or an ingested particle is a layer of about 50–80 nm thickness arising from protein interactions with the surface. This is surprisingly similar in thickness to the surface layer created by the much smaller oligosaccharide-rich reduced tryptic fragments composing the main body of the mucin subunit (40 nm). These fragments contain only a short hydrophobic sequence at their N-terminal ends and appear to be grafted to the surface by this means. These carbohydrate-rich nanolayers appear to be structured and in particular can absorb beads below 100 nm in size and in doing so can change their dissipative behavior.
|
Materials and methods |
|---|
|
TopAbstractIntroductionResultsDiscussionConclusionsMaterials and methodsConflict of interest statementReferences |
|---|
Quartz crystal mass detection with dissipation
QCM-D (Q-sense, Sweden) has been developed to measure both mass and structural properties of the absorbed molecular layers formed on quartz crystals (Rodahl et al. 1995
; Hook et al. 1998). These crystals can be coated with an appropriate material surface of interest such as gold, polystyrene, silicone dioxide hydroxyapatite, etc., to which the molecules absorb. Due to the piezoelectric properties of quartz, it is possible to excite the crystal to oscillation by applying a voltage pulse across its electrodes. The crystal has a natural frequency F0 and also overtones F3, F5, F7, when pulsed electrically. On loading the crystal surface with an undeformable mass the frequency changes in proportion to the loading mass and there is no dissipation change. The mass of the adhering layer is calculated by using the Sauerbrey equation: |
|
It is also possible to get an estimation of the thickness (d) of the adhering layer from the formula
|
|
eff is the effective density of the layer.
In many situations the adsorbed film is not rigid but floppy and the Sauerbrey relation becomes invalid. A film that is "soft" (viscoelastic) will not be fully coupled to the oscillation of the crystal and the surface mass will be underestimated. A soft film dampens the crystal's oscillation. The damping or, dissipation (D), of the crystal's oscillation reveals the film's softness (viscoelasticity) D as defined by
|
|
The dissipation of the crystal is measured by recording the response of a freely oscillating crystal that has been vibrated at its resonance frequency. This also gives the opportunity to jump between the fundamental frequency and overtones (e.g., 15, 25, and 35 MHz). By measuring at multiple frequencies and applying a viscoelastic model (the so-called Voight model) the properties of the adhering soft film, i.e. viscosity, elasticity, and thickness, can be estimated when certain assumptions are made. From a biological perspective our goal is to specify the nature and mechanism of the molecular attachment to the surface, i.e., to create specific matrices for studying their structure and interactions. A variety of such studies relevant to biology may be found in Andersson et al. (2003, 2005), Ayela et al. (2007), Rawle et al. (2007, 2008), and Schofield et al. (2007).
Sensor crystals coated with pure gold were employed in this study. All protein and mucin solutions were made up in the 200 mM NaCl solution with 10 mM EDTA using high-purity Milli-Q water. The buffer filtered through 0.2 µm filters and degassed by sonication before use. All experiments were conducted at a constant temperature of 26°C. Solutions were flowed over the crystal at 50–100 µL/min at concentrations typically between 50 and 100 µg/mL.
Measuring mass and size of mucin solutions by MALLS
MALLS was performed in-line with gel permeation chromatography to measure weight average molecular weight (Mw) and hydrodynamic radius of gyration (Rg) distributions of isolated whole and tryptically digested mucins. This approach yields absolute values of the concentration and molecular weight and size distributions (Thornton et al. 1996). Briefly the mucin preparations were chromatographed either on a Sephacryl 1000 (15 cm x 2.5 cm) or a Sepharose CL-2B (15 cm x 2.5 cm) column eluted with 0.2 M NaCl at a flow rate of 500 µL/min. The column effluent was passed through an in-line Dawn DSP laser photometer coupled to a Wyatt/Optilab 903 (CA) inferometric refractometer to measure light scattering and sample concentration, respectively. Light scattering measurements were taken continuously at 18 angles between 15° and 151°; the data were analyzed according to Zimm (1948).
Mucin preparations
The Intact MUC5B Mucin
The intact MUC5B mucin used in this study was isolated from saliva essentially as previously described (Mehrotra et al. 1998; Thornton et al. 1999). Saliva was collected from an individual healthy male donor. Saliva was stimulated by chewing on Parafilm and collected into an equal volume of 200 mM NaCl on ice. The solution was centrifuged at 3000 x gav for 10 min at 4°C. The supernatant was decanted and stored at 4°C until processed further. MUC5B from saliva was isolated both by a single-step gel chromatography procedure and a two-step procedure involving chromatography fractionation followed by density gradient fractionation (Thornton et al. 1999). In brief aliquots of the supernatant chromatographed on a column of Sephacryl 1000 (50 cm x 2.5 cm) eluted with 200 mM NaCl with 10 mM EDTA, pH 7, at a flow rate of 1 mL/min. The mucins eluted in the void volume of the column (MUC5B) were then subjected to CsCl/4 M guanidinium chloride density gradient centrifugation at a starting density of 1.4 g/mL in a Beckman Ti45 angle rotor at 40,000 rpm for 67 h at 15°C. The mucin (MUC5B) containing fractions were determined by a polyclonal antibody against MUC5B (MAN5BI, Thornton et al. 1997) and subsequently pooled and dialyzed into 200 mM NaCl with 10 mM EDTA, pH 7.0.
Reduced Mucin Subunits
Reduced mucin subunits were obtained by the treatment of the purified mucins in 6 M guanidinium chloride/0.1 M Tris, pH 8.0, with 10 mM dithiothreitol for 2 h at 37°C. Iodoacetamide was then added to a final concentration of 25 mM, and the mixture was left in the dark for 30 min at room temperature. Reduced mucin subunits were dialyzed into 200 mM NaCl with 10 mM EDTA and stored at 4°C.
Reduced Tryptic Glycopeptides (T-Domains)
Reduced tryptic glycopeptides (t-domains) were obtained from reduced mucin subunits. These were dialyzed into 50 mM ammonium hydrogen carbonate, pH 8.0, and digested overnight with trypsin at 37°C. The digest was chromatographed on a Superdex 200 HR 10/30 column in 50 mM ammonium hydrogen carbonate at a flow rate of 0.3 mL/min. The void fraction was collected and contained the large glycopeptides/T-Domains.
Dynamic Light Scattering Measurements (Quasielastic Scattering)
These experiments were conducted on the reduced tryptic glycopeptides and whole mucins to give alternative estimates of the mucin surface layer thickness. The prediction from previous electron microscopy studies that colloidal gold bound only to one end of the reduced tryptic fragments was confirmed here by dynamic light scattering experiments performed on glycopeptides bound to 30 nm (nominal value) colloidal gold particles (BBInternational). The thickness of the glycopeptide and whole mucin coats on an open hydrophobic surface was tested using polystyrene beads (380 nm as a nominal value, Molecular probes). These experiments provided an alternative estimate of a surface coat thickness and the likely nature of the glycopeptide and mucin binding to hydrophobic surfaces to compare with estimates made by QCM-D. From previous electron microscopy experiments we have shown that a number of such fragments bind avidly to a single gold nanoparticle by one end. The glycopeptides, prepared as described above, were added dropwise to a solution of gold nanoparticles in water and complexes prepared as previously described (Sheehan and Carlstedt 1990). The 380 nm polystyrene-glycopeptide and intact mucin complexes were prepared similarly by adding glycopeptides or mucins to a dilute suspension of these beads in water. The mucin-bead complexes were then purified from excess mucins by zonal centrifugation through a 10% sucrose step layered upon a 20% sucrose step. After brief centrifugation in a ti-rotor for 30 min at 20 K rpm the beads were recovered at the 20% sucrose interface and diluted (100–200 times) to yield an optimum scattering intensity for dynamic light scattering measurements which were performed on a Malvern 4700 autosizer employing a 20 mW helium neon laser (Malvern Instruments Ltd., UK). Samples are studied at a constant temperature of 25°C. Light scattering from the sample was detected by a photomultiplier tube placed at 90° to the incident laser beam. The translational diffusion coefficient of the solutions was calculated from the time autocorrelation of the scattered light intensity and the translational diffusion coefficient was extracted from the correlogram using the method of cumulants as applied in the proprietory Malvern software. The diameter of the particles with and without the adsorbed mucins was obtained from the application of Stokes–Einstein equations (Almond et al. 1998).
|
Conflict of interest statement |
|---|
|
TopAbstractIntroductionResultsDiscussionConclusionsMaterials and methodsConflict of interest statementReferences |
|---|
None declared.
|
Acknowledgements |
|---|
This work was supported by a gift from an anonymous donor for research targeted to proteomics of cystic fibrosis lung disease, by the Cystic Fibrosis Foundation and by NHLBI/NIH (HL 084934-02). We are thankful to our colleagues at UNC Chapel Hill, particularly the members of the Virtual Lung Group, for useful discussions.
|
Footnotes |
|---|
The figures have been updated.
|
Abbreviations |
|---|
CF, cystic fibrosis; CsCl, caesium chloride; GuHcl, guanidium hydrochloride; MALLS, multiangle laser light scattering; Rg, radii of gyration; QCM-D, quartz crystal mass balance with dissipation
|
References |
|---|
|
TopAbstractIntroductionResultsDiscussionConclusionsMaterials and methodsConflict of interest statementReferences |
|---|
Almond A, Brass A, Sheehan JK. Deducing polymeric structure from aqueous molecular dynamics simulations of oligosaccharides: Predictions from simulations of hyaluronan tetrasaccharides compared with hydrodynamic and X-ray fibre diffraction data. J Mol Biol (1998) 284(5):1425–1437.[CrossRef][Web of Science][Medline]
Andersson AS, Glasmastar K, Sutherland D, Lidberg U, Kasemo B. Cell adhesion on supported lipid bilayers. J Biomed Mater Res A (2003) 64(4):622–629.[Medline]
Andersson M, Andersson J, Sellborn A, Berglin M, Nilsson B, Elwing H. Quartz crystal microbalance—with dissipation monitoring (QCM-D) for real time measurements of blood coagulation density and immune complement activation on artificial surfaces. Biosens Bioelectron (2005) 21(1):79–86.[CrossRef][Web of Science][Medline]
Ayela C, Roquet F, Valera L, Granier C, Nicu L, Pugniere M. Antibody-antigenic peptide interactions monitored by SPR and QCM-D. A model for SPR detection of IA-2 autoantibodies in human serum. Biosens Bioelectron (2007) 22(12):3113–3119.[CrossRef][Web of Science][Medline]
Carlstedt I, Lindgren H, Sheehan JK. The macromolecular structure of human cervical-mucus glycoproteins. Studies on fragments obtained after reduction of disulphide bridges and after subsequent trypsin digestion. Biochem J (1983) 213(2):427–435.[Web of Science][Medline]
Degennes PG. Conformations of polymers attached to an interface. Macromolecules (1980) 13:1069.[CrossRef][Web of Science]
Desseyn JL, Guyonnet-Duperat V, Porchet N, Aubert JP, Laine A. Human mucin gene MUC5B, the 10.7-kb large central exon encodes various alternate subdomains resulting in a super-repeat. Structural evidence for a 11p15.5 gene family. J Biol Chem (1997) 272(6):3168–3178.
Desseyn JL, Aubert JP, Van Seuningen I, Porchet N, Laine A. Genomic organization of the 3' region of the human mucin gene MUC5B. J Biol Chem (1997) 272(27):16873–16883.
Desseyn JL, Buisine MP, Porchet N, Aubert JP, Laine A. Genomic organization of the human mucin gene MUC5B. cDNA and genomic sequences upstream of the large central exon. J Biol Chem (1998) 273(46):30157–30164.
Feiler AA, Sahlholm A, Sandberg T, Caldwell KD. Adsorption and viscoelastic properties of fractionated mucin (BSM) and bovine serum albumin (BSA) studied with quartz crystal microbalance (QCM-D). J Colloid Interface Sci (2007) 315(2):475–481.[CrossRef][Web of Science][Medline]
Gerken TA, Dearborn DG. Carbon-13 NMR studies of native and modified ovine submaxillary mucin. Biochemistry (1984) 23(7):1485–1497.[CrossRef][Web of Science][Medline]
Harden JL, Cates ME. Deformation of grafted polymer layers in strong shear flows. Phys Rev E Stat Phys Plasmas Fluids Relat Interdiscip Topics (1996) 53(4):3782–3787.[Medline]
Holmen JM, Karlsson NG, Abdullah LH, Randell SH, Sheehan JK, Hansson GC, Davis CW. Mucins and their O-glycans from human bronchial epithelial cell cultures. Am J Physiol Lung Cell Mol Physiol (2004) 287(4):L824–L834.
Hook F, Rodahl M., Brzezinski P., Kasemo B. Measurements using the quartz crystal microbalance technique of ferritin monolayers on methyl-thiolated gold: Dependence of energy dissipation and saturation coverage on salt concentration. J Colloid Interface Sci (1998) 208(1):63–67.[CrossRef][Web of Science][Medline]
Kirkham S, Sheehan JK, Knight D, Richardson PS, Thornton DJ. Heterogeneity of airways mucus: Variations in the amounts and glycoforms of the major oligomeric mucins MUC5AC and MUC5B. Biochem J (2002) 361(Pt 3):537–546.[CrossRef][Web of Science][Medline]
Mehrotra R, Thornton DJ, Sheehan JK. Isolation and physical characterization of the MUC7 (MG2) mucin from saliva: Evidence for self-association. Biochem J (1998) 334(Pt 2):415–422.[Web of Science][Medline]
Nielsen PA, Bennett EP, Wandall HH, Therkildsen MH, Hannibal J, Clausen H. Identification of a major human high molecular weight salivary mucin (MG1) as tracheobronchial mucin MUC5B. Glycobiology (1997) 7(3):413–419.
Offner GD, Nunes DP, Keates AC, Afdhal NH, Troxler RF. The amino-terminal sequence of MUC5B contains conserved multifunctional D domains: Implications for tissue-specific mucin functions. Biochem Biophys Res Commun (1998) 251(1):350–355.[CrossRef][Web of Science][Medline]
Rawle RJ, Selassie CR, Johal MS. Creation of mammalian single- and double-stranded DNA surfaces: A real-time QCM-D study. Langmuir (2007) 23(19):9563–9566.[CrossRef][Web of Science][Medline]
Rawle RJ, Johal MS, Selassie CR. A real-time QCM-D approach to monitoring mammalian DNA damage using DNA adsorbed to a polyelectrolyte surface. Biomacromolecules (2008) 9(1):9–12.[CrossRef][Web of Science][Medline]
Rodahl F, Hook A, Krozer P, Brzezinski, Kasemo B. Quartz crystal microbalance setup for frequency and Q-factor measurements in gaseous and liquid environments. Rev Sci Instruments (1995) 66:3924–3930.[CrossRef][Web of Science]
Schofield AL, Rudd TR, Martin DS, Fernig DG, Edwards C. Real-time monitoring of the development and stability of biofilms of Streptococcus mutans using the quartz crystal microbalance with dissipation monitoring. Biosens Bioelectron (2007) 23(3):407–413.[CrossRef][Web of Science][Medline]
Sheehan JK, Oates K, Carlstedt I. Electron microscopy of cervical, gastric and bronchial mucus glycoproteins. Biochem J (1986) 239(1):147–153.[Web of Science][Medline]
Sheehan JK, Carlstedt I. Electron microscopy of cervical-mucus glycoproteins and fragments therefrom. The use of colloidal gold to make visible ‘naked’ protein regions. Biochem J (1990) 265(1):169–177.[Web of Science][Medline]
Sheehan JK, Richardson PS, Fung DC, Howard M, Thornton DJ. Analysis of respiratory mucus glycoproteins in asthma: A detailed study from a patient who died in status asthmaticus. Am J Respir Cell Mol Biol (1995) 13(6):748–756.[Abstract]
Sheehan JK, Howard M, Richardson PS, Longwill T, Thornton DJ. Physical characterization of a low-charge glycoform of the MUC5B mucin comprising the gel-phase of an asthmatic respiratory mucous plug. Biochem J (1999) 338(Pt 2):507–513.[CrossRef][Web of Science][Medline]
Sheehan JK, Kesimer M, Pickles R. Innate immunity and mucus structure and function. Novartis Found Symp (2006) 279:155–166. discussion 167–169, 216–219.[Medline]
Shori DK, Kariyawasam HH, Knight RA, Hodson ME, Genter T, Hansen J, Koch C, Kalogeridis A. Sulphation of the salivary mucin MG1 (MUC-5B) is not correlated to the degree of its sialylation and is unaffected by cystic fibrosis. Pflugers Arch (2001) 443(Suppl_1):S50–S54.[CrossRef][Web of Science][Medline]
Thornton DJ, Devine PL, Hanski C, Howard M, Sheehan JK. Identification of two major populations of mucins in respiratory secretions. Am J Respir Crit Care Med (1994) 150(3):823–832.[Abstract]
Thornton DJ, Carlstedt I, Howard M, Devine PL, Price MR, Sheehan JK. Respiratory mucins: Identification of core proteins and glycoforms. Biochem J (1996) 316(Pt 3):967–975.[Web of Science][Medline]
Thornton DJ, Howard M, Khan N, Sheehan JK. Identification of two glycoforms of the MUC5B mucin in human respiratory mucus. Evidence for a cysteine-rich sequence repeated within the molecule. J Biol Chem (1997) 272(14):9561–9566.
Thornton DJ, Khan N, Mehrotra R, Howard M, Veerman E, Packer NH, Sheehan JK. Salivary mucin MG1 is comprised almost entirely of different glycosylated forms of the MUC5B gene product. Glycobiology (1999) 9(3):293–302.
Thornton DJ, Gray T, Nettesheim P, Howard M, Koo JS, Sheehan JK. Characterization of mucins from cultured normal human tracheobronchial epithelial cells. Am J Physiol Lung Cell Mol Physiol (2000) 278(6):L1118–L1128.
Troxler RF, Iontcheva I, Oppenheim FG, Nunes DP, Offner GD. Molecular characterization of a major high molecular weight mucin from human sublingual gland. Glycobiology (1997) 7(7):965–973.
Veerman EC, Van Den Keijbus PA, Nazmi K, Vos W, Van Der Wal JE, Bloemena E, Bolscher JG, Amerongen AV. Distinct localization of MUC5B glycoforms in the human salivary glands. Glycobiology (2003) 13(5):363–366.
Zimm BH. The scattering of light and the radial distribution function of high-polymer solutions. J Chem Phys (1948) 16:1099–1116.[CrossRef]
CiteULike 
Connotea 
Del.icio.us What's this?
| ||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||