Secreted human conjunctival mucus contains MUC5AC glycoforms

Roger B.Ellingham1, Monica Berry, David Stevenson2, Anthony P.Corfield3

Division of Ophthalmology, University of Bristol Department of Hospital Medicine, Bristol Eye Hospital, Lower Maudlin Street, Bristol, BS1 2LX, UK, 2Oxford GlycoSciences, 10 The Quadrant, Abingdon Science Park, Abingdon, Oxon, OX14 3YS, UK, and 3University of Bristol Department of Hospital Medicine, Dorothy Crowfoot Laboratories, Mucin Research Group, Bristol Royal Infirmary, Bristol, BS2 8HW, UK

Received on November 5, 1998; revised on February 12, 1999; accepted on May 12, 1999

This study addresses the extent of variation in secreted end-product mucins in human conjunctival mucus. The aim was to determine whether the variety of mucin species found was encompassed by the mucin genes which have been cloned to date. Extraction into guanidine hydrochloride and separation of mucin constituents, by a combination of cesium chloride density gradient centrifugation, size separation on Sepharose CL-2B, MonoQ ion exchange chromatography and agarose gel electrophoresis, demonstrates a complex mixture of mucins. Sample size limitations precluded compositional amino acid analysis. MUC 5AC and MUC1, 2, and 4 are all detected in the buoyant density range 1.3-1.5 g/ml by antibody binding. The mucins vary in size from >40 × 106 to <97 × 103 Da. A wide range of molecular size was confirmed using rate zonal centrifugation. The presence of smaller species contrasts with other mucous secretions similarly studied. In each size range are low, medium, and high charge mucins. Sialylation predominates in the medium charge and sulfate in the high charge. Only MUC5AC cross-reactivity is maintained throughout the analysis. It is detected in large and medium sized mucins but accounts for only the least mobile mucins within copurified species of similar density, size, and charge resolved using agarose electrophoresis. MUC5AC cross-reactivity is also detected in both medium and high charge species, indicating the presence of glycoforms.

Key words: mucin/glycoform/conjunctiva/MUC5AC

INTRODUCTION

The eye shares a feature with the other wet, exposed surfaces of the body: it possesses a mucosal epithelium, the conjunctiva, which at its center merges with the epithelium of the transparent cornea. During eye closure the conjunctiva lining the upper lid is in intimate contact with the cornea and it is believed that the mucous secretions of the conjunctival goblet cells contribute to the mucous gel of the cornea. Vision requires that ocular mucins spread smoothly over the eye as transparent mucus. Evidence for a secreted precorneal mucin gel more than 30 µm thick (Prydal and Campbell, 1992; Prydal et al., 1992), and continuous with the conjunctival mucus layer, is now widely accepted. The gel probably adheres through interaction with the epithelial glycocalyx (Nichols et al., 1985) and with membrane bound mucins synthesized by the squamous epithelial cells of the cornea and conjunctiva (Gipson and Inatomi, 1997). The cornea does not possess goblet cells but, in addition to its apical cell surface bearing membrane mucin (Inatomi et al., 1995; Watanabe et al., 1995), the cornea may shed membrane mucin or secrete mucin through a non-goblet cell pathway of the type described for the conjunctiva (Dilly, 1985; Greiner et al., 1985). The majority of mucins secreted into both the conjunctival and corneal mucous gels, however, are likely to be of goblet cell origin, from the conjunctiva, hydrated by aqueous tears from the lacrimal glands.

Northern blot, in situ hybridization, and immunohistochemistry have provided evidence for the expression, in human conjunctiva, of the cloned mucin genes MUC1, MUC4, and MUC5AC (Inatomi et al., 1995; Inatomi et al., 1996). MUC1 and MUC4 are detected in the squamous epithelial cells while MUC5AC is limited to goblet cells. Northern blotting (Jumblatt et al., 1995), RT-PCR (McKenzie et al., 1998), and immunochemistry (Bolis et al., 1995) also identified low levels of MUC2 products in the conjunctiva. MUC1, the only cloned mucin gene identified in the cornea (Inatomi et al., 1995), is ubiquitously expressed as a membrane bound mucin although it may be cleaved from the surface (Hilkens et al., 1992). The uncloned membrane-bound sialo-mucin complex, ASGP (Rossi et al., 1996), from cornea and conjunctiva (Pflugfelder et al., 1998), also contributes to surface mucus. Recent evidence suggests MUC4 and ASGP might share the same identity (Moniaux et al., 1998). The relative contribution of each gene is unknown as is the tissue specific glycosylation of the constituent mucins. These can only be determined by analysis of the end product ocular secretion. We have reported (Berry et al., 1996) that the secreted mucins of human conjunctiva are similar to those of other mucous secretions in that they are polydisperse and some reach very great size. Individual components could not be resolved by electrophoresis alone. An unusual feature was the predominance of very short oligosaccharide side chains, in particular GalNAc[alpha]-O-Ser/Thr (Tn) and NeuAc[alpha]2-6GalNAc[alpha]-O-Ser/Thr (sialyl-Tn).

Complex cross-fractionation is necessary to reveal all components in a mixed secretion. To untangle this mixture, with the tiny samples obtainable from human conjunctival tissue, we aimed to ensure maximal homogeneity within each subfraction analyzed. Samples of similar buoyant density and molecular size were fractionated according to charge and separated by mobility on agarose gel electrophoresis. Our aim was to determine if the variety of mucin species found is fully encompassed by the small number of candidate mucin genes described in ocular tissues.

RESULTS

Mucin purification

Mucins banded in the density range 1.3-1.5 g/ml during cesium chloride density gradient centrifugation in 4 M guanidine hydrochloride (Figure 1a). This is characteristic of mucins dispersed in this chaotropic solvent (Carlstedt and Sheehan, 1984). Fractions in this range bound the anti-MUC5AC antibody LUM5-1 and the anti-MUC1 antibody BC2. Anti-MUC4 crossreactivity (M4.171/M4.275) peaked later in this range between 1.4-1.5 g/ml. Anti-MUC2 crossreactivity was the weakest. Binding of WGA (Figure 1b) extended to 1.22 g/ml which is consistent with the presence of non-mucin glycoproteins. Crossreaction with the anti-mucin antibodies in these lighter buoyant densities (Figure 1a) is consistent with binding to nonglycosylated mucin precursors. MLL (Figure 1b) showed two peaks of crossreaction, suggesting a concentration of the Tn or Sialyl-Tn epitopes in the density ranges 1.3-1.4 and 1.4-1.5 g/ml. Three density ranges 1.3-1.35 (low, L), 1.35-1.4 (medium, M), and 1.4-1.5 g/ml (high, H) were kept separate in the subsequent reduction and alkylation, Sepharose CL-2B gel filtration, and MonoQ ion exchange.


Fig. 1. Cesium chloride density gradient profiles of human conjunctival extract.(a) Dot blots of fractions from a density gradient in 4 M guanidine hydrochloride were probed with the anti-mucin peptide antibodies BC2 (MUC1), M4.171/M4.275 (MUC4), LUM5-1 (MUC5AC), and LUM2-3 (MUC2). (b) The same fractions probed with lectins MLL and WGA. For subsequent analyses the density range containing mucins was divided into: (L) low density mucins, 1.3-1.35 g/ml; (M) medium density mucins, 1.35-1.4 g/ml; (H) high density mucins 1.4-1.5 g/ml. Proteins, glycoproteins, and mucin precursors (<1.3 g/ml) and fractions containing nucleic acids (>1.5 g/ml) were discarded. Densitometry was obtained with Scan Analysis 2.50 and plotted against the measured density of the fractions from the gradient. Quantitative comparisons of staining intensity between different reagents is not possible. The data illustrate relative positions of peaks of detection.

Size fractionation of reduced and alkylated mucin subunits by Sepharose CL-2B

Following reduction and alkylation, each of the mucin-rich density ranges yielded a triphasic cross-reaction with WGA on Sepharose CL-2B gel filtration (Figure 2). Cross-reaction with the anti-mucin antibodies could not be reliably detected due to sample dilution. The first peak (fractions 9-16) was in the volume (V0)excluded by the column matrix suggesting a molecular weight >4-5 × 106. Material in the size range resolved by the column (molecular weight range 7 × 104 to 4-5 × 106) eluted as a broad peak (fractions 17-25, Vi). The last peak (fractions 26-32) eluted in the total volume of the column (Vt) consisting of material too small to be resolved. V0, Vi, and Vt were applied to MonoQ ion exchange chromatography separately.


Fig. 2. Separation of mucin populations by hydrodynamic volume. Mucins of low (L), medium (M), and high (H) buoyant density from a cesium chloride density gradient (see Figure 1) were reduced and alkylated, then separately applied to Sepharose CL-2B. Dot blots of the eluted fractions were probed with WGA. Fractions were pooled as V0 (fractions 9-16), Vi (fractions 17-25), and Vt (fractions 26-32) for subsequent analyses.

Rate zonal centrifugation of mucins

In parallel with size analysis on Sepharose CL-2B, mucin size was analyzed by rate zonal centrifugation. Native and reduced/alkylated mucins 1.3-1.5 g/ml in 4 M guanidine hydrochloride were further purified in a cesium chloride density gradient in 0.5 M guanidine hydrochloride then subjected to rate zonal centrifugation.

Rate zonal centrifugation of the density range 1.3-1.4 g/ml showed a mixture of sizes detected by all four anti-mucin antibodies (Figure 3a). Peaks of detection coincide between antibodies. A shift to smaller molecules was apparent on reduction and alkylation of WGA- (Figure 3b), MUC5AC-, and MUC2-positive material (not shown). No shift was detected in MUC1 or MUC4 binding patterns. Mucins of density 1.4-1.5 g/ml behaved similarly.


Fig. 3. Rate zonal centrifugation of native and reduced/alkylated conjunctival mucins. (a) Reduced/alkylated mucins of buoyant density 1.3-1.4 g/ml in 0.5 M guanidine hydrochloride were analyzed by rate zonal centrifugation in a 4-6 M guanidine hydrochloride gradient. Dot blots were probed with the anti-mucin peptide antibodies BC2 (MUC1), M4.171/M4.275 (MUC4), LUM5-1 (MUC5AC), and LUM2-3 (MUC2). (b) Reduced/alkylated mucins of buoyant density 1.3-1.4 g/ml analyzed by rate zonal centrifugation and probed with WGA show a shift to smaller sizes when compared with native mucins of the same density. Densitometry was performed using Scan Analysis 2.50. Sedimentation rate (size) increases with fraction number along the abscissa. Quantitative comparisons can be made between traces in (b) since equivalent amounts of material were used.

Charge fractionation by ion exchange chromatography

Each size range (Vt after extensive dialysis against MonoQ buffer) was applied to MonoQ ion exchange chromatography. The MonoQ eluates were too dilute for antibody detection. All WGA positive material bound to the MonoQ column since none eluted from the column during sample loading or the initial column wash. In Figure 4, the WGA elution profiles of V0, Vi, and Vt are vertically aligned for mucins of low, L, (Figure 4a); medium, M, (Figure 4b); and high, H, buoyant densities (Figure 4c). Vacuum blots of agarose electrophoresis gels are shown under the respective sample numbers.

   A
   B
   C

Fig. 4. MonoQ elution profiles and agarose electrophoresis of reduced/alkylated mucin subunits. The graphs represent MonoQ mucin elution profiles detected by WGA and the concentration of lithium perchlorate in the eluting buffer (similar in each graph set). The figure shows (a) low, (b) medium, and (c) high density mucins, of the CL2B pools V0 (top), Vi (middle), and Vt (bottom). Lithium perchlorate concentration rises linearly from 0 to 0.4 M before stepping up to the 0.8 M wash. Below each abscissa (the labels denote fraction number from the MonoQ column) are WGA probed vacuum blots of agarose electrophoresis of every other fraction. The lowest charged mucins elute first. Many MonoQ fractions contain more than one mucin species. Mucin species in each MonoQ fraction have similar charge density. Electrophoresis lanes marked by [lambda] are equivalent to the lanes in blots probed with LUM5-1 in Figure 5. Quantitative comparison between the MonoQ mucin elution profiles is not possible. The data illustrate the relative positions of peaks of detection.

The MonoQ elution profiles show that most species, in each size range of each buoyant density, were of medium charge (Figure 4, fractions 28-44) eluting with 0.1-0.2 M lithium perchlorate. The low density V0 mucins contained the majority of the higher charged species (Figure 4a, top) eluting at lithium perchlorate concentrations above 0.2 M. A small proportion of the material from this pool remained bound to the column until the 0.8 M lithium perchlorate wash. In both the low and medium density ranges the smallest (Vt) mucins (Figure 4a,b, bottom) had an early peak of low charge eluting below 0.1 M lithium perchlorate.

Electrophoresis resolved most MonoQ fractions into two or three bands of different mobility, best illustrated in Figure 4b, V0 and Vi. At similar charge, the higher mobility bands were more prominent in the smaller (Vt ) than the larger (V0 ) material. Low charge mucins in Vt of low and medium density (Figure 4a,b, bottom) were not visible after agarose electrophoresis, but were seen on SDS-PAGE (not shown) as bands of 97 × 103 Da.

Analysis of MonoQ separated mucin species

Sample size precluded amino acid and sugar analysis of individual electrophoresis bands. Antibody probing with BC2, LUM2-3, M4.275, and M4.171 showed no cross-reaction after electrophoresis (not shown). However, MonoQ fractions, indicated by a dot in Figure 4, cross reacted with LUM5-1 and MLL. Antibody LUM5-1 bound weakly. Nevertheless, in low density V0 and high density Vi mucins, it highlighted distinct bands of different charge (Figure 5). This cross-reaction included some, but not all the bands highlighted by WGA. MLL binding was very similar to the anti-MUC5AC pattern (not shown).


Fig. 5. MUC5AC detection in MonoQ fractions. MonoQ fractions representing mucin subunits of large (V0) and medium (Vi) hydrodynamic volume and low (L) and medium (M) buoyant density were separated by agarose gel electrophoresis and the blots probed with antibody LUM5-1 against MUC5AC. WGA binding of equivalent blots can be compared by reference to Figure 4a,b using the fraction number indicated above electrophoresis lanes on the respective blots. Only the slow migrating bands are detected by LUM5-1.

Sialic acid, sulfate, and protein determinations were performed in four charge ranges of mucins, pooled across all size and buoyant densities. The charge ranges corresponded to the peaks eluting at lithium perchlorate concentrations of (A) 0-0.1 M, (B) 0.1-0.2 M, (C) 0.2-0.325 M, and (D) 0.325-0.8 M. Reverse phase HPLC profiles of DMB labeled sialic acids (not shown) demonstrated Neu5Ac and a small quantity of Neu5,7Ac2 in all samples except pool B where only Neu5Ac was detected. Sialylation, expressed as the ratio of total sialic acids to optical density at 260 nm, peaks in pool B and is lowest in pools C and D (Figure 6). Pools C and D contain relatively more sulfate than pools A and B when detected in dot blots using Alcian blue, pH 1.0 and high iron diamine stains (Figure 6, inset). Levels of sulfation, however, cannot be quantified by these methods.


Fig. 6. Ratio of total sialic acid content to absorbance at 260 nm in mucin samples of increasing charge density. MonoQ separated mucins eluting at lithium perchlorate concentrations of (A) 0-0.1 M, (B) 0.1-0.2 M, (C) 0.2-0.325 M, and (D) 0.325-0.8 M were pooled. The absolute molar content of sialic acids and absorbance at 260 nm were determined for each pool. The ratio is used as a guide to sialylation. Charge density increases from A to D, but sialylation does not. The inset shows 10 µl dots of each pool in triplicate stained with HID (top) and Alcian blue, pH 1.0 (bottom) as a guide to the presence of sulfate. The less-sulfated mucins in A and B block background staining of the membrane. The numerical bar labels indicate absorbance at 260 nm in arbitrary units, as a guide to the amount of mucin in each sample, for comparison with the dot blot.

DISCUSSION

It is likely that the functions of the preocular gel depend critically on the mucin peptide and oligosaccharide side chain structures it contains. We show here that conjuctival mucins vary in size from >4-5 × 106 (Figure 2) to <97 × 103 Da and that within each size range there are low to high charge species. Sialylation predominates in the moderately charged and sulfation in the highly charged species (Figure 6). MUC5AC mucins are present as glycoforms of different charge (Figure 4), but do not account for all mucin species detected.

Tissue extracted mucins include a mixture of precursors, mature secreted mucins and breakdown products. Our samples may also contain physiologically cleaved membrane-associated mucins accounting for the cross-reactivity with antibodies against MUC1 and MUC4 (Figures 1, 3). Cesium chloride density gradient centrifugation is an important initial step in mucin separation since small mucins are not selectively lost with the protein fraction as they tend to be when initial fractionation is made on the basis of molecular size. In a density gradient the highly glycosylated mucins, regardless of their size, are separated from proteins and glycoproteins, which have a lower carbohydrate content (Carlstedt and Sheehan, 1984). Nonglycosylated mucin precursors, seen cross-reacting with the anti-mucin antibodies at densities <1.3 g/ml (Figure 1a), will be removed with the proteins. Cross-reaction with anti-mucin peptide antibodies supports the presence of mucins in the density range 1.3-1.5 g/ml in our 4 M guanidine preparations, as do the characteristic migration patterns seen on subsequent agarose electrophoresis (Figure 4).

The large species eluting in the V0 and Vi of Sepharose CL-2B, shown to be mucins by their anti-MUC5AC antibody binding after concentration on MonoQ columns, form lubricating gels supporting the hydrated environment required for the health of the ocular surface. Ocular mucin subunit preparations also include a surprisingly rich pool of small molecules eluting as Vt fractions on Sepharose CL-2B, in addition to the expected molecules in V0 (Figure 2). This is in contrast to other mucous secretions prepared the same way (Carlstedt et al., 1983; Thornton et al., 1994b). These small mucins, including the 97 × 103 Da bands seen on SDS-PAGE, might be unusual mature mucins, breakdown products or proteolytically cleaved fragments of larger molecules released by reduction and alkylation.

Reduction and alkylation had little effect on the electrophoretic mobility of human conjunctival mucins (Berry et al., 1996). Rate zonal centrifugation, however, shows that MUC5AC and MUC2 mucins do become smaller on reduction and alkylation. This suggests a subunit structure with disulfide bridges as in other mucosal systems (Carlstedt et al., 1985; Strous and Dekker, 1992; Forstner and Forstner, 1994). MUC1 and MUC4 mucins are unaffected by reduction and alkylation, in keeping with the expected structure of membrane bound mucins.

An Alcian-blue/periodic acid Schiff (AB/PAS) pH dependent staining method has suggested the presence of neutral mucins in conjunctival subsurface vesicles (Greiner et al., 1985). We did not detect neutral mucins eluting from MonoQ columns even after concentration on PVDF membranes. The results obtained with WGA detection precisely mirror those obtained with 14C-radio-alkylated subunits (Berry et al., 1996). If neutral mucins are present in ocular secretions then they must constitute a minor fraction.

Each MonoQ fraction in Figure 4 is a mixture of at least two species, precluding protein sequencing. There was insufficient material extractable from agarose electrophoresis blots for amino acid analysis. Using antibody detection, we have identified some of the bands as MUC5AC gene products. These vary in charge within one size range suggesting the presence of glycoforms. The remaining bands could not be identified. They may contain material below the detection thresholds of the antibodies employed because cross-reaction was observed at earlier stages of purification. MUC1 and MUC4 are likely to exist as membrane associated mucins in the conjunctiva and have been detected using BC2, M4.171, and M4.275 in detergent extracted membrane preparations (Berry et al., unpublished observations). We await development of reliable antibodies against reduced and alkylated secreted mucins to recognize the unidentified peptide cores we have purified.

We have published evidence for a predominance of short oligosaccharide side chains including Tn and sialyl-Tn in mature conjunctival mucins in humans (Berry et al., 1996; Corfield et al., 1997). The colocalization of MLL and LUM5-1 binding after cross-fractionation supports this finding, but larger carbohydrate epitopes have also been detected, including the sialylated Lewis blood group antigens (Garcher et al., 1994; Bolis et al., 1995). The presence of sialylated structures is of interest because they could modulate immune cell adhesion (Majuri et al., 1995; Zhang et al., 1996) in closed eye tears, and interact with cell adhesion sites on ocular pathogens, e.g., Pseudomonas aeruginosa (Fleiszig et al., 1994; Hazlett et al., 1986). Sulfation may have a role in blocking bacterial degradation of the oligosaccharide side-chains (Corfield and Warren, 1996).

We do not yet know how physiological turnover of the preocular gel is achieved. Perhaps some of the smaller mucin species are destined for disposal, or function coating particles for removal by watery tears. Such a system would be unusual for most mucosal surfaces (Strous and Dekker, 1992; Gum, 1995) but is similar to the salivary system which comprises the small soluble MUC7 (MG2) mucin (Bobek et al., 1993) and the large gel forming MUC5B (MG1) mucin (Nielsen et al., 1997). Small sulfated salivary mucins have been shown to aggregate bacteria and protect against dental caries (Slomiany et al., 1996). Glycoforms would also ensure a diversity of binding sites for microorganisms. Identification of the protein and oligosaccharide structures in human ocular mucins should provide major clues about their functions at the ocular surface.

MATERIALS AND METHODS

Materials

SeaKem LE agarose was manufactured by FMC BioProducts, Rockland, ME. Acrylamide/bis 40% (19:1) was purchased from Bio-Rad, Hemel Hempstead, UK. High molecular weight rainbow markers and streptavidin-horseradish peroxidase were from Amersham International, Amersham, UK. HRP-WGA was purchased from Vector Laboratories, Peterborough, UK. PVDF membrane (Immobilon-P) was from Millipore, Watford, UK; nitrocellulose membrane from Bio-Rad, Hercules, CA; and Sepharose CL-2B from Pharmacia, Uppsala, Sweden. Sialic acid determination was contracted from Oxford GlycoSciences, Abingdon, UK. Benzamidine, BSA, cesium chloride, CHAPS, DAB, N-ethylmaleimide, guanidine hydrochloride, phenylmethylsulfonylfluoride, piperazine, SDS, soybean trypsin inhibitor, and the general chemicals of reagent grade were purchased from Sigma, Poole, UK. Antibodies LUM2-3 and LUM5-1 were given by Ingemar Carlstedt, Lund University, Lund, Sweden. Michael McGuckin, University of Queensland, Australia gave the antibodies BC2, M4.171, and M4.275. Biotinylated MLL was a gift from Nathan Sharon, Weizmann Institute of Science, Rehovot, Israel.

Collection of tissue

Approval by the local ethics committee was obtained for the use of human tissue in research. Human conjunctiva was obtained from 16 eye bank eyes (8 male and 8 female) following donation of the eyes for transplantation and research. The donors aged 17-78 years. Conjunctival tissue was stored immediately at 4°C in 4 M guanidine hydrochloride in PBS with protease inhibitors (5 mM EDTA disodium salt, 5 mM N-ethylmaleimide, 1 mM phenylmethylsulfonylfluoride, 0.01mg/ml soybean trypsin inhibitor, and 10 mM benzamidine). When tissue is left for several weeks in this solution the chaotropic guanidine hydrochloride solubilizes mucins and prevents their reaggregation.

Mucin detection reagents

Reagents used to identify mucins are described in Table I.

Table I. Mucin detection reagents
Reagent Specificity Comments
Antibody LUM5-1 MUC5AC peptide C-terminus Polyclonal antibody raised against the recombinant peptide RNQDQQGFPKMC (Carlstedt et al., 1995)
Antibody LUM2-3 MUC2 peptide C-terminus Polyclonal antibody raised against the recombinant peptide NGLQPVRVEDPDGC (Carlstedt et al., 1995; Hovenberg et al., 1996)
Antibody BC2 Recombinant MUC1 tandem repeat domain Xing et al., 1992
Antibodies M4.171 & M4.275 Recombinant MUC4 tandem repeat domain Monoclonal antibodies (Xing et al., 1994)
Wheatgerm agglutinin Sialic acids and N-acetylglucosamine An effective general reagent for glycosylated material including conjunctival mucins
Moluccella laevis lectin Tn and sialyl-Tn epitopes 20× greater affinity for GalNAc-O-Ser/Thr than forNeuAc-[alpha]2-6-GalNAc-O-Ser/Thr (Lis et al., 1994)
Periodic acid-Schiff Vicinal glycol groups  
Alcian blue, pH 1.0 Sulfated glycoproteins Thornton et al., 1994a
High iron diamine Sulfated glycoproteins Thornton et al., 1994a

Prevention of sample losses

Normal human ocular mucin is not available in large quantities. Care was taken to minimize sample losses, which may be selective, at all stages of mucin preparation and analysis. Glass labware was used where possible. Guanidine hydrochloride and urea kept mucins in solution and plastic labware was first rinsed with 0.5% PEG compound to block mucin binding plastic in the absence of a chaotropic solvent. At no stage were mucin samples lyophilized or concentrated by ethanol precipitation since these procedures produce insoluble aggregates. MonoQ ion exchange chromatography concentrated the charged mucins, optimizing their detection.

Mucin purification

Extracted mucins were isolated from proteins, glycoproteins, and nucleic acids by cesium chloride density gradient centrifugation. Insoluble tissue was removed from solubilized material by centrifugation at 12,000 r.p.m. at 16°C for 30 min. Cesium chloride was added to a final density of 1.4 g/ml then centrifuged at 150,000 × g in 13.5 ml sealed tubes in a Beckman 70.1 Ti rotor at 10°C for 24 h. The density of 0.5 ml fractions was established by weighing. Mucin-rich fractions were located by dot blotting on PVDF membrane (Immobilon-P) and probing with mucin detection reagents. Fractions were pooled in the density ranges 1.3-1.35, 1.35-1.4, 1.4-1.5 g/ml.

Preparation of mucin subunits

Mucin samples were adjusted to pH 8.0 with 1 M sodium hydroxide and reduced with 10 mM dithiothreitol at 37°C for 5 h, and then alkylated by addition of 25 mM iodoacetamide and incubation at room temperature for 14 h in the dark.

Size fractionation of reduced and alkylated mucin subunits by Sepharose CL-2B gel filtration

Columns (30 cm × 1.2 cm) were packed with Sepharose CL-2B and equilibrated with a buffer containing 10 mM piperazine, 6 M urea and 1% CHAPS, pH 8 (MQ buffer). Reduced and alkylated mucin samples in pooled density ranges were applied directly to the columns in 1 ml volumes and eluted with MQ buffer. Thirty-two 1 ml fractions were collected and pooled as follows: V0 (Mr > 4-5 × 106 Da, fractions 9-16), Vi (fractions 17-25) and Vt (Mr < 7 × 104 Da, fractions 26-32). The pooled Vt samples were dialyzed against MQ buffer to remove salts.

Ion exchange chromatography

Pooled V0,Vi, and Vt mucin subunits from the three buoyant density ranges were concentrated separately by adsorption to a 2 ml MonoQ resin column equilibrated with MQ buffer. The loaded column was then washed with 2.5 ml of MQ buffer. Mucin subunits were eluted from the column with a linear lithium perchlorate gradient (0-0.4 M in MQ buffer) before a final wash with 2.5 ml of 0.8 M lithium perchlorate in MQ buffer. The column eluate, including washings, was collected as 80 sequential 250 µl fractions. Mucin-rich fractions were detected in dot blots on PVDF membranes probed with mucin detection reagents.

Agarose gel electrophoresis

Electrophoresis was performed on 1% agarose gels in 40 mM Tris acetate 1 mM EDTA pH 8.0 containing 0.1% (w/v) sodium dodecyl sulfate (SDS), at room temperature for 18 h at 18V. No sample buffer was required and samples were not heated prior to loading. Western blotting onto polyvinylidene difluoride (PVDF) membranes was achieved under vacuum (125 mmHg for 60 min ) with 3 M sodium chloride, 300 mM sodium citrate, pH 7 (20 × SSC).

Polyacrylamide gel electrophoresis

Gels (8.5 cm × 7.5 cm × 1 mm) for a Bio-Rad Mini-Protean II were cast as 8.5% acrylamide/bis in 0.4 M TrisHCl pH 8.8 with a stacking gel of 4.0% acrylamide/bis in 0.1 M TrisHCl pH 6.8. Sample buffer containing 0.1 M TrisHCl pH 6.8, 10% glycerol, 2% SDS, 5% mercaptoethanol, and 0.05% bromophenol blue was added, and mixed without heating, to samples prior to electrophoresis. Electrophoresis was performed in 25 mM Tris pH 8.3 with 0.2 M glycine and 1% SDS, for 45 min at 200 V. Transfer to PVDF membranes was achieved with a Bio-Rad Trans-Blot SD semidry electrophoretic transfer cell with a buffer containing 44.5 mM Tris borate and 1mM EDTA (0.5 × TBE) at a constant 150 mA for 1 h.

Determination of sialic acid content of mucins

Sialic acids were released by acid hydrolysis with 2 M acetic acid at 80°C for 2 h. They were labeled by heating in the dark at 50°C for 2.5 h with 7 mM 1,2 diamino-4,5-methylenedioxybenzene (DMB) in a solution containing 18 mM sodium hydrosulfite, 1.4 M acetic acid, and 0.75 M [beta]-mercaptoethanol; 5 µl aliquots were separated by reverse phase C18 HPLC (GlycoSepR 4.6 × 150 mm) in acetonitrile:methanol:water (9:7:84). Detection was achieved with an on-line fluorimeter ([lambda]ex = 373 nm, [lambda]em = 448 nm) (Hara et al., 1989). The system was calibrated with known amounts of Neu5Ac and a reference panel containing six different sialic acids (Oxford GlycoSciences RP-2503).

Protein quantification

Protein was assayed by its absorbance at 260nm in a SpectraMAX Plus spectrophotometer (Molecular Devices, Sunnyvale, CA).

Rate zonal centrifugation

Mucins for rate zonal centrifugation were prepared from a collection of small pieces of conjunctiva. Mucins were extracted into 4 M guanidine hydrochloride in PBS with protease inhibitors. Half the collection was reduced and alkylated prior to cesium chloride density gradient centrifugation while the other half was not. The buoyant density range 1.3-1.5 g/ml was collected and dialyzed to reduce the guanidine hydrochloride concentration to 0.5 M. CsCl was added to the dialyzed sample producing a density of 1.38 g/ml before a second density gradient centrifugation as detailed above producing a gradient from 1.2-1.6 g/ml. Both native and reduced/alkylated gradients were collected in 0.5 ml fractions and dot blotted. Mucin-rich fractions were divided in the density ranges 1.2-1.3, 1.3-1.4, and 1.4-1.5 g/ml. Both removal of cesium chloride and volume reduction were achieved by dialysis against 2 M guanidine hydrochloride in PBS containing 10% PEG compound. Five hundred microliters of the samples so prepared were layered onto 12 ml gradients of 4-6 M guanidine hydrochloride and centrifuged at 40,000 r.p.m. for 150 min at 10°C in a Beckman SW40 Ti swing-out rotor (Sheehan and Carlstedt, 1987); 0.5 ml fractions were collected from the top of the gradient, and mucin-rich fractions identified in dot-blots probed with WGA and anti-mucin peptide antibodies.

Dialysis

Dialysis tubing was prepared by boiling in saturated sodium hydrogen carbonate for 30 min rinsing with distilled water and soaking for 30 min in 10 mM EDTA before a final rinse in distilled water containing 0.5% PEG compound. If sample concentration was required then samples were dialyzed against an appropriate solution containing PEG compound. Small volume dialyses were performed in Slide-a-lyzer cassettes [Pierce & Warriner (UK) Ltd., Chester, UK].

Dot blotting

Dot blots were made onto PVDF membrane using a 96 well format Bio-Rad vacuum dot blotter. After probing with mucin detection reagents, densitometry was performed using an HP Scanjet flatbed scanner and ScanAnalysis 2.50 (Biosoft, Cambridge, UK).

Lectin and antibody probing

After a 15 min incubation of PVDF membranes with 1% H2O2 in 30% methanol to inhibit endogenous peroxidase, nonspecific binding was blocked with 1% bovine serum albumin (BSA) in phosphate-buffered saline (PBS) pH 7 at room temperature for 4 h. Lectin binding was performed at room temperature overnight in blocking solution. Horseradish peroxidase-conjugated wheat germ agglutinin (HRP-WGA) was used at a concentration of 0.4 µg/ml. Biotinylated Moluccella laevis lectin was used at a concentration of approximately 0.1-1 µg/ml with a subsequent incubation with HRP-linked streptavidin at a dilution of 1:500 for 120 min at room temperature. Antibodies BC2, M4.171, and M4.275 were used at a dilution of 1:1000 in blocking solution. Second layer antibody was HRP-linked porcine anti-mouse immunoglobulin. Antibodies LUM5-1 and LUM2-3 were used at a dilution of 1:1000 rabbit serum in blocking solution. Second layer antibody was HRP-linked porcine anti-rabbit immunoglobulin. HRP-linked reagents were visualized with diaminobenzidine (DAB).

Sulfate detection

Dot blots of mucin samples on nitrocellulose membrane were stained with Alcian blue, pH 1.0, or High Iron Diamine (Thornton et al., 1994a).

ACKNOWLEDGMENTS

We thank Ingemar Carlstedt, Lund, Sweden, for LUM5-1 and LUM2-3; Michael McGuckin, University of Queensland, Australia for BC2 and M4.171 and M4.275; and Nathan Sharon, Weizmann Institute of Science, Rehovot, Israel for his gift of MLL. Rate zonal centrifugation was done with the kind help and facilities provided by David Thornton, Wellcome Center for Cell Matrix Research, Manchester, UK. Staff of the Eye Bank, Bristol Eye Hospital, Bristol, UK, provided a constant supply of small pieces of conjunctival tissue. Without their assistance this work would not have been possible. The project is supported by The Guide Dogs for the Blind Association and The Wellcome Trust UK (046381).

ABBREVIATIONS

BSA, bovine serum albumin; CHAPS, 3-[(3-cholamidopropyl)dimethylammonio]-1-propane-sulfonate; DAB, diaminobenzidine; DMB, 1,2 diamino-4,5-methylenedioxybenzene; EDTA, ethylenediaminetetraacetic acid; HRP, horseradish peroxidase; MLL, Moluccella laevis lectin; PAGE, polyacrylamide gel electrophoresis; PBS, 10 mM phosphate-buffered saline; PEG, polyethylene glycol; PVDF, polyvinylidene difluoride; SDS, sodium dodecyl sulfate; SSC, standard sodium citrate buffer; TBE, Tris-borate-EDTA buffer; WGA, wheatgerm agglutinin.

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