2Wellcome Trust Centre for Cell-Matrix Research, Division of Biochemistry, School of Biological Sciences, 2.205 Stopford Building, University of Manchester, Manchester, M13 9PT, UK; 3Mucosal Biology Group, Department of Cell and Molecular Biology, Lund University, BMC, C-13, SE-22184, Lund, Sweden; 4St. Georges Hospital Medical School, Department of Physiology, Cranmer Terrace, London, SW17 ORE, UK.
Received on April 30, 2001; revised on June 28, 2001; accepted on July 3, 2001.
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Abstract |
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Key words: Gp-340/mucus/MUC5AC/MUC5B
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Introduction |
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The mucus gel provides a physical barrier to chemical and invasive biological agents and in addition contains a host of protective agents, which include lysozyme, lactoferrin, transferrin, proteinases, proteinase inhibitors and secretory IgA (for a review see Rayner and Wilson, 1997). These proteins are vital to the sterility of the airways and act to destroy bacteria or prevent their colonization of the mucosal surface and to protect against host and bacterial proteinases. These components may associate with the gel-forming mucins and thus become part of the gel network and provide an immobilized reservoir of protective factors. In support of this, previous studies have reported that proteins are released from purified respiratory mucins after treatment with reducing agents (Tabachnik et al., 1981
; Ringler et al., 1988
; Naziruddin et al., 1990
; Gupta and Jentoft, 1992
; Thornton et al., 1994
), but their identity was not determined. Furthermore, recent investigations on saliva have identified a number of proteins involved in heterotypic associations with the gel-forming mucins, which in this case are the product of the MUC5B gene (Iontcheva et al., 1997
; Wickstrom et al., 2000
).
In a previous study of asthmatic mucins we noted that after reduction of a highly purified respiratory mucin preparation, a protein-rich fraction was generated (Thornton et al., 1994). In the present study we present data to suggest that a high-molecular weight glycoprotein (gp-340), previously identified in bronchioalveolar lavage fluid (Holmskov et al., 1997
, 1999), is present in this fraction. It is likely that due to its chemical composition that this glycoprotein was present in the mucin fraction due to copurification. However, our data suggest that in respiratory mucus this glycoprotein is involved in high-molecular-weight complexes that may involve the gel-forming mucins and in particular a subpopulation of the MUC5B mucins.
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Results |
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Discussion |
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Previously we have shown that a protein-rich fraction was generated after reduction of a respiratory mucin preparation that was isolated by extraction with 6 M guanidinium chloride followed by three stages of isopycnic density-gradient centrifugation (Thornton et al., 1994). The results of the present investigation clearly show that this fraction contained a component of nonmucin origin. Its broad buoyant density range in 4 M guanidinium chloride/CsCl overlaps with that of the MUC5AC and MUC5B mucins, explaining why a proportion of it was found in the original mucin preparation. Therefore to remove this glycoprotein from mucin preparations it will be necessary to add a "dissociative" gel chromatography step on a porous media (e.g., Sepharose CL-2B) to the current CsCl density-gradient protocol. Further study of the complex pool of peptides generated by trypsin treatment of the protein-rich fraction is ongoing to ascertain the molecular identity of the parent proteins. It cannot be ruled out that this fraction may also contain or even be dominated by peptides derived from the mucins.
The isolated peptide was identical to a 14-amino-acid sequence that is repeated 12 times within the deduced primary sequence of gp-340, which is an alternatively spliced form of the DMBT1 gene (Mollenhauer et al., 1997; Holmskov et al., 1999
). Gp-340 was previously isolated from bronchioalveolar lavage fluid, where it was shown to bind surfactant protein-D, which is a member of the collectin family (Holmskov et al., 1997
). More recently it has been shown that the polypeptide of gp-340 is likely to be identical to that of the salivary agglutinin, this latter glycoprotein has been demonstrated to bind to oral bacteria (Prakobphol et al., 2000
). Immunostaining for gp-340 has been shown in the lung on alveolar macrophages and epithelial cells of the small intestine and ducts of the salivary glands (Holmskov et al., 1999
). In the present study, using a polyclonal antiserum raised to the isolated 14 amino acid peptide, we have localised immunoreactivity to the serous cells of the submucosal glands.
The gp-340 preparation is polydisperse (Mr 2.5 x 1051 x 106) with an average Mr (in NaCl) of 5 x 105, and there is no evidence for heterogeneity across the molecular size distribution. This is in broad agreement with previous estimates from gel chromatography under nondissociating conditions, which report a value > 1000 kDa (Holmskov et al., 1997). The average mass determined by multi-angle laser light scattering (MALLS) is higher than the mass of 258 kDa that is predicted from the deduced sequence (Holmskov et al., 1999
), and the difference may be largely explained by glycosylation. The broad buoyant density range observed for the glycoprotein is consistent with variable glycosylation. However, it is also possible that the molecule has a tendency to self-associate and the chromatographic profile on Superose 6 may reflect an equilibrium of interacting species under the conditions used. There was no detectable measurement of the radius of gyration (RG) across the entire chromatographic distribution and this indicates that putative complexes would have to have RG < 15nm, because this is the size of the smallest particles that can be detected with this technique. These findings strongly suggest that there is no tendency toward an open-ended self-association for this molecule.
A single species of gp-340 was observed after extraction of mucus with guanidinium chloride and subsequent Sepharose CL-2B gel chromatography in this "dissociative" solvent. This contrasts with the same mucus sample extracted with PBS prior to gel chromatography under "associative" conditions. In this case there is evidence for more than one population of the gp-340, one in the void volume and others included on the column. These data provide evidence that gp-340 may exist as part of a complex in mucus. The MUC5AC and MUC5B mucins also chromatograph in the void volume of Sepharose CL-2B, thus the distribution of the gp-340 may have resulted from its direct or indirect association with one or both of these gel-forming mucins. Sucrose gradient centrifugation of selected fractions from the Sepharose CL-2B gel chromatography of the "associative" mucus extracts confirms the presence of an association, which is consistent with the involvement of mucins. In particular the sedimentation data suggest that a subpopulation of the MUC5B mucins may be involved, whereas the MUC5AC mucins are not. Further studies are required to distinguish between self-association and complex formation with the MUC5B mucins. However, in support of our data it has been demonstrated that the salivary agglutinin (gp-340) (Prakobphol et al., 2000) is enriched in the gel phase of salivary mucus (Holmskov et al., 1999
) where the predominant gel-forming mucin in this secretion is MUC5B (Thornton et al., 1999
). It is interesting to speculate on the consequences of an interaction of gp-340 with the mucin network. Gp-340 clearly has an important antibacterial role in mucus either via its direct binding to bacteria (Prakobphol et al., 2000
) or its association with the bacterial-binding collectin surfactant protein-D (Holmskov et al., 1997
). Thus binding of gp-340 to the gel network increases the functionality of the mucus by facilitating clearance of sequestered bacteria from the respiratory tract via the mucocilliary escalator or cough.
The peptide sequence reported here is repeated exactly 12 times in the deduced gp-340 sequence and is part of a larger motif known as the SRCR domain. The function of this motif is still not defined however; the known mammalian SRCR domaincontaining proteins are found on immune cell surfaces or are secreted and suspected of being involved in host defense (Resnick et al., 1994). Other SRCR-containing proteins contain repeated sequences that are homologous to the peptide sequenced here; the three most similar, murine CRP-ductin (Cheng et al., 1996
), rat ebnerin (Li and Snyder, 1995
), and bovine gallbladder mucin (Nunes et al., 1995
), are all expressed at epithelial surfaces. It seems likely from the data presented in this study and previously by Holmskov and colleagues (1999) that the putative gallbladder mucin is not a classical mucin but a bovine form of gp-340 or a closely related molecule.
In addition to 14 SRCR domains the deduced gp-340 polypeptide contains 2 CUB (complement subcomponents C1r/C1s, Uegf protein, and bone morphogenic protein) domains and a zona pellucida (ZP) domain (Bork and Sander, 1992; Bork and Beckmann, 1993
). All of these motifs are found in molecules that are secreted or located at the cell surface (Mollenhauer et al., 1997
) and are potential sites for binding proteins, carbohydrates and lipids (Moestrup et al., 1998
). Recent crystal structure data on seminal fluid spermadhesins indicates that the CUB motif may provide sites for strong self-interaction (Varela et al., 1997
; Romao et al., 1997
). It is possible that any or all of these different domains (SRCR, CUB, and ZP) are sites by which the glycoprotein may interact with itself or other molecules and may provide the basis for the complexes identified by gel chromatography and rate-zonal centrifugation. By the nature of its known and potential binding abilities this glycoprotein may have numerous protective functions in the mucus. For example, the repeated SRCR motifs may provide multiple sites for interaction with toxins or pathogens. Therefore it will be important in the future to specify its functions and assay its relative level in health and disease.
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Materials and methods |
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Preparation of 14C-radiolabeled reduced mucin preparation
Mucins were extracted with 4 M guanidinium chloride containing proteinase inhibitors from the mucus gel plug obtained post mortem from the lungs of an individual who died in status asthmaticus and purified by CsCl density-gradient centrifugation as described previously (Thornton et al., 1994). The mucin preparation was treated with 10 mM dithiothreitol to reduce disulfide bonds, and these were subsequently blocked with 14C-radiolabeled iodoacetamide as described previously (Thornton et al., 1994
).
Rate-zonal centrifugation
The 14C-radiolabeled reduced mucin preparation was fractionated by centrifugation on preformed 68 M guanidinium chloride gradients using a SW40 Ti swing bucket rotor for 7.5 h at 40,000 r.p.m. as described previously (Thornton et al., 1994). Samples from gel chromatography of associative extracts of mucus were analyzed by centrifugation on preformed 1030% (w/v) sucrose gradients using a SW40 Ti swing bucket rotor for 1.5 or 2.5 h at 40,000 r.p.m..
Isolation of tryptic peptides
The protein-rich fraction from the rate-zonal gradient was dialyzed against water and lyophilized. The material was dissolved in 0.1 M ammonium hydrogen carbonate and treated with modified trypsin for 16 h at 37°C as described previously (Thornton et al., 1997b). Tryptic peptides were chromatographed at a flow rate of 240 µl/min on a µRPC C2/C18 PC 3.2/3 column using the Pharmacia SMART system. The column elution program was 0.1% (v/v) trifluoracetic acid (TFA) (5 min) followed by a linear gradient of 040% (v/v) acetonitrile in 0.1% (v/v) TFA (30 min). The column eluent was monitored for absorbance at 215 nm and fractions were analyzed by MALDI-TOF MS. Peptides were purified to homogeneity by rechromatography on the column employing shallower gradients centered on their elution point.
MALDI-TOF MS
Samples (1 µl) in 0.1% TFA containing various proportions of acetonitrile were mixed with an equal volume of 50 mM -cyano-4-hydroxycinnamic acid and applied to a TOFspec target. Samples were analyzed by MALDI-TOF MS in positive ion mode using a VG TOFSpec-E with Substance-P (M+ 1348.7) and bovine insulin (M+ 5734.5) as internal standards. The data generated were processed using the OPUSTM peak detection program.
Gel electrophoresis
SDSPAGE was performed with a discontinuous gel system in a 5% (w/v) resolving gel with a 3% (w/v) stacking gel. Alternatively samples were electrophoresed in 1.0% (w/v) agarose gels in 40 mM Tris-acetate/1mM EDTA, pH 8.0, containing 0.1% (w/v) SDS. Gels were analyzed by transfer of molecules to nitrocellulose by vacuum blotting prior to detection using antibodies (Thornton et al., 1995, 1996, 1997b).
Isolation of the gp-340
Respiratory secretions solubilized in 6 M guanidinium chloride were brought to 4 M guanidinium chloride and solid CsCl was added to a density of 1.4 g/ml. The sample was subjected to centrifugation in a Beckman Ti 70 rotor at 40,000 r.p.m. for 64 h at 15°C. The gp-340-containing fractions were pooled (see Figure 5a), dialyzed into 4 M guanidinium chloride, and chromatographed on Sepharose CL-2B. The column (88 cm x 2.6 cm) was eluted with 4 M guanidinium chloride at a flow rate of 22 ml/h. The gp-340-containing fractions were pooled (see Figure 5b), dialyzed into 6 M urea containing 0.02% CHAPS and subjected to anion exchange chromatography on a Mono Q HR 5/5 column. The column was eluted at a flow rate of 0.5ml/min with a linear gradient of 00.4 M lithium perchlorate/10 mM piperazine, pH 5.0, in 6 M urea containing 0.02% CHAPS (Thornton et al., 1996). The gp-340-containing fractions were pooled (see Figure 5c), dialyzed into 6 M urea containing 0.02% CHAPS, and rechromatographed on the Mono Q column under identical conditions. The gp-340-containing fractions were pooled, dialyzed against 6 M urea, and stored at 4°C.
Monosaccharide analysis
Samples were hydrolyzed in 2 M TFA at 100°C for 5 h, then rotary evaporated to dryness, redissolved in water, and the monosaccharides separated on a Dionex CarboPac PA1 column eluted at 1 ml/min with 21 mM sodium hydroxide. Monosaccharides were detected using a Dionex PAD II detector.
Molecular weight determination by MALLS
The gp-340 preparation was chromatographed on a Pharmacia Superose 6 HR 10/30 column eluted with 0.2 M NaCl at a flow rate of 200 µl/min. The column effluent was passed through an in-line Dawn DSP laser photometer coupled to a Wyatt/Optilab 903 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).
Analytical gel chromatography
Respiratory secretions were chromatographed on a Sepharose CL-2B (29 cm x 1.6 cm) column eluted with either 4 M guanidinium chloride or PBS at a flow rate of 3 ml/h.
Polyclonal antisera
The polyclonal antisera MAN-5ACI and MAN-5BI, raised against sequences within the MUC5AC and MUC5B mucins, respectively, were employed and these have been described previously (Thornton et al., 1997b; Sheehan et al., 1999
). A polyclonal antiserum (MAN-gp-340) was raised to the following peptide FGQGSGPIVLDDVR conjugated to keyhole limpet hemocyanin. The antiserum was used at a dilution of 1:2000 for immunoblots and western blots and was more effective against reduced samples. Inhibition of antiserum reactivity in both immunoblots and western blots could be achieved by preincubation with the peptide antigen (2 µg/ml for 1 h at room temperature).
Immunolocalization
Human trachea was fixed in 10% neutral buffered formal saline and embedded in paraffin prior to the preparation of 4-µm sections. The slides were dewaxed, rehydrated, and treated for 10 min with 10 mM sodium citrate buffer, pH 6, at 100°C in a microwave oven. Endogenous peroxidase activity was quenched with 3% H2O2 for 30 min, and the sections were then blocked with normal goat serum (diluted in 1:5 0.15 M NaCl/0.05 M TrisHCl buffer, pH 7.4) for 1 h. Endogenous biotin was blocked by treatment with the Dako biotin blocking kit used according to the manufacturers instructions. Sections were then incubated with the MAN-gp-340 antiserum (1:2000 in 0.15 M NaCl/0.05 M TrisHCl buffer, pH 7.4 containing 0.05% bovine serum albumin) for 1 h in Coverplate immunostaining chambers (Shandon, Pittsburgh, PA). After the detection of antibody binding using the Dako Strept ABComplex/HRP kit with diaminobenzidine as the substrate, sections were counterstained with Mayers hematoxylin.
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Acknowledgments |
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Abbreviations |
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Footnotes |
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References |
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