Identification of Two Glycoforms of the MUC5B Mucin in Human Respiratory Mucus
EVIDENCE FOR A CYSTEINE-RICH SEQUENCE REPEATED WITHIN THE MOLECULE*

(Received for publication, November 18, 1996, and in revised form, January 24, 1997)

David J. Thornton Dagger , Marj Howard , Nagma Khan § and John K. Sheehan

From The Wellcome Trust Centre for Cell-Matrix Research, University of Manchester, 2.205, Stopford Building, Manchester M139PT, United Kingdom

ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
FOOTNOTES
ACKNOWLEDGEMENTS
REFERENCES


ABSTRACT

It has been demonstrated previously that respiratory secretions contain three oligomeric, gel-forming mucins; one of these was identified as the product of the MUC5AC gene (1). Here we demonstrate that the other two mucins are glycoforms of the MUC5B gene product. This was accomplished by trypsin treatment of the purified reduced mucin subunit populations and N-terminal sequencing of the liberated peptides. The products of trypsin digestion were separated by gel filtration into high molecular weight mucin glycopeptides and low molecular weight tryptic peptides. The latter were fractionated by reverse phase chromatography, and four of the major peptides were sequenced. Three of these peptides were identical to and contiguous within a 51-amino acid sequence deduced from a cDNA clone (JER57) encoding a portion of the MUC5B mucin. The other peptide is also present within this sequence but showed identity in only 9 of its 10 residues. A polyclonal antiserum raised against one of these peptides was reactive with the two putative MUC5B glycoforms. Analysis of the high molecular weight glycopeptides indicated that the MUC5B subunit contained different types and lengths of glycosylated domains; one domain of Mr 7.3 × 105, two domains of Mr 5.2 × 105, and a third domain of Mr 2.0 × 105. The amino acid composition of the larger two glycopeptides was similar in serine, threonine, and proline content but distinct from that of the smallest glycopeptide. Each of these domains in the mucin subunit is separated by a trypsin-sensitive region, and the relative abundance of the major peptides derived by proteolysis of these regions and their occurrence in a contiguous sequence suggest that they contain a common cysteine-rich motif.


INTRODUCTION

Respiratory tract mucus is the principal barrier in the lung against chemical and pathological insult. The physical properties of this gel-like secretion are due solely to high molecular weight O-linked glycoproteins termed mucins. Respiratory mucins are polydisperse in mass (Mr 2-40 × 106) and length (0.5-10 µm) and can be fragmented into their constituent subunits (Mr 2-3 × 106) by reduction (1-6). Proteinase treatment of reduced subunits yields high molecular weight glycopeptides (Mr 300,000-500,000), and these fragments contain the majority of the O-linked glycans. The core protein of the reduced mucin subunit is thus composed of alternating oligosaccharide-rich proteinase-resistant domains and proteinase-sensitive "naked" domains.

Northern blot and in situ hybridization analyses have shown that at least eight mucin genes (MUC1, MUC2, MUC3, MUC4, MUC5AC, MUC5B, MUC7, and MUC 8) are expressed in the respiratory tract (7-14). However, biochemical analysis of respiratory secretions demonstrates that only three major mucin populations comprise the bulk of the gel-forming species (1). These mucins, which differ in charge density and electrophoretic mobility, are all polymeric species that can be fragmented into subunits by reduction (1, 15), and using mucin-specific antisera, one of these has been identified as the MUC5AC mucin (1, 16). Immunohistochemistry demonstrated that this mucin was a product of the goblet cells (17), and this is in agreement with in situ hybridization data (11). Another of the major species, the least charged and least electrophoretically mobile, was shown to be a product of the submucosal glands (16). In situ hybridization has demonstrated that this is the site of synthesis of the MUC5B mucin in the respiratory tract (11), and thus it is likely that this mucin may be the product of the MUC5B gene.

In this study we have generated tryptic peptides from the proteinase-sensitive naked regions of the core proteins of the two previously unidentified mucin populations (1) (termed here mucins X and Y) in an attempt to determine if they are the products of novel or identified MUC genes. In addition we have also investigated the structural organization of their subunits.


EXPERIMENTAL PROCEDURES

Materials

Trypsin, modified by reductive alkylation to reduce autolysis, was purchased from Promega (Southampton, United Kingdom). alpha -Cyano-4-hydroxycinnamic acid, substance P, and insulin (bovine pancreas) were from Sigma. Sequencing-grade trifluoroacetic acid was from Applied Biosystems (Warrington, United Kingdom), and acetonitrile (high performance liquid chromatography grade) was purchased from Rathburn Chemicals (Walkerburn, United Kingdom).

Preparation of Reduced Mucin Subunits

Reduced subunits were prepared from mucins extracted from the mucus gel plug obtained postmortem from the lungs of an individual who died in status asthmaticus as described previously (1, 18). The reduced mucin subunit populations were purified by anion exchange chromatography on Mono Q as described previously (1) and then dialyzed against water and lyophilized.

Agarose Gel Electrophoresis

Reduced mucin subunits were electrophoresed in 1.0% (w/v) agarose gels in 40 mM Tris acetate, 1 mM EDTA, pH 8.0, containing 0.1% (w/v) SDS and then transferred to nitrocellulose by vacuum-blotting before detection using antibodies (1, 19).

Preparation of High Molecular Weight Glycopeptides and Tryptic Peptides

Reduced mucin subunits (3.5 mg) were dissolved in 450 µl of 0.1 M ammonium hydrogen carbonate, pH 8.0, and 1 µg of trypsin (50 µl) was added; after 24 h at 37 °C the digest was chromatographed on a Superose 12 HR 10/30 column in 0.1 M ammonium hydrogen carbonate, pH 8.0, at a flow rate of 0.4 ml/min. Fractions from the column were taken into five pools (TR-I to TR-V; see Fig. 2) that were further analyzed.


Fig. 2. Superose 12 chromatography of trypsin digestion products of mucin population X reduced subunits. The reduced subunit preparation from mucin population X was treated with trypsin and subsequently chromatographed on a Superose 12 HR 10/30 column. The column eluent was monitored for absorbance at 280 nm, and the fractions from the column were pooled as follows: 20-29 (TR-I), 30-35 (TR-II), 36-41 (TR-III), 42-52 (TR-IV), and 53-59 (TR-V). These poolings are indicated by the bars. The void and total volumes of the column are in fraction numbers 24 and 55, respectively.
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Reverse Phase Chromatography of Tryptic Peptides

Tryptic peptides were chromatographed at a flow rate of 240 µl/min on a µRPC C2/C18 PC 3.2/3 column eluted with 0.1% (v/v) trifluoroacetic acid (5 min) followed by a linear gradient of 0-30% (v/v) acetonitrile in 0.1% (v/v) trifluoroacetic acid (30 min) using the Pharmacia SMART system. Major peaks were analyzed by matrix-assisted laser desorption time of flight mass spectrometry (MALDI-TOF MS),1 and peptides were purified to homogeneity by re-chromatography on the column using shallower gradients centered around their elution point.

MALDI-TOF MS

Samples (1 µl) in 0.1% trifluoroacetic acid containing various proportions of acetonitrile were mixed with an equal volume of 50 mM alpha -cyano-4-hydroxycinnamic acid, applied to a TOFSpec target, and analyzed by MALDI-TOF MS in positive ion mode using a VG TOFSpec-E with substance P (mass, 1348.7 Da) and bovine insulin (mass, 5734.5 Da) as internal standards. The data generated were processed using the OPUSTM peak detection program.

N-terminal Sequencing

N-terminal amino acid sequencing was performed on isolated peptides using an Applied Biosystems 476A protein microsequencer.

Polyclonal Antisera

As a general nonspecific mucin probe, a polyclonal antiserum raised against cervical mucin reduced subunits that recognizes protein epitopes on a range of reduced mucins was used (20). A polyclonal antiserum (MAN-5BI) was raised against a synthetic peptide, corresponding to the sequence of peptide TR-IV-C (ELGQVVECSLDFGLVCR), conjugated with keyhole limpet hemocyanin. In an enzyme-linked immunosorbent assay with the free peptide on the solid phase at 1 µg/well, the antiserum (incubated for 1 h at room temperature) had a titer of 1:1500. The titer is defined as the antiserum dilution giving an A405 of 0.5 (the midpoint of the sigmoidal curve) using a horseradish peroxidase-labeled secondary antibody (1 h at room temperature) with O-phenylenediamine (10 min) as the substrate. The antisera were used at a dilution of 1:1000 for Western blots.

Fractionation and Molecular Weight Determination of Mucin Glycopeptides (TR-I)

High molecular weight glycopeptides (pool TR-I) were chromatographed on a Superose 6 HR 10/30 column eluted with 4 M guanidinium chloride 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 (1), and the data were analyzed according to Zimm (21). Fractions across the glycopeptide distribution were taken into three pools (GP-I to GP-III; Fig. 9) that were re-chromatographed on the column and then desalted on a Hi-Trap column and lyophilized before determination of their amino acid compositions.


Fig. 9. Superose 6 chromatography of high molecular weight mucin glycopeptides (TR-I). a, the high molecular weight mucin glycopeptide fraction TR-I (900 µg) was chromatographed on a Superose 6 HR 10/30 column. The column effluent was monitored in-line for light scattering to determine molecular weight (dotted line, logarithmic scale), and the refractive index was measured to determine sample concentration (solid line). Three populations of distinct size are apparent, and fractions across the distribution were pooled as indicated by the bars to yield three fractions (GP-I, GP-II, and GP-III) with average molecular weights of 7.3 × 105, 5.2 × 105, and 2.0 × 105, respectively. The void volume of the column is 7.8 ml. b, the relationship between size and molecular weight is shown across the distributions of the larger two glycopeptide populations (GP-I and GP-II).
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Amino Acid Analysis

Samples were hydrolyzed under nitrogen in 3 M HCl at 105 °C for 16 h, and the resulting amino acids were derivatized with phenylisothiocyanate and then separated by reverse phase chromatography using a 3-µm ODS2 column (1).


RESULTS

We and others have demonstrated that respiratory secretions from normal and a variety of hypersecretory conditions contain two or three major mucin species (1, 15-17). The reduced mucin subunits corresponding to each of these populations were purified from mucus gel obtained from an asthmatic individual by anion exchange chromatography as described previously (1). The mucin subunit populations had different electrophoretic mobilities on agarose gel electrophoresis (Fig. 1), and one of these was previously identified as the MUC5AC mucin (1). The object of this investigation was to identify the other two mucin populations (termed here mucin X and mucin Y).


Fig. 1. 1% (w/v) agarose gel electrophoresis of reduced mucin subunits. Reduced mucin subunits (2 µg/lane) were electrophoresed on a 1% (w/v) agarose gel and blotted onto nitrocellulose, and the blot was probed with an antiserum raised against reduced mucin subunits. Lane A, mucin X; Lane B, MUC5AC; and Lane C, mucin Y reduced subunits.
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Mucin Population X

To determine the identity of mucin population X its reduced subunits were fragmented with trypsin, and the resulting tryptic peptides were fractionated, and major peptides were sequenced by automated Edman degradation. Trypsin treatment yielded five main peaks (TR-I-V) after gel filtration chromatography on Superose 12 (Fig. 2). Fraction TR-I contained high molecular weight mucin glycopeptides (Mr 300,000-700,000), and fractions TR-II and TR-III contained lower molecular weight glycosylated peptides (Mr 10,000-50,000). MALDI-TOF MS revealed that the majority of the low molecular weight tryptic peptides (Mr 1,000-10,000) were present in fractions TR-IV and TR-V (Fig. 3, a and b). The spectra show that although there are a large number of peptides generated by proteolysis, there are a few major peptides present, i.e. those peptides with masses 1038, 1129, 1147, 1685, and 1975 Da (Fig. 3). TR-IV was the major peptide-containing fraction and, as expected from the elution position on Superose 12, had higher molecular weight components than TR-V.


Fig. 3. MALDI-TOF MS of tryptic peptide fractions TR-IV (a) and TR-V (b).
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The tryptic peptides in fractions TR-IV and TR-V were separated by reverse phase chromatography, and both samples showed a complex series of peaks, however, it was evident that there was similarity in the major peaks in the chromatograms (Fig. 4, a and b). Four of these peptides (TR-IV-A-C and TR-V-D; see Fig. 4) were purified by re-chromatography on the reverse phase column, and their homogeneity was ascertained by MALDI-TOF MS (data not shown). The mass of each of the four peptides A-D was 1038, 1685, 1975, and 1129 Da, respectively. It can be seen that the major peaks in the reverse phase separation correspond to some of the major peaks observed in the mass spectra of the unfractionated peptides (Fig. 3), indicating that these four peptides are major products of trypsin digestion of this mucin population. Peptides TR-IV-A, TR-IV-B, and TR-IV-C are also present in the TR-V chromatogram, showing that the initial size fractionation (Fig. 2) was not totally effective.


Fig. 4. Reverse phase chromatography of tryptic peptide fractions TR-IV (a) and TR-V (b). Fractions TR-IV and TR-V were chromatographed on a µRPC C2/C18 PC 3.2/3 column, the eluent was monitored for absorbance at 215 nm (solid line), and the nominal gradient is indicated (broken line). The elution position of four major peptides, labeled A-D, is highlighted.
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The primary sequence of each of the four peptides was determined by automated Edman degradation, and the data are presented in Table I. A search of the protein sequence data bases revealed a 100% sequence identity for peptides TR-IV-B, TR-IV-C, and TR-V-D and a 1-amino acid difference (arginine for glycine) in 10 residues for peptide TR-IV-A within a 51-amino acid sequence deduced from the JER57 cDNA clone (Fig. 5a), which codes for a part of the MUC5B mucin (22). This region of the MUC5B mucin and our peptides also seem homologous but not identical to two other human mucins, MUC2 and MUC5AC (Fig. 5b). In summary these data indicate that the core protein of mucin population X is encoded by the MUC5B gene.

Table I.

Amino acid sequences of tryptic peptides


Peptide Sequence

TR-IV-A AQAQPGVPLR
TR-IV-B AAGGAVCEQPLGLE
TR-IV-C ELGQVVECSLDFGLVCR
TR-V-D MCFNYEIR


Fig. 5. Alignment of tryptic peptides with a portion of the deduced amino acid sequence of the MUC5B cDNA clone JER57 (a) and homology with other human mucins (b). a, the four tryptic peptides TR-IV A-C and TR-V-D are found within a contiguous region of a portion of the deduced amino acid sequence of the MUC5B cDNA clone JER57 (22). In addition the first 13 residues of the TR-IV-B peptide were identical in sequence to that of a tryptic peptide (TR-3D) derived from a human tracheobronchial mucin preparation (23). The arrows indicate trypsin cleavage sites in the deduced MUC5B protein sequence. b, alignment of the 51-amino acid sequence of MUC5B with the human mucins MUC5AC (NP3a) (24) and MUC2 (25). The asterisks indicate identity with the MUC5B sequence, and the dashes represent single-residue gaps.
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Mucin Y

Trypsin treatment of this mucin subunit population yielded a similar gel filtration profile (data not shown) to that observed for mucin population X (Fig. 2). The low molecular weight tryptic peptides were pooled into a single fraction corresponding to TR-IV and TR-V and analyzed by MALDI-TOF MS (Fig. 6). The spectrum is similar to those presented for mucin X peptides (Fig. 3), and four of the major peptides (A-D) with masses of 1038, 1685, 1975, and 1129 Da, respectively, are also present (Fig. 6). In contrast, other major signals (with masses of 1457 and 1549 Da) are observed that were absent from the spectra for mucin X. However, we have previously shown that this sample contains a minor amount of the MUC5AC mucin (1), and these peptides may arise from this mucin protein. Reverse phase chromatography (Fig. 7) revealed a complex pattern of peaks; three of the major peaks occur in the same fractions as observed for the mucin X peptide chromatograms (Fig. 4). In addition, MALDI-TOF MS on these fractions reveals peptides of identical mass to the mucin X peptides (data not shown). In summary these data indicate that mucin Y is a different glycoform of the MUC5B gene product.


Fig. 6. MALDI-TOF MS of the low molecular weight tryptic peptides derived from mucin population Y. Peaks corresponding to peptides A-D from mucin population X (MUC5B) are highlighted. The signal at M+ 1129 corresponding to peptide TR-V-D is low in this sample.
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Fig. 7. Reverse phase chromatography of tryptic peptides derived from mucin population Y reduced subunits. Tryptic peptides from mucin population Y corresponding to fractions TR-IV and TR-V were chromatographed on a µRPC C2/C18 PC 3.2/3 column, the eluent was monitored for absorbance at 215 nm (solid line), and the nominal gradient is indicated (broken line). Three of the major peaks (labeled A, B, and C) occur in the same fractions as the mucin X peptides TR-IV-A-C (Fig. 4a), and MALDI-TOF MS revealed that these contained peptides of identical mass to peptides TR-IV-A-C (data not shown).
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To confirm this a polyclonal antiserum (MAN-5BI) raised against a synthetic peptide corresponding to TR-IV-C was used to probe a Western blot of an agarose gel separation of the three mucin subunit populations (Fig. 8). It is apparent that subunit bands corresponding to mucin X and mucin Y (Fig. 1) are both reactive with this antiserum, whereas the MUC5AC subunit is not.


Fig. 8. 1% (w/v) agarose gel electrophoresis of reduced mucin subunits. Reduced mucin subunits (3 µg/lane) were electrophoresed on a 1% (w/v) agarose gel and blotted onto nitrocellulose, and the blot was probed with an antiserum (MAN-5BI) raised against a synthetic peptide corresponding to peptide TR-IV-C. Lane A, mucin X; Lane B, MUC5AC; and Lane C, mucin Y reduced subunits. The arrowhead represents the migration position of the MUC5AC subunit band.
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Structural Organization of the MUC5B Reduced Subunit

The high molecular weight mucin glycopeptides (TR-I, see Fig. 2) were separated into three components (GP-I-III) by gel filtration chromatography on Superose 6 (Fig. 9a), and their amino acid compositions are presented in Table II. Each glycopeptide has a high content of serine, threonine, and proline, and the data demonstrate that samples GP-I and GP-II are similar but different from GP-III, which has a much higher content of serine relative to threonine. The molecular weight distribution for the glycopeptides was determined by light scattering (Fig. 9a), and the average molecular weight values calculated for GP-I, GP-II, and GP-III were 7.3 × 105, 5.2 × 105, and 2.0 × 105, respectively. From the light scattering data we were also able to deduce radii of gyration across the glycopeptide distribution (data not shown), and the relationship between the radius of gyration and molecular weight is presented (Fig. 9b). The slope of this plot is a shape-sensitive parameter, and the value of 0.78 is consistent with an almost rod-like structure for the glycopeptides. From integration of the refractive index increments across the three peaks, the amount of each glycopeptide was deduced, and in conjunction with their measured average molecular weight, a molar ratio of 1:2:1 was determined for the three components GP-I, GP-II, and GP-III, respectively.

Table II.

Amino acid composition of high molecular weight mucin glycopeptides (GP-I-III)a


Amino acid High molecular weight mucin glycopeptide fraction
GP-I GP-II GP-III

Asx 10 25 29
Glx 34 48 87
Ser 172 158 135
Gly 56 66 186
Thr 290 253 141
Ala 129 117 103
Pro 180 171 121
His ND ND ND
Arg 19 21 14
Tyr 4 11 15
Val 35 40 52
Met ND ND ND
Ile 16 21 9
Leu 39 48 71
Phe 14 12 34
Lys 2 9 3

a Values are expressed per 1000 residues. ND, not determined.


DISCUSSION

Previously we have shown that three oligomeric mucin populations comprise the bulk of the gel-forming species in human respiratory secretions (1). After reduction these mucins can be separated by anion exchange chromatography and agarose gel electrophoresis (1, 15). One of these mucins was identified as the product of the MUC5AC gene, but the genetic identity of the other two was not ascertained (1). The mucin preparation studied here was shown to contain all three populations, and the aim of this study was to assign genetic identities to the two unknowns. Amino acid sequence data were obtained from N-terminal sequencing of four tryptic peptides, and these identical sequences were found within a 51-amino acid sequence deduced from the cDNA clone (JER57) that encodes a small segment of the MUC5B mucin. In contrast, only partial similarity in sequence is observed between these peptides and regions of the human mucins MUC2 (24) and MUC5AC (25). On the basis of these findings and the similarity in the pattern of their tryptic peptides and their reactivity with an antipeptide antiserum we conclude that the two mucin populations X and Y are products of the MUC5B gene, but they represent different glycoforms. In support of this conclusion we have shown previously that these two mucin populations have different carbohydrate compositions and charge densities (1). The latter finding explained the difference in their electrophoretic mobility. The existence of two glycoforms of the MUC5B gene product raises questions as to whether they are both present in the normal situation, whether their level is changed between normal and diseased conditions, and if they are the product of the same or different cells.

A mucin population corresponding to the least-charged variant of the MUC5B mucin was shown to be enriched in mucin preparations derived from human respiratory tract submucosal tissue (16), which, according to in situ hybridization data, is the site of synthesis of MUC5B mucin in the airways (11). This mucin was virtually absent from epithelial surface (i.e. goblet cell) mucin preparations that are enriched in the MUC5AC mucin (16), a finding that is also consistent with in situ hybridization data (11).

Hydrodynamic and electron microscopy studies led us to propose a model for mucin subunits as containing proteinase-resistant, oligosaccharide-rich domains flanked by proteinase-sensitive, cysteine-containing, naked-protein regions (3, 4, 15, 20, 26, 27). The oligosaccharide-rich domains correspond to the high molecular weight mucin glycopeptides released by proteolysis of the reduced mucin subunit. Here we show that the high molecular weight mucin glycopeptides derived from the least-charged glycoform of the MUC5B mucin can be separated on the basis of size into three different components with an average Mr of 7.3 × 105, 5.2 × 105, and 2 × 105 and a relative molar ratio of 1:2:1. If present in this ratio in the intact reduced MUC5B subunit, then the glycosylated domains would account for approximately 80% of the mass (Mr 2.5 × 106 (1)). Thus, we can suggest a model for the MUC5B mucin subunit (Fig. 10) in which these glycosylated proteinase-resistant domains are flanked by cysteine-containing regions of the protein core less substituted with glycan chains and thus more susceptible to proteolysis after reduction. These naked domains account for approximately 20% of the mass of the subunit (i.e. 500,000) and can be fragmented by trypsin into smaller peptides and glycopeptides. From the dramatic relative abundance of a few major peptides and the surprising fact that these peptides are found in a contiguous sequence one may propose that this cysteine-rich motif is repeated with identical sequence numerous times in the molecule. Interestingly a homologous motif is found repeated twice within the MUC2 mucin core protein flanking the smaller of the two glycosylated regions (25). By analogy we propose that this cysteine-rich motif flanks the glycosylated domains in the MUC5B mucin subunit. Thus, our data provide an important new insight into the structure of the proteinase-sensitive naked domains (Fig. 10).


Fig. 10. Proposed model for MUC5B mucin subunit structure. The model depicts an average structure for the MUC5B mucin subunit, which would be composed of four glycosylated domains, three of which are similar in amino acid composition and distinct from the smaller fourth domain. The glycosylated domains are separated by proteinase-sensitive domains containing a common cysteine-rich motif. The order of the glycosylated regions and the position and number of the cysteine-rich motifs are speculative.
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Biochemical analyses of airway secretions have demonstrated that the MUC5AC (1) and MUC5B mucins are responsible for the respiratory mucus gel. Northern blot and in situ hybridization indicate that at least six other mucins, namely MUC1, MUC2, MUC3, MUC4, MUC7, and MUC8, are expressed in the airways (7, 9, 10, 12-14), but only the MUC2 mucin has been demonstrated to be of the large gel-forming type (28, 29). However, we and others are unable to find significant quantities of the MUC2 mucin in respiratory tract secretions (1, 16, 18). Thus, the level of mRNA expression may be a poor indicator of the amount of a particular mucin in the gel, and our findings demonstrate the necessity of determining the amount of mucin in the secretion with biochemical methods, rather than relying solely on the level of mucin mRNA expression.


FOOTNOTES

*   This work was supported by the Wellcome Trust.The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
Dagger    To whom correspondence should be addressed. Tel.: 0161-275-6954; Fax: 0161 275 5082; E-mail: Dave.Thornton{at}man.ac.uk.
§   Recipient of a Biotechnology and Biological Sciences Research Council Cooperative Award in Science and Engineering studentship in collaboration with Beckman Instruments Ltd.
1   The abbreviation used is: MALDI-TOF MS, matrix-assisted laser desorption time of flight mass spectrometry.

ACKNOWLEDGEMENTS

We thank Prof. Tim Hardingham for reading of the manuscript and the Wellcome Trust for support.


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