Mucin production is controlled by mucin genes (MUCs). To date, 21 human mucin genes (MUCs 1, 2, 3A, 3B, 4, 5AC, 5B, 6–9, 12, 13, and 15–22) have been identified by cDNA cloning [
Mucins represent the major constituent responsible for mucus viscoelastic properties [
As sinus mucus is made up from multiple mucin gene products, it is important to understand the structure and composition of sinus mucins and how their distribution varies in sinus mucus. Alteration of mucus quantity and/or quality could alter mucus viscoelastic properties leading to alterations of the mucociliary clearance. From the clinical point of view, this alteration could aggravate patients’ symptoms of mucus rhinorrhea and difficulty in mucus clearance with the development of other nasal, pharyngeal, and laryngeal symptoms. From the pathopharmacological point of view, altered mucus physical and/or biological properties could affect the penetration of mucus by pathogenic and/or therapeutic agents. We studied the sinus mucin fragmentation as this will help in the understanding of the physical and biological properties of sinus mucins in CS and could help to develop therapeutic modalities to alter these properties and facilitate mucus drainage and relieve relevant CS symptoms such as rhinorrhea, chronic cough, and globus pharyngeus.
Sinus mucus was collected from 8 patients undergoing functional endoscopic sinus surgery. Chronic sinusitis is defined as persistence of sinus symptoms (nasal obstruction, discharge, reduced sense of smell, headache, and facial pain) for more than 3 months (with topical steroids used for at least 3 months) [
The patients’ group included 5 males and 3 females, aged from 19 to 78 years with mean age of 43 years. Mucus samples were collected from the maxillary sinuses through the middle meatal antrostomy (MMA) during functional endoscopic sinus surgery (FESS). Native/polymeric mucins were isolated by 2 rounds of caesium chloride density gradient centrifugation [
Polymeric sinus mucins were fragmented by reduction with dithiothreitol (DTT) or proteolytic digestion with trypsin or papain.
2 mg of polymeric mucin was incubated with 10 mM DTT, 20 mM tris/HCl, and 6 M guanidinium chloride, pH 8, 37°C, for 5 h. Solid iodoacetamide 25 mM was then added to block the free-SH groups and the incubation continued overnight at room temperature in the dark.
2 mg of mucin from purified sinus mucin sample (S1) rich in MUC5AC mucin was incubated with either porcine trypsin 0.5
After extensive dialysis, polymeric and fragmented mucins were fractionated by gel filtration chromatography using a Sepharose CL-2B column (125 × 1.5 cm) and eluted by upward flow with 0.2 M NaCl and 0.02% (w/v) NaN3 and 2 mL fractions were collected. Glycoprotein contents were measured by the periodic acid Schiff (PAS) assay. The Kav values were calculated for the glycoprotein rich peaks.
After gel chromatography, polymeric and fragmented mucins were subjected to SDS-PAGE on 4–15% gradient gels. Mucin rich peaks from gel filtration were pooled and dialysed against distilled water for 48 h and then freeze-dried and 1
This was performed by a modified version of the method of Carlton and Morgan [
Four sinus mucin samples had MUC5AC mucin as the major mucin (S1–4) and the other four samples had MUC2 mucin as the major mucin (S5–8).
Gel filtration was performed on MUC5AC rich sinus mucin sample (S1). The elution profiles are shown in Figure
Chromatographic profile of the purified fractionated sinus mucin sample (S1). Sepharose CL-2B gel column (125 × 2.5 cm) was eluted by upward flow with 0.2 M sodium chloride containing 0.02% (w/v) sodium azide and flow rate 18 mL/h. Calibration was firstly performed using 1% (w/v) dextran blue solution containing 0.05% (w/v) methyl orange. The glycoprotein content was estimated by the PAS solution assay.
Electrophoretic patterns of polymeric and fragmented sinus mucins reflected those noted after gel chromatography. Polymeric mucin remained in the stacking gel phase whereas fragmented mucin migrated into the running gel phase to a distance dependent on its hydrodynamic size with a progressive diminishing of size from reduced to digested conditions (results not shown). Furthermore, SDS-PAGE of a polymeric MUC5AC rich mucin sample without gel chromatography showed that 76% of mucin remained at the point of application and 24% migrated from the point of application compared to 80% excluded and 20% included on gel filtration (results not shown). This indicated that SDS-PAGE can be used as an alternative method to gel chromatography to study sinus mucin fragmentation patterns.
Two sinus mucin samples, one rich in MUC5AC (S1) and one rich in MUC2 (S5) mucins, were subjected to SDS-PAGE. In the 2 samples, polymeric sinus mucins gave similar profiles, with the majority of the mucin at the point of application in the stacking gel and a small amount migrating to the interface between the stacking and running gels (Figures
Densitometric scans of SDS-PAGE of polymeric and fragmented MUC5AC and MUC2 rich sinus mucins. The
Protein content of the mucins varied between 14.2% and 24.8% by weight. MUC5AC rich mucins had higher protein content than the MUC2 rich mucins, 21.1% ± 1.4% and 15.4% ± 0.8%, respectively. The general amino acid composition of sinus mucins showed the characteristic mucin analysis rich in serine, threonine, and proline. Total content of these 3 amino acids represented 29.7% to 49.6% by weight of the protein core. MUC5AC and MUC2 rich mucins contained total serine, threonine, and proline of 43.6% ± 3.7% and 40.1% ± 3.7%, respectively, the main difference being in serine content which was 7.8 ± 0.5% in MUC5AC rich mucins compared to 16.8 ± 2.1% for the MUC2 rich mucins (
Amino acid composition of native/polymeric chronic sinusitis mucins.
Amino acid | S1 | S2 | S3 | S4 | S5 | S6 | S7 | S8 |
---|---|---|---|---|---|---|---|---|
MUC5AC rich samples | MUC2 rich samples | |||||||
% content of amino acids | % content of amino acids | |||||||
His | 5.7 ± 0.2 | 9.9 ± 0.1 | 6.3 ± 0.7 | 8.5 ± 0.4 | 11.0 ± 1.8 | 1.8 ± 0.4 | 3.1 ± 0.1 | 4.1 ± 0.1 |
Arg | 14.9 ± 1.9 | 16.8 ± 1.8 | 11.5 ± 1.4 | 14.6 ± 1.0 | 6.6 ± 0.6 | 13.7 ± 1.6 | 11.2 ± 1.7 | 24.5 ± 0.5 |
Ser | 14.7 ± 0.2 | 18.5 ± 1.3 | 21.0 ± 2.1 | 12.2 ± 1.3 | 16.7 ± 2.3 | 35.7 ± 2.7 | 27.4 ± 3.5 | 23.7 ± 0.5 |
Thr | 25.2 ± 0.7 | 33.2 ± 3.2 | 35.8 ± 0.8 | 24.6 ± 0.5 | 12.1 ± 0.5 | 25.2 ± 1.1 | 17.4 ± 1.8 | 20.1 ± 1.1 |
Asp | 32.3 ± 0.3 | 19.5 ± 0.3 | 22.8 ± 0.2 | 21.3 ± 1.2 | 12.5 ± 1.5 | 11.4 ± 0.4 | 7.3 ± 1.0 | 6.2 ± 0.2 |
Glu | 23.9 ± 0.4 | 19.5 ± 1.3 | 23.5 ± 0.4 | 12.8 ± 0.9 | 20.0 ± 1.2 | 15.6 ± 1.4 | 7.8 ± 1.2 | 10.3 ± 0.8 |
Gly | 12.4 ± 0.1 | 7.8 ± 0.1 | 13.3 ± 1.2 | 7.2 ± 0.2 | 10.3 ± 0.4 | 3.9 ± 0.9 | 6.8 ± 0.4 | 5.3 ± 0.7 |
Ala | 18.1 ± 0.8 | 23.6 ± 0.6 | 25.0 ± 0.9 | 31.0 ± 3.4 | 12.2 ± 1.4 | 22.9 ± 2.7 | 18.0 ± 1.2 | 19.1 ± 0.1 |
Pro | 21.2. ± 2.8 | 26.9 ± 1.9 | 32.3 ± 3.1 | 27.9 ± 0.1 | 17.7 ± 0.3 | 23.1 ± 1.6 | 15.4 ± 2.3 | 12.8 ± 0.8 |
Val | 9.5 ± 0.5 | 12.3 ± 0.3 | 16.5 ± 0.4 | 9.7 ± 0.7 | 7.2 ± 0.7 | 7.0 ± 0.8 | 6.6 ± 0.4 | 3.0 ± 1.1 |
Ile/Leu/Phe | 16.4 ± 0.2 | 16.6 ± 0.7 | 23.8 ± 0.1 | 18.2 ± 0.8 | 15.0 ± 1.8 | 11.2 ± 1.3 | 15.6 ± 1.7 | 9.7 ± 2.3 |
Lys | 13.0 ± 1.3 | 11.9 ± 1.8 | 16.5 ± 1.4 | 5.0 ± 1.5 | 11.6 ± 1.5 | 3.9 ± 0.2 | 5.8 ± 1.2 | 4.1 ± 0.9 |
Calc. ptn | 207.3 ± 1.1 | 216.5 ± 4.5 | 248.3 ± 5.6 | 193.0 ± 3.6 | 152.9 ± 8.3 | 175.4 ± 14.3 | 142.2 ± 8.2 | 142.9 ± 4.0 |
The values quoted are of the % amino acid contents in polymeric mucins. The samples were arranged according to the proportions of the relevant predominant mucin. In the first 4 samples MUC5AC (S1–S4) was predominant and MUC2 was predominant in the other 4 samples (S5–S8). Amino acid content was calculated in quadruplicate samples and the mean content of amino acid in each sample was calculated. Calc. ptn: calculated total protein in ug/mg of freeze-dried mucin.
Following reduction, a seventh and a fifth of the protein content were lost for the MUC5AC and MUC2 rich mucins, respectively (Table
Amino acid composition of reduced chronic sinusitis mucins.
Amino acid | S1 | S2 | S3 | S4 | S5 | S6 | S7 | S8 |
---|---|---|---|---|---|---|---|---|
MUC5AC rich samples | MUC2 rich samples | |||||||
% content of amino acids | % content of amino acids | |||||||
His | 3.8 ± .0.7 | 1.6 ± 0.4 | 2.1 ± 0.1 | 5.5 ± 0.4 | 7.8 ± 0.7 | 2.3 ± 0.3 | 3.5 ± 0.1 | 1.5 ± 0.3 |
Arg | 20.4 ± 1.4 | 13.2 ± 1.7 | 7.2 ± 0.5 | 9.7 ± 0.7 | 4.2 ± 0.2 | 9.9 ± 1.0 | 5.9 ± 0.4 | 7.3 ± 0.7 |
Ser | 27.9 ± 0.1 | 12.4 ± 1.3 | 22.5 ± 1.5 | 14.3 ± 0.7 | 11.1 ± 1.1 | 27.8 ± 0.8 | 25.5 ± 1.6 | 18.6 ± 1.6 |
Thr | 30.6 ± 0.5 | 25.4 ± 2.4 | 32.9 ± 1.2 | 25.8 ± 2.1 | 21.2 ± 0.2 | 19.4 ± 1.2 | 17.8 ± 0.8 | 9.4 ± 0.4 |
Asp | 32.4 ± 0.4 | 19.7 ± 1.6 | 30.5 ± 2.2 | 20.6 ± 0.6 | 11.2 ± 0.3 | 7.2 ± 0.2 | 4.9 ± 0.4 | 20.2 ± 1.6 |
Glu | 9.6 ± 1.4 | 9.8 ± 0.2 | 21.2 ± 1.2 | 11.1 ± 1.0 | 17.2 ± 0.2 | 8.2 ± 1.3 | 8.3 ± 0.3 | 4.5 ± 0.6 |
Gly | 5.7 ± 1.2 | 6.8 ± 0.4 | 17.0 ± 1.3 | 14.5 ± 0.8 | 3.4 ± 0.2 | 6.6 ± 0.6 | 7.0 ± 0.2 | 1.0 ± 0.1 |
Ala | 25.2 ± 1.0 | 22.0 ± 2.2 | 24.2 ± 1.1 | 24.2 ± 1.9 | 5.8 ± 0.8 | 14.7 ± 1.2 | 15.4 ± 1.5 | 10.7 ± 0.1 |
Pro | 26.5 ± 2.7 | 27.9 ± 0.9 | 24.8 ± 2.2 | 16.1 ± 1.7 | 9.5 ± .0.5 | 24.2 ± 1.2 | 11.3 ± 0.2 | 11.4 ± 0.3 |
Val | 4.6 ± 0.6 | 3.4 ± 0.6 | 9.1 ± 0.1 | 3.7 ± 0.2 | 2.9 ± 0.3 | 5.3 ± 0.3 | 4.6 ± 0.8 | 1.6 ± 0.4 |
Ile/Leu/Phe | 14.6 ± 1.8 | 0.9 ± 1.5 | 11.0 ± 1.1 | 13.0 ± 1.2 | 6.9 ± 0.3 | 15.1 ± 1.3 | 20.8 ± 0.8 | 13.7 ± 1.5 |
Lys | 5.4 ± 1.0 | 5.5 ± 0.4 | 7.6 ± 1.1 | 2.6 ± 0.2 | 8.8 ± 0.2 | 2.9 ± 0.5 | 3.6 ± 0.7 | 00 ± 00 |
Calc. ptn | 206.7 ± 10.2 | 156.7 ± 9.9 | 210.0 ± 13.4 | 161.1 ± 3.8 | 110.0 ± 2.2 | 143.6 ± 3.7 | 128.0 ± 3.4 | 99.7 ± 7.6 |
The values quoted are of the % amino acid contents in reduced sinus mucins. The samples were arranged according to the proportions of the relevant predominant mucin. In the first 4 samples MUC5AC (S1–S4) was predominant and MUC2 was predominant in the other 4 samples (S5–S8). Amino acid content was calculated in quadruplicate samples and the mean content of amino acid in each sample was calculated. Calc. ptn: calculated total protein in ug/mg of freeze-dried mucin.
Proteolytic digestion of sinus mucins produced a greater loss of protein than reduction with approximately one-third of the protein content lost. Digested mucins still had different protein contents which represented 14.4 ± 1.1% in MUC5AC rich mucin and 10.0 ± 0.9% in MUC2 rich mucin. The content of serine, threonine, and proline increased in both sinus mucin groups after proteolytic digestion to account for almost half the remaining protein, 48.8 ± 1.0% and 48.8 ± 2.3% for MUC5AC and MUC2 rich mucins, respectively. There was a large loss of acidic amino acids on proteolysis, with the MUC5AC rich mucins decreasing from 20% in the polymeric mucins to 8% in the digested mucins and the MUC2 rich mucins decreasing from 15% in the polymeric mucins to 9.5% in the digested mucins (Table
Amino acid composition of papain digested chronic sinusitis mucins.
Amino acid | S1 | S2 | S3 | S4 | S5 | S6 | S7 | S8 |
---|---|---|---|---|---|---|---|---|
MUC5AC rich samples | MUC2 rich samples | |||||||
% content of amino acids | % content of amino acids | |||||||
His | 2.3 ± 0.2 | 7.8 ± 0.5 | 7.4 ± 1.5 | 2.4 ± 0.3 | 5.2 ± 1.4 | 0.0 ± 0.0 | 3.6 ± 0.1 | 1.1 ± 0.1 |
Arg | 9.8 ± 0.3 | 8.4 ± 0.4 | 6.2 ± 0.7 | 6.6 ± 2.6 | 4.1 ± 0.1 | 7.5 ± 0.1 | 3.9 ± 0.4 | 8.3 ± 0.6 |
Ser | 26.2 ± 0.2 | 32.8 ± 2.8 | 34.9 ± 1.9 | 17.2 ± 2.7 | 14.3 ± 2.0 | 30.3 ± 0.6 | 19.8 ± 0.1 | 22.6 ± 1.6 |
Thr | 17.1 ± 0.5 | 19.6 ± 1.5 | 30.3 ± 0.5 | 16.9 ± 0.9 | 18.0 ± 0.9 | 20.3 ± 0.9 | 12.5 ± 0.5 | 14.1 ± 1.3 |
Asp | 4.8 ± 0.3 | 2.8 ± 0.1 | 4.6 ± 0.6 | 6.6 ± 1.1 | 4.9 ± 0.2 | 3.1 ± 0.1 | 2.5 ± 0.4 | 3.1 ± 0.1 |
Glu | 3.9 ± 0.8 | 3.6 ± 0.3 | 12.8 ± 0.2 | 5.6 ± 0.7 | 9.5 ± 0.2 | 3.0 ± 0.6 | 5.6 ± 0.9 | 4.4 ± 0.5 |
Gly | 00 ± 00 | 0.0 ± 00 | 3.4 ± 1.3 | 0.1 ± 0.1 | 7.2 ± 0.1 | 2.2 ± 0.3 | 7.3 ± 0.3 | 4.7 ± 0.2 |
Ala | 27.7 ± 2.5 | 19.6 ± 0.6 | 22.4 ± 1.4 | 18.4 ± 3.2 | 3.6 ± 0.3 | 28.4 ± 0.3 | 14.8 ± 1.9 | 10.3 ± 1.3 |
Pro | 23.1 ± 1.1 | 18.2 ± 0.3 | 20.7 ± 1.9 | 24.1 ± 4.1 | 5.8 ± 2.0 | 17.5 ± 2.1 | 13.2 ± 0.8 | 8.5 ± 0.1 |
Val | 3.1 ± 0.1 | 4.2 ± 0.7 | 4.6 ± 0.1 | 6.0 ± 1.0 | 1.9 ± 0.4 | 2.8 ± 0.1 | 1.9 ± 0.1 | 5.6 ± 0.2 |
Ile/Leu/Phe | 23.00 ± 1.5 | 16.2 ± 1.0 | 17.8 ± 2.8 | 12.6 ± 1.0 | 7.1 ± 0.7 | 11.4 ± 1.4 | 4.5 ± 0.2 | 7.8 ± 0.9 |
Lys | 2.8 ± 0.5 | 7.0 ± 0.4 | 6.0 ± 2.4 | 3.6 ± 0.9 | 7.4 ± 0.2 | 0.1 ± 0.1 | 1.5 ± 0.4 | 2.5 ± 0.2 |
Calc. ptn | 143.8 ± 2.2 | 140.2 ± 2.6 | 171.1 ± 6.9 | 120.1 ± 0.5 | 89.0 ± 0.2 | 126.6 ± 3.3 | 91.1 ± 2.7 | 93.0 ± 1.0 |
The values quoted are of the % amino acid contents in papain digested sinus mucins. The samples were arranged according to the proportions of the relevant predominant mucin. In the first 4 samples MUC5AC (S1–S4) was predominant and MUC2 was predominant in the other 4 samples (S5–S8). Amino acid content was calculated in quadruplicate samples and the mean content of amino acid in each sample was calculated. Calc. ptn: calculated total protein in ug/mg of freeze-dried mucin.
This study does not include normal sinus mucin as a control. It would be more informative to have such a control to find out if there were differences in its antigenic identity, polymeric structure, and fragmentation behaviour compared to that in chronic sinusitis. There are ethical and technical difficulties to obtain normal sinus mucus for mucin extraction. This would entail an unnecessary invasive procedure to obtain maxillary sinus mucus from healthy sinuses. Furthermore, normal sinus mucus production is extremely small and it would not be possible to do the required biochemical analyses on such small amounts. This would then necessitate pooling different normal sinus mucus samples together to obtain adequate mucin material for these tests. This would not then allow the normal sinus mucin gene profile for each patient to be identified.
Mucins present in paranasal sinus mucus have been identified predominantly as MUC2, MUC5AC, and MUC5B [
Due to limited available amount of mucin which remained after other mucin analyses including mucin identity studies (ELISA), the sinus mucin sample subjected to fragmentation and analysis on gel filtration was rich in MUC5AC. Not enough mucin was available to study gel chromatographic behavior of MUC2 rich samples. However, as the SDS-PAGE distribution of MUC5AC rich polymeric and fragmented mucins mirrored the chromatographic pattern of these mucins on Sepharose 2B gel filtration, SDS-PAGE allowed the study of mucin fragmentation in small quantities of mucin samples. This finding is similar to what was reported in other studies on gastric [
Polymeric MUC5AC and MUC2 rich mucins are of large molecular size with the majority of the sample remaining at the point of application on the polyacrylamide gel. As the polymeric structure of MUC2 and MUC5AC mucin molecules is based on disulphide bridges, reduction produced a size change consistent with a subunit structure. However, MUC2 rich mucin showed little change compared to MUC5AC rich mucin. This is similar to previous finding in reduced pig colonic mucin, rich in MUC2, which was largely excluded on Sepharose Cl-2B gel chromatography [
Papain digestion of the MUC2 rich mucins did not produce distinct glycosylated units in the running gel whereas MUC5AC rich mucins produced four distinct species in the running gel after papain and trypsin digestion. The antigenic identity of these peaks was not determined but these could reflect different sized glycosylated domains in the MUC5AC molecule that are different in size and/or charge. It is unlikely that these bands represent MUC5B mucin as both the MUC2 and the MUC5AC rich samples contained MUC5B. Furthermore, it has also been reported that MUC5AC may be more susceptible to proteolytic degradation than MUC5B [
The integrity of the polymeric structure is important for the gel-formation character of mucins. The polymeric structure of MUC2 and MUC5AC is based on disulphide bridges between mucin subunits and peptide bonds between mucin glycopeptides [
Amino acid analysis of the polymeric mucins demonstrated that the two groups of mucins were rich in serine, threonine, and proline similar to reported levels of these amino acids in other well characterized polymeric mucins, that is, human gastric, cervical, respiratory, and middle ear [
On reduction nearly similar amounts of protein were lost in both groups which meant that the reduced mucin groups still contained different amounts of protein. This amount of protein lost on reduction has previously been shown for pig colonic mucin [
Digestion of human sinus mucins results in approximately one-third of the protein content being lost. The proportions of serine, threonine, and proline have increased to almost one-half of the protein core; similar increases have been demonstrated for human middle ear and gastric mucins [
Detailed characterization of amino acid composition of sinus mucins could help identify potential amino acid sites for reduction and proteolytic activity to break down the mucin molecule and hence reduce its viscosity and facilitate its mucociliary clearance.
MUC2 and MUC5AC expressed in chronic sinusitis mucus behave differently on reduction and proteolytic digestion. Proteolytic digestion of MUC5AC rich mucins produced mucin glycopeptides which, on SDS-PAGE, produced 4 distinctive bands which could be used as finger prints for MUC5AC. These mucins have different amino acid composition which, however, is similar to the general amino acid composition profile of known mucins.
Further characterisation of sinus mucins in health and disease conditions could help in the understanding of biological importance of polymeric sinus mucin molecules and its subspecies and might help in the invention of medical treatment modalities for chronic sinusitis.
This study does not include normal sinus mucin as a control. It would be more informative to have such a control to identify the differences in its polymeric structure and fragmentation behaviour compared to that in chronic sinusitis. There are ethical and technical difficulties to obtain normal sinus mucus. Normal nasal mucus might represent an alternative for sinus mucin. However, we have found that nasal and sinus mucin expression are not similar in disease [
The authors declare that there is no conflict of interests regarding the publication of this paper.