Glycobiology Advance Access originally published online on April 6, 2005
Glycobiology 2005 15(8):791-804; doi:10.1093/glycob/cwi059
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MUC5B glycosylation in human saliva reflects blood group and secretor status
2 Department of Medical Biochemistry, Göteborg University, Box 440, 405 30 Göteborg, Sweden; and 3 Proteome Systems Limited, Locked Bag 2073, North Ryde, Sydney, New South Wales 1670, Australia
1 To whom correspondence should be addressed; e-mail: niclas.karlsson{at}proteomesystems.com
Received on November 9, 2004; revised on February 24, 2005; accepted on March 28, 2005
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Abstract |
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This study aimed to characterize human salivary glycoforms and the natural glycosylation variation of the major ABO blood group bearing high molecular weight glycoprotein fraction MG1, which mainly consists of MUC5B mucin. Reduced and alkylated mucins from individuals of blood group A, B, and O were purified by sodium dodecyl sulfate-agarose/polyacrylamide composite gel electrophoresis (SDS–AgPAGE), blotted to polyvinylidene fluoride (PVDF) membranes, and visualized with alcian blue. O-linked oligosaccharides were released from MUC5B glycoform bands by reductive ß-elimination and analyzed by liquid chromatography (LC) electrospray ion trap mass spectrometry (MS). Slow electrophoretically migrating MUC5B components (sm) were found to be dominated by neutral oligosaccharides, and fast-migrating (fm) components were dominated by sulfated oligosaccharides. ABO blood group-specific sequences were found on all glycoforms, and novel oligosaccharides containing blood group A and B type sequences were sequenced. This is the first molecular description of the influence of the blood group ABO system on salivary MUC5B oligosaccharides. Expanding these results from the three A, B, and O individuals into larger population (29 individuals), we found oligosaccharide sequences corresponding to the blood group of the donor on MUC5B from 23 individuals. The remaining six individuals were characterized by a high degree of sialylation. These individuals were assigned as nonsecretors, whereas blood group-expressing individuals were assigned as secretors. Western blot assays with antibodies confirmed increased expression of Sialyl Lewis a (Si-Lea) in the nonsecretors. Our results highlight that salivary MUC5B consists of glycoforms with distinct glycosylation that vary extensively between individuals and that some of this variation is owing to blood group and secretor status.
Key words: glycoforms / mass spectrometry / MUC5B / saliva
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Introduction |
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Salivary secretions are essential for the protection and lubrication of the oral surfaces in the mouth. The high turnover rate of the saliva promotes clearance of trapped bacteria, and the individual salivary protein components regulate the oral flora by providing an antimicrobial activity. Mucins are major protein components in saliva. Mucins are multimerising macromolecules with an extensive O-linked glycosylation (50–80 wt %) of Ser/Thr/Pro rich domains (Moniaux et al., 2001)
. Mucin oligosaccharides contribute to the viscous and adhesive properties of saliva, as well as providing interaction sites for bacteria and for leukocyte interactions (reviewed by Zalewska et al., 2000).
Two populations of mucins with distinct features are present in saliva: MG1, a high molecular mass component (Mr >106), and MG2 (Mr 1.2–1.5 x 105) (van Nieuw Amerongen et al., 1995). The major protein component of MG1 is encoded by the MUC5B gene (Nielsen et al., 1997; Wickström et al., 1998; Thornton et al., 1999) and is a large, gel-forming, multimerising mucin, produced by mucosal cells (Nielsen et al., 1996). MG2 consists of a small, nonmultimerising protein encoded by MUC7 (Bobek et al., 1993) and is secreted by serous cells (Nielsen et al., 1996). Both mucin populations appear to make up part of the salivary pellicle that coats the tooth surface and interacts with different species of bacteria (Murray et al., 1992). The two mucin populations both appear as glycoforms (Ramasubbu et al., 1991; Bolscher et al., 1995) and carry different glycosylation, with MUC5B containing on average longer and more diverse oligosaccharides than MUC7 (Prakobphol et al., 1998; Thomsson et al., 2002). A previous study performed on the high molecular weight fraction of saliva suggests that MUC5B is the main mucin carrier of blood group antigens (Prakobphol et al., 1993). Different 
1,2fucosyltransferase gene products in blood and body secretions are responsible for the production of the main blood group antigen saccharide precursor, the H epitope (Fuc1-2Gal) (reviewed by Henry et al., 1995). The Secretor (Se) fucosyltransferase is expressed in salivary glands, and individuals screened for ABH determinants in their saliva using immunological methods have been named "secretors" or "nonsecretors" depending on the presence or absence of these epitopes. In the Caucasian population, the distribution between secretors and nonsecretors is 
4:1.
The aim of this study was to characterize MUC5B glycoforms and to investigate to what extent salivary MUC5B glycosylation varies between individuals. This was performed by using an approach described in recent works (Schulz et al., 2002a,b), involving gel electrophoresis of reduced and alkylated saliva, electroblotting of protein components to polyvinylidene fluoride (PVDF) membrane, release of oligosaccharides, and analysis with liquid chromatography–mass spectrometry (LC-MS) and LC-tandem MS (LC-MS/MS).
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Gel electrophoresis of salivary MUC5B and screening of oligosaccharides from blood group A, B, and O individuals
Fresh saliva from six individuals was reduced, alkylated, separated with 1 D sodium dodecyl sulfate-agarose/polyacrylamide composite gel electrophoresis (SDS–AgPAGE) gradient gels, blotted onto PVDF membranes, and stained with alcian blue, which stains acidic oligosaccharides. MUC5B appeared as one or more bands at 1–2 MDa apparent molecular weight (Figure 1), and its position in the gel varied somewhat. However, the differences in the migration were not correlated with the ABO phenotype of the individuals. O-linked oligosaccharides were released by ß-elimination from the slow- (sm) and fast-migrating (fm) MUC5B components from one blood group A, one blood group B, and one blood group O individual according to Figure 1. The oligosaccharides were analyzed with LC-MS and MS/MS. The total ion chromatograms from the six analyses are shown in Figure 2, where major peaks are annotated with pseudomolecular [M–xH]x– and oligosaccharide composition. The sm MUC5B from individuals with different ABO blood groups (Figure 2: upper panels) contained glycoforms dominated by neutral oligosaccharides, whereas fm MUC5B glycoforms (Figure 2: lower panels) were dominated by sulfated oligosaccharides. All major peaks were fucosylated.
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MUC5B neutral glycoforms from blood group A, B, and O individuals
Further analysis of the glycosylation of MUC5B from three individuals (from Figure 1) representing each of the three blood groups were performed to elucidate blood group-specific variations. The major oligosaccharide components from the sm MUC5B (Figure 1: bands labeled sm; Figure 2: upper panels) were shown to differ between the three samples. The composition of detected molecular ions together with sequences are listed in Table I. The major differences in the sm MUC5B from different individuals were owing to an extensive expression of neutral oligosaccharides corresponding to the individuals’ ABO blood type. The O individual expressed blood group H epitopes on the oligosaccharides, whereas the blood group A and blood group B individuals in addition to H epitopes expressed blood group A and blood group B antigens on MUC5B, respectively. The molecular basis for the blood group-related epitopes and the corresponding sequences detected by MS are summarized in Table II.
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MUC5B sulfated glycoforms from blood group A, B, and O individuals
The oligosaccharide profiles obtained of the fm "sulfated" glycoforms from the three different ABO individuals (Figure 1: bands labeled fm, Figure 2: lower panels) consisted mainly of monosulfated oligosaccharides, though disulfated and sialic acid-containing oligosaccharides were also detected. The compositions of identified molecular ions are listed together with elucidated sequences in Table I.
MS/MS and MS3 were used to fully sequence the dominating sulfated oligosaccharides (such as the [M–H]– ion of m/z 1340 and m/z 1381) and to assign the sulfate to a monosaccharide unit. Figure 3 shows the MS/MS spectrum of m/z 1340 (Figure 3A) and MS3 spectrum of the daughter ion at m/z 667 (Figure 3B) from the blood group B individual. Figure 3A shows the series of Yi fragments and one B2 fragment, from which part of the two branches can be elucidated: one branch carrying a blood group B sequence linked to a core N-acetylhexosamine (HexNAc) residue and the second branch consisting of a Hexose (Hex) and a HexNAc unit with a sulfate group attached to either sugar. The exact arrangement of the second branch was determined by using MS3 of the Y fragment at m/z 667 (Figure 3B), giving an intense fragment at m/z 241, diagnostic for sulfate linked to a Hex residue. The lack of a fragment indicating the loss of a terminal HexNAc residue at m/z 1137 indicates that the sequence contains a core 4 arrangement, where the full sequence is HSO3-Hex-HexNAc-(Hex-(Fuc-)Hex-HexNAc-)HexNAcol. In addition to the sequence corresponding to m/z 1340, two additional sulfated oligosaccharides containing blood group B sequences from the blood group B individual (Figure 2) were identified, one having the sequence Hex-(Fuc-)Hex-(HSO3-)HexNAc-HexNAcol (m/z 975) and the other at m/z 1121 with the same sequence containing a Fuc residue linked to the sulfated HexNAc residue.
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Using this approach, we have characterized two MUC5B glycoforms derived from each of three individuals with distinct glycosylation (Table I), in part due to the presence of blood group-specific sequences. The sulfation of MUC5B was shown to be linked to both Hex (galactose) and HexNAc (N-acetylglucosamine). Because similar oligosaccharide sequences were found in both sm and fm MUC5B glycoforms from the same individual, it can be hypothesized that the faster migration was caused by the addition of sulfate (and increased charge) to neutral oligosaccharides.
ABH blood group sequences and secretor status
Saliva from 29 individuals with known ABO blood group was collected and analyzed as described above. In the initial screening, to correlate blood group with expressed MUC5B oligosaccharides, we used the retention time and MS/MS spectra of the diagnostic structures HexNAc-(Fuc)-Hex-3HexNAcol ([M–H]– ion of m/z 733, blood group A), Hex-(Fuc-)Hex-4HexNAc-HexNAcol ([M–H]– ion of m/z 895, blood group B), Fuc-Hex-3HexNAc-HexNAcol ([M–H]– ion of m/z 733, blood group H), and Fuc-Hex-(Hex-HexNAc-) HexNAcol ([M–H]– ion of m/z 895, blood group H). In 23 of 29 individuals, we identified sequences that corresponded to the blood group of the individual (Table III). Figure 4A shows the MS/MS spectrum of the blood group A-specific tetrasaccharide. Diagnostic fragments are found at m/z 512 (Z2), which is indicative of loss of a HexNAc in a terminal position, m/z 625 ([M–H]– –108), and m/z 510 (B2), where the latter two are indicative of a singly branched HexNAcol. The fragment at m/z 625 originates from a cleavage within the HexNAcol moiety (Karlsson et al., 2004). Figure 4B shows the MS/MS spectrum of the blood group B-specific pentasaccharide, (Hex-(Fuc-)Hex-4HexNAc-HexNAcol), where m/z 715 origins from loss of a Hex in terminal position, and the two fragments at m/z 787 ([M–108]–) and m/z 672 (B3) are diagnostic for a singly branched HexNAcol. The fragment at m/z 571 results from a cleavage within the HexNAc moiety followed by loss of H2O and is diagnostic for a type 2 branch.
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The presence of ABO blood group-specific sequences on salivary MUC5B matching the blood group status suggests that 23 individuals were secretors. The six remaining individuals did not express the blood group-related sequences of m/z 733 or m/z 895, nor did we find any additional blood group sequences on other oligosaccharides in these samples. This finding is consistent with a nonsecretor status of these six individuals.
Increased sialylation on MUC5B from individuals mass spectrometrically identified as nonsecretors
The alcian blue-stained MUC5B component from a blood group B individual identified as a nonsecretor by MS is shown in Figure 5. Oligosaccharides were released from the positions marked in the inserted picture, and the total ion mass spectra from the upper (sm) and lower (fm) band are shown in Figure 5A and B, respectively. The sm components were dominated by sialylated oligosaccharides, whereas the fm glycoform contained mainly sulfated oligosaccharides, of which several abundant peaks contained two sulfate groups. The composition of detected molecular ions and the elucidated oligosaccharide sequences corresponding to the sm component in Figure 5A are listed in Table IV. All major peaks contained sialic acid and Fuc. Five neutral and two sialylated sequences were deduced. The highly abundant sialylated ion at m/z 1477 eluted as a single peak in the chromatogram indicating that it was only present as one component. It was identified as a doubly sialylated heptasaccharide with the sequence NeuAc-Hex-(NeuAc-Hex-(Fuc-)HexNAc)HexNAcol. The sequence indicates that Sialyl Lewis x (Si-Lex) [NeuAc2-3Galß1-4(Fuc1-3)GlcNAcß1-] and/or Sialyl Lewis a (Si-Lea) [NeuAc2-3Galß1-3(Fuc1-4)GlcNAcß1-] epitopes are expressed on MUC5B. Four secretors and four nonsecretors were screened for binding of antibodies against these two epitopes. Western blots showed that binding to Si-Lex and Si-Lea was found both among secretors and nonsecretors where MUC5B positive bands were found, and that the binding to Si-Lea was much stronger in nonsecretors compared with secretors (Figure 6). We could therefore conclude that the increased sialylation among nonsecretor individuals was linked to an increased expression of Si-Lea epitopes. The western blots also indicate that MUC7 and other lower molecular weight glycoproteins preferentially expressed Si-Lex and were less affected by the secretor status.
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To verify the observation that individuals identified as nonsecretors carried more sialylated oligosaccharides compared with secretors, we reanalyzed oligosaccharides from 13 secretors and four nonsecretors, this time obtained from PVDF strips containing the complete MUC5B bands. The relative amounts of Fuc, Hex, HexNAc, NeuAc, and sulfate in each sample were calculated based upon the identification of the composition of molecular ions and their relative peak intensity. The results are shown in Figure 7 and support our observation that the amounts of sialic acid residues are increased relative to other saccharide residues on nonsecretor MUC5B.
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Discussion |
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The presence of MUC5B glycoforms in saliva has previously been established with biochemical methods and antibodies (Bolscher et al., 1995
; Wickström et al., 1998; Thornton et al., 1999). The two salivary MUC5B bands found in this study from three individuals of blood group A, B, and O contained predominantly either neutral or sulfated oligosaccharides as judged from relative intensities in the mass spectra and always had sialylated components present. The total ion spectra were different between the individuals, and our data suggest that one explanation for this is that the different blood group-specific sequences were frequently expressed. The observation of ABO blood group-specific epitopes on salivary MUC5B is consistent with other studies using immunochemical techniques (Prakobphol et al., 1993). Mixtures of neutral and sialylated MUC5B oligosaccharides from blood group O secretor individuals have been previously characterized (Klein et al., 1992; Thomsson et al., 2002) and were described as large, extensively fucosylated and with frequently expressed H epitopes (Fuc1-2Galß1-), in agreement with our results. The presence of sulfated glycoforms and epitopes has also been determined with immunochemical methods, by using an antibody that recognizes the HSO3-3Galß1-3GlcNAcß1- moiety of sulfo Lewis a (Su-Lea) epitopes (Veerman et al., 1997).
We used a mass spectrometric approach to screen for the presence of blood group sequences in MUC5B. Six of 29 individuals did not express any ABH blood group-related epitopes. They were all of Caucasian origin and made up 27% of the Caucasians that took part in the study. This figure is close to the reported 20% of nonsecretors in the Caucasian population not encoding for the Se 2fucosyltransferase (FUT2) that makes the majority of the H epitopes (and consequently A and B) in saliva (reviewed by Henry et al., 1995), and suggest that MS can be used as a supplementary technique for determining the secretor status of an individual.
Mass spectra of MUC5B oligosaccharides obtained from the nonsecretors suggested that these individuals expressed more sialylated oligosaccharides than secretors. We found that increased sialylation of oligosaccharides obtained from nonsecretors was linked to increased expression of the saccharide epitope Si-Lea. This finding is supported by a previous study, where whole saliva was analyzed from individuals genotyped for secretor status, by using a monoclonal antibody (mA) against Si-Lea (Brockhaus et al., 1985). Their hypothesis for increased expression of Si-Lea epitopes was that when the Se gene product (FUT2) is lacking, Galß1-3GlcNAc precursor chains that otherwise would have been converted into H type 1 chains [Fuc1-2Galß1-3GlcNAc] are instead available for conversion into Si-Lea [NeuAc2-3Galß1-3(Fuc1-4)GlcNAc]. This hypothesis is supported by this study. Further support that a sialyltransferase substitute for the fucosyltransferase in nonsecretors comes from the analysis of two Asian participants in this study, who were identified as B secretors, but whose MUC5B oligosaccharide mass profile resembled the characteristic nonsecretor profile of predominantly sialylated oligosaccharides. Semiquantitative analyses performed on sm and fm bands from these individuals gave values of above 0.5 NeuAc per average oligosaccharide, which were typical of nonsecretors (Figure 7). The "partial" secretor phenotype Le(a+b+) involving a weak secretor gene is rare in Caucasians, but common in other populations such as Asians (Broadberry and Lin-Chu, 1991).
Our results highlight that there is extensive natural variation in the glycosylation of MUC5B oligosaccharides, which is partly dependent on inherited factors such as blood group and secretor status. There are further known inherited factors that could be expected to influence the individual variation of MUC5B glycosylation, which we have not investigated in this work. One factor is ABO polymorphism, with six known, common phenotypes in Caucasians (A1, A2, B, A1B, A2B, and O), giving rise to ABO blood group-specific glycosyltransferases of varying efficiency. Also, the Lewis histo-blood group antigens are found in secretions, and the phenotype can be confirmed in saliva with antibodies against the Lewis antigens Lea [Galß1-3(Fuc1-4)GlcNAcß1-] and Leb [Fuc1-2Galß1-3(Fuc1-4)GlcNAcß1-]. The Lewis fucosyltransferase (FUT3) is expressed in 93% of the Caucasian population. In the approach presented in this study, we cannot mass spectrometrically distinguish between the structural isomers Lea/Lex and Leb/Ley.
We have noticed that salivary MUC5B oligosaccharide profiles can be heterogenous even among individuals of the same blood group. The statistical data presented in this work suggest that the relative amount of sulfation on the MUC5B glycoform can vary extensively between individuals and may account for some of the heterogeneity between individuals with the same blood group. Different MUC5B glycoforms are produced by salivary glands that are physically separated in the oral cavity (Veerman et al., 1992; Bolscher et al., 1995; Wickström et al., 1998; Thornton et al., 1999), so it is possible that there could be subsets of cells specialized to produce and secrete either neutral or sulfated MUC5B glycoforms.
The clinical impact of carrying blood group-dependent glycosylation on mucosal surfaces has been investigated by many researchers. ABO blood group, secretor phenotype, and/or Lewis blood group-status have been linked with predisposition to bacterial infections by, for example Helicobacter pylori and Candida sp., and with chronic urinary tract infection (reviewed by D’Adamo and Kelly, 2001). Further evidence for a protective role for the Se gene is in FUT2 knockout mice, which show increased susceptibility to cervical yeast infection (Hurd and Domino, 2004). The analytical approach applied in this work permits potential screening of groups of individuals to investigate if a predisposition to a specific disease can be linked not only to known phenotypes such as ABO blood group and secretor phenotype, but also to combinations of phenotypes resulting from the activity of other glycosyltransferases such as fucosyl-, sialyl- or sulfotransferases.
The variation in salivary MUC5B glycosylation may have profound effects on the microenvironment in the oral cavity of different individuals. We propose that the glycosylation variation of MUC5B between individuals is due to territorial and historical diversity of the biological pressure from pathogens. This would suggest that salivary MUC5B is a key molecule in the initial interaction between host and invading pathogen and is important for understanding the mechanism of mucosal defense. In addition, our results also provide a warning that when comparative studies are performed on mucin glycosylation or bacterial infection in healthy and diseased states, the possibility that individuals can vary considerably even among the healthy controls should be considered carefully.
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Materials and methods |
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Sample collection and preparation
Twenty-nine human volunteers (22 Caucasian, 7 Asian) with known ABO blood group status were encouraged to spit saliva (300–600 µL) produced by the glands under the tongue (submandibular/sublingual glands) into a test tube. Aliquots of 200 µL saliva were reduced within 60 min after collection with 200 µL of sample buffer containing dithiothreitol (20 mM), 20% glycerol, Tris–HCl (0.75 M, pH 8.1), 2% SDS, and 0.01% bromophenol blue, for 20 min at 100°C. The samples were alkylated with 25 mM of iodoacetamide overnight, in the dark and with gentle shaking. The samples were centrifuged at 10,000 x g for 10 min to precipitate nonreduced material (usually no pellet was observed) and then concentrated to 15–40 µL on 100 kD cut-off membranes (Millipore, Bedford, MA). Dilution of each sample was performed by adding 120 µL of sample loading buffer (as above, but with 30% glycerol), and 40 µL loaded onto 0–7% acrylamide/1% agarose gels (Schulz et al., 2002a)
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The gels were run in boronate/Tris buffer (192 mM boric acid, pH adjusted to 7.6 with Tris, 1 mM EDTA, and 0.1% SDS) at 30 mA/gel for 3–3.5 h until the dye front ran out of the gel. The gels were blotted to PVDF membranes and stained with alcian blue as described by Schulz et al. (2002a). The presence and identification of MUC5B as the dominating mucin in the 1–2 MDa fraction was done by using western blotting (Thomsson et al., 2002) and peptide mass fingerprinting (Schulz et al., 2004).
Release of O-linked oligosaccharides
Reductive ß-elimination of oligosaccharides from mucins on blots was done essentially as described by Schulz et al. (2002a), with minor alterations. Briefly, alcian blue stained PVDF membrane bands were cut out either as strips across the whole MUC5B band (2 mm wide and 15 mm long) for semiquantitative analyses or as square bands (10–20 mm2) for glycoform analyses. The oligosaccharides were released overnight in 50 mM NaOH and 0.5M NaBH4 and desalted on 25 µL cation exchange resin in a C18 ZipTip (Millipore), followed by vacuum evaporation and removal of borate with repetitive acidic methanol extraction.
LC/MS, MS/MS, and MS3 of released oligosaccharides
Sample injection and LC was performed by using a Surveyor autosampler and LC pump (Thermo Finnigan, San Jose, CA). Oligosaccharides were resuspended in 10 µL of water and injected on graphitized carbon columns, either commercially packed columns (100 x 0.32 mm or 100 x 0.18 mm id, 7 µm Hypercarb particles, Thermo-Hypersil, Runcorn, UK) or homepacked with the same particles. Oligosaccharides were eluted with an H20/acetonitrile gradient containing 10 mM NH4HCO3 (0–34% acetonitrile in 27 min, followed by 34–90% acetonitrile in 6 min, then a 7-min wash with 90% acetonitrile). MS was performed by using an LCQ Deca XP mass spectrometer (Thermo Finnigan) in negative mode. The capillary temperature was 340°C, the capillary voltage 59 V, and the electrospray voltage 3.5 V. For MS/MS and MS3 experiments, the normalized collision energy was 40% with an activation time of 30 min. For MS/MS experiments, MS was performed with four scan events: full scan with mass range m/z 350–2000, followed by successive MS/MS scans after collision induced fragmentation for the three most intense ions in each full scan. For MS3 experiments, a three-scan method was used, full scan m/z 350–2000, the data-dependent MS/MS scan on the most intense peak, followed by data-dependent MS3 of the most intense fragment in the mass range m/z 400–840.
Semiquantitative analyses using MS
For semiquantitation experiments, alcian blue-stained PVDF membrane strips were cut out across the whole MUC5B band, 2 mm wide and 15 mm long, and the oligosaccharides released as described above. The LC-MS conditions were as described above, except for that a single scan method was applied, with a full scan in the mass range 350–2000 m/z. Thirteen secretors and four nonsecretors were analyzed, with the following blood group distribution: O : A : B : AB, 3:5:3:2 (secretors) and 2:1:1:0 (nonsecretors). The chromatograms were processed by using the program ACD/Spec Manager (ACD/Laboratories, Toronto, Ontario) for peak picking. Identification of oligosaccharide ions and calculation of relative amounts of monosaccharide residues were performed in an application program developed at Proteome Systems Limited (Sydney, Australia). The matches were manually examined, and in cases when different molecular ion compositions were possible, the correct composition was chosen based upon MS/MS data from previous analyses.
Western blot with antibodies raised against carbohydrate epitopes Si-Lea and Si-Lex
Reduced and alkylated saliva (equivalent of 0.06 µL saliva in 5–10 µL sample buffer for the Si-Lea assay and equivalent of 6 µL saliva in 5–10 µL sample buffer for the Si-Lex assay) was analyzed on gels and blotted to PVDF membranes as described above. The blots were wetted in methanol and blocked overnight at 4°C in 1% bovine serum albumin (BSA) and in phosphate buffered saline-T (PBS-T; PBS + 0.1% Tween-20), followed by incubation for 1–2 h at room temperature with antibodies against either Si-Lea (C241, Nilsson et al., 1987) or Si-Lex (CSLEX-1 from A.T.C.C., Manassas, VA), both diluted 1 µg/mL in blocking solution. After three washes with PBS-T, the membranes were incubated with horse radish peroxidase-conjugated goat anti-mouse IgG (Chemicon, Victoria, Australia) diluted 1:10,000 in blocking solution for 60 min at room temperature. The membranes were washed five times for 5 min in PBS-T, followed by incubation (2 min) in Supersignal West Femto Maximum Sensitivity Substrate (Pierce Biotechnology, Rockford, IL). The blots were exposed to Hyperfilm ECL (Amersham Bioscience, Little Chalfont, UK) for 1–30 s and developed by using Phenisol developer and Hypam rapid fixer solution from Ilford Imaging (Cheshire, UK).
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Acknowledgments |
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Dr. Mark Bayliss is thanked for allowing the use of the MS-manager software for analysis of LC-MS data. Support for this work was provided by The Postgenomic Research and Technology Programme in Western Sweden (Swegene) and the Swedish Medical Research Council (7461).
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Abbreviations |
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Fuc, fucose; Hex, Hexose; HexNAc, N-acetylhexosamine; HexNAcol, N-acetylhexosaminitol; LC, liquid chromatography; MS, mass spectrometry; NeuAc, N-acetylneuraminic acid; PBS, phosphate buffered saline; SDS–AgPAGE, sodium dodecyl sulfate-agarose/polyacrylamide composite gel electrophoresis; Si-Lea, Sialyl Lewis a; Si-Lex, Sialyl Lewis x
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References |
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Bobek, L.A., Tsai, H., Biesbrock, A.R., and Levine, M.J. (1993) Molecular cloning, sequence, and specificity of expression of the gene encoding the low molecular weight human salivary mucin (MUC7). J. Biol. Chem., 268, 20563–20569.
Bolscher, J., Veerman, E., van Nieuw Amerongen, A., Tulp, A., and Verwoerd, D. (1995) Distinct populations of high-Mr mucins secreted by different human salivary glands discriminated by density-gradient electrophoresis. Biochem. J., 309, 801–806.
Broadberry, R.E. and Lin-Chu, M. (1991) The Lewis blood group among chinese in Taiwan. Hum. Hered., 41, 290–294.[ISI][Medline]
Brockhaus, M., Wysocka, M., Magnani, J.L., Steplewski, Z., Koprowski, H., and Ginsburg. V. (1985) Normal salivary mucin contains the gastrointestinal cancer-associated antigen detected by monoclonal antibody, 19–9 in the serum mucin of patients. Vox Sang., 48, 34–38.[ISI][Medline]
D’Adamo, P.J. and Kelly, G.S. (2001) Metabolic and immunological consequences of ABH secretor and Lewis subtype status. Altern. Med. Rev., 6, 390–405.[Medline]
Henry, S., Oriol, R., and Samuelsson, B. (1995) Lewis histo-blood group system and associated secretory phenotypes. Vox Sang., 69, 166–182.[ISI][Medline]
Hurd, E.A. and Domino, S.E. (2004) Increased susceptibility of secretor factor gene Fut2-null mice to experimental vaginal candidiasis. Infect. Immun., 72, 4279–4281.
Karlsson, N.G., Schulz, B.L., and Packer, N.H. (2004) Structural determination of neutral O-linked oligosaccharide alditols by negative ion LC-electrospray-MSn. J. Am. Soc. Mass. Spectrom., 15, 660–673.
Klein, A., Carnoy, C., Wieruszeski, J.-M., Strecker, G., Strang, A.-M., van Halbeek, H., Roussel, P., and Lamblin, G. (1992) The broad diversity of neutral and sialylated oligosaccharides derived from human salivary mucins. Biochemistry, 31, 6152–6165.[CrossRef][Medline]
Moniaux, N., Escande, F., Porchet, N., Aubert, J.P., and Batra, S.K. (2001) Structural organisation and classification of the human mucin genes. Front. Biosci., 6, D1192–D1206.[ISI][Medline]
Murray, P.A., Prakobphol, A., Lee, T., Hoover, C.I., and Fisher, S.J. (1992) Adherence of oral streptococci to salivary glycoproteins. Infect. Immun., 60, 31–38.
Nielsen, P.A., Mandel, U., Therkildsen, M.H., and Clausen, H. (1996) Differential expression of human high-molecular-weight salivary mucin (MG1) and low-molecular-weight salivary mucin (MG2). J. Dent. Res., 75, 1820–1826.
Nielsen, P.A., Bennett, E.P., Wandall, H.H., Therkildsen, M.H., Hannibal, J., and Clausen, H. (1997) Identification of a major human high molecular weight salivary mucin (MG1) as tracheobronchial mucin MUC5B. Glycobiology, 7, 413–419.
van Nieuw Amerongen, A., Bolscher, J.G.M., and Veerman, E.C.I. (1995) Salivary mucins: protective functions in relation to their diversity. Glycobiology, 5, 733–740.
Nilsson, L.A., Gisslen, H., Lindholm, A., Lindholm, L., and Mobacken, H. (1987) Identification of basal cell carcinomas by means of monoclonal antibodies C50 and C241. J. Dermatol. Surg. Oncol., 1, 49–51.
Prakobphol, A., Leffler, H., and Fisher, S.J. (1993) The high-molecular-weight mucin is the primary salivary carrier of ABH, Le (a), and Le (b) blood group antigens. Crit. Rev. Oral Biol. Med., 4, 325–333.
Prakobphol, A., Thomsson, K.A., Singer, M., Hansson, G., Leffler, H., and Fisher, S. (1998) Human low-molecular-weight salivary mucin expresses the sialyl Lewisx determinant and has L-selectin ligand activity. Biochemistry, 37, 4916–4927.[CrossRef][Medline]
Ramasubbu, N., Reddy, M.S., Bergey, E.J., Haraszthy, G.G., Soni, S.D., and Levine, M.J. (1991) Large-scale purification and characterization of the major phosphoproteins and mucins of human submandibular-sublingual saliva. Biochem. J., 280, 341–352.
Schulz, B.L., Packer, N.H., and Karlsson, N.G. (2002a) Small-scale analysis of O-linked oligosaccharides from glycoproteins and mucins separated by gel electrophoresis. Anal. Chem., 74, 6088–6097.[Medline]
Schulz, B.L., Oxley, D., Packer, N.H., and Karlsson, N.G. (2002b) Identification of two highly sialylated human tear-fluid DMBT1 isoforms: the major high-molecular-mass glycoproteins in human tears. Biochem. J., 366, 511–520.[CrossRef][ISI][Medline]
Schulz, B.L., Sloane, A.J., Robinson, L.J., Sebastian, L.T., Glanville, A.R., Song, Y., Verkman, A.S., Harry, J.L., Packer, N.H., and Karlsson, N.G. (2004) Mucin glycosylation changes in cystic fibrosis lung disease are not manifest in submucosal gland secretions. Biochem. J., in press.
Thomsson, K.A., Prakobphol, A., Leffler, H., Reddy, M.S., Levine, M.J., Fisher, S.J., and Hansson, G. (2002) The salivary mucin MG1 (MUC5B) carries a repertoire of unique oligosaccharides that is large and diverse. Glycobiology, 12, 1–14.
Thornton, D.J., Khan, N., Mehrotra, R., Howard, M., Veerman, E., Packer, N.H., and Sheehan, J.K. (1999) Salivary mucin MG1 is comprised almost entirely of different glycosylated forms of the MUC5B gene product. Glycobiology, 9, 293–302.
Veerman, E.C.I., van den Keybus, P.A.M., Valentijn Benz, M., and van Nieuw Amerongen, A. (1992) Isolation of different high-Mr mucin species from human whole saliva. Biochem. J., 283, 807–811.
Veerman, E.C., Bolscher, J.G., Appelmelk, B.J., Bloemena, E., van den Berg, T.K., and van Nieuw Amerongen, A. (1997) A monoclonal antibody directed against high M (r) salivary mucins recognizes the SO3–3Gal beta, 1–3GlcNAc moiety of sulfo-Lewis (a): a histochemical survey of human and rat tissue. Glycobiology, 7, 37–43.
Wickström, C., Davies, J.R., Eriksen, G.V., Veerman, E.C.I., and Carlstedt, I. (1998) MUC5B is a major, gel-forming, oligomeric mucin from human salivary gland, respiratory tract and endocervix: identification of glycoforms and C-terminal cleavage. Biochem. J., 334, 685–693.
Zalewska, A., Zwierz, K., Zolkowski, K., and Gindzienski, A. (2000) Structure and biosynthesis of human salivary mucins. Acta Biochim. Pol., 47, 1067–1079.[ISI][Medline]
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