The salivary mucin MG1 (MUC5B) carries a repertoire of unique oligosaccharides that is large and diverse

Kristina A. Thomsson2, Akraporn Prakobphol3, Hakon Leffler4, Molakala S. Reddy5, Michael J. Levine5, Susan J. Fisher3,6 and Gunnar C. Hansson1,2

2Department of Medical Biochemistry, Göteborg University, Medicinaregatan 9A, 413 90 Gothenburg, Sweden; 3Department of Stomatology, University of California San Francisco, San Francisco, CA 94143, USA; 4Department of Molecular Medicine, University of Lund, Lund, Sweden; and 5Department of Oral Biology, State University of New York at Buffalo, Buffalo, NY 14214, USA; and 6Departments of Anatomy, Pharmaceutical Chemistry, and Obstetrics, Gynecology and Reproductive Sciences, University of California San Francisco, San Francisco, CA 94143, USA

Received on March 8, 2001; revised on August 16, 2001; accepted on August 22, 2001.


    Abstract
 Top
 Abstract
 Introduction
 Results
 Discussion
 Material and methods
 Acknowledgments
 Abbreviations
 References
 
The high-molecular-mass salivary mucin MG1, one of two major mucins produced by human salivary glands, plays an important role in oral health by coating the tooth surface and by acting as a bacterial receptor. Here this mucin was purified from the submandibular/sublingual saliva of a blood group O individual. The presence of MUC5B as the major mucin in this preparation was confirmed by amino acid analysis and its reactivity with the monoclonal antibody PAN H2. To structurally characterize MG1 carbohydrates the O-glycans were released by reductive ß-elimination. Nuclear magnetic resonance spectroscopy of the nonfractionated mixture showed that (1) fucose was present in blood group H, Lea, Lex, Leb, and Ley epitopes; (2) NeuAc was mainly linked {alpha}2-3 to Gal or {alpha}2-6 to GalNAcol; and (3) the major internal structures were core 1 and core 2 sequences. After this preliminary analysis the released oligosaccharides were separated into neutral (56%), sialylated (26%), and sulfated (19%) fractions, with an average length of 13, 17, and 41 sugar residues, respectively. Gas chromatography–mass spectrometry and matrix-assisted laser desorption/ionization time-of-flight mass spectrometry of mixtures of neutral and sialylated oligosaccharides revealed at least 62 neutral and 25 sialylated oligosaccharides consisting of up to 20 monosaccharide residues. These results showed that the MG1-derived oligosaccharides were much longer than those of MG2, and only a few species were found on both molecules. Thus, these two mucins create an enormous repertoire of potential binding sites for microorganisms at one of the major portals where infectious organisms enter the body.

Key words: mass spectrometry/mucin/oligosaccharide/proton NMR spectroscopy/salivary gland


    Introduction
 Top
 Abstract
 Introduction
 Results
 Discussion
 Material and methods
 Acknowledgments
 Abbreviations
 References
 
Mucins are major glycoprotein components of mucus, the viscous gel that covers the epithelial layers of the body and protects the underlying epithelia. These molecules are highly glycosylated; the oligosaccharides (50–80% of the weight) are linked to serine and threonine residues clustered in mucin domains on the protein backbone. Currently, twelve mucin protein cores have been identified (Perez-Vilar and Hill, 1999Go). There are regional differences in expression, as particular mucin subsets variably contribute to the mucus coat that covers the specialized epithelial layers of the body. The presence of cell type– and tissue-specific glycoforms suggests distinct functions of mucin O-glycans, such as variations in viscosity and adhesivity, including bacterial receptor activity.

Two major, physically distinct mucin populations have been identified in saliva—the high-molecular-mass (MG1) and the low-molecular-mass (MG2) mucins. The MG1 mucin core, a large oligomeric protein composed of disulfide-linked subunits, is encoded by the MUC5B gene (Nielsen et al., 1997Go; Wickström et al., 1998Go; Desseyn et al., 1997aGo, 1998). MUC5B is produced by all salivary glands except the parotid (Veerman et al., 1996Go; Nielsen et al., 1996Go). The existence of different glycoforms has been demonstrated by the isolation of differently charged subpopulations (Wickström et al., 1998Go; Bolscher et al., 1995Go; Thornton et al., 1999Go). MUC5B has also been identified as one of the two major mucin components of the mucus layer in the respiratory tract and endocervix (Wickström et al., 1998Go; Thornton et al., 1997Go). It is interesting to note that viruses (e.g., HIV-1) and bacteria (e.g., Helicobacter pylori) can bind to MG1, suggesting that this mucin plays a role in establishment of the oral ecology (Bergey et al., 1994Go; Veerman et al., 1997Go).

MG2, a small, nonmultimerizing protein that is exclusively found in salivary secretions, is encoded by the MUC7 gene (Bobek et al., 1993Go). This mucin is produced by the submandibular (SM), sublingual (SL), and palatine glands (Bolscher et al., 1999Go). Two glycoforms with different levels of fucose and sialic acid have been distinguished (Ramasubbu et al., 1991Go). MG2 interacts with a large number of oral microorganisms (Nieuw Amerongen et al., 1995Go) and can mediate leukocyte rolling and tethering (Prakobphol et al., 1999Go). Together, MG1 and MG2 are the major glycosylated components of the acquired pellicle which coats and protects the tooth surface (Fisher et al., 1987Go).

Previous descriptions of glycans expressed on MG1 and MG2 suggested that MG1-derived oligosaccharides are more heterogeneous than those of MG2 (Reddy et al., 1985Go; Levine et al., 1987Go). In a recent study using nuclear magnetic resonance (NMR) and mass spectrometry (MS) techniques, we demonstrated that glycans isolated from highly purified MG2 carry diverse structures that are largely variations of the T antigen and Lex motifs (Prakobphol et al., 1998Go). In the study presented here, a similar strategy was applied to elucidate the structure of glycans derived from highly purified MG1. The results showed a remarkable degree of heterogeneity, even greater than that observed among the MG2 oligosaccharides. A comparison of the MG1 and MG2 repertoire, with respect to size, diversity, and expression (semiquantitative) of glycans and terminal epitopes, showed a high level of mucin-specific glycosylation that could have important implications for the specialized biological functions these molecules play in the oral cavity.


    Results
 Top
 Abstract
 Introduction
 Results
 Discussion
 Material and methods
 Acknowledgments
 Abbreviations
 References
 
Characterization of the MG1 preparation
Purified salivary MG1 isolated from the SM/SL saliva of a blood group O individual was analyzed for carbohydrate and amino acid composition. The results are shown in Tables I and II. The mucin was composed of 19% protein and 81% carbohydrate, with high amounts of Ser, Thr, and Pro (44 weight %). With regard to carbohydrate composition, NeuAc, Fuc, Gal, GlcNAc, and GalNAc were present in the following molar ratios: 1:5:4:3:1 (GalNAc set to 1), respectively. As expected, only trace amounts of mannose were detected. The amino acid composition was almost identical to the theoretical estimate based on translating the MUC5B nucleic acid sequence (Desseyn et al., 1997aGo,b, 1998). These data suggested that MUC5B was the major component. The binding of monoclonal antibody PAN H2 (Nielsen et al., 1997Go) to partially deglycosylated MG1 confirmed the identity of the purified mucin as MUC5B (data not shown).


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Table I. Chemical composition of MG1
 

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Table II. Amino acid analysis
 
Experimental strategy
The MG1 glycans were released by treatment with alkaline borohydride. During this procedure the GalNAc attached to Ser/Thr of the peptide core was converted to GalNAcol. The percent of saccharides that were recovered after release from the protein backbone was estimated based on monosaccharide compositional analyses of the whole mucin and of the neutral, sialylated, and sulfated fractions. The yield was approximately 50%, typical for this procedure. After the desalting step, the MG1 oligosaccharide mixture was first analyzed by NMR spectroscopy. Then the oligosaccharides were fractionated into pools of neutral, sialylated, and sulfated species, from which aliquots were withdrawn for monosaccharide compositional analyses. Neutral and sialylated oligosaccharides were permethylated and analyzed by gas chromatography (GC), matrix-assisted laser desorption/ionization time-of-flight (MALDI-TOF) MS, and GC-MS. Finally, the sulfated oligosaccharides were subjected to gel chromatography as an additional method for estimating their molecular weight.

NMR spectroscopy
Proton NMR spectra of a large number of O-linked glycans have been compiled (Kamerling and Vliegenthart, 1992Go) and gathered in a database (Sugabase) of the proton chemical shifts. Typically, these chemical shifts are very sensitive to the presence of the immediate saccharide neighbors and relatively less sensitive to more distant residues. In the current study, we have used 1D and 2D double quantum-filtered correlated spectroscopy (DQF-COSY) and total correlated spectroscopy (TOCSY) proton NMR spectroscopy to analyze the unfractionated mixture of O-linked glycans released from MG1. The 1D NMR spectra provided the highest sensitivity, but in some areas crowding precluded positive identification of important signals. DQF-COSY, which is less sensitive, resolved these signals; groups of cross-peaks with characteristic fine structures permitted identification of the same type of structural reporter group within multiple environments.

The results showed that the MG1 NMR spectra (Figures 1 and 2) were far more complex than those obtained from analysis of the MG2 oligosaccharides (Prakobphol et al., 1998Go). Despite this complexity, different classes of NMR signals (Table III) could be readily distinguished and interpreted in light of previously published spectra of purified reference compounds (see Materials and methods). In agreement with the composition analysis (Table I), {alpha}-linked fucose residues were prominent components of the MG1 glycans. They were identified by downfield shifted H1 signals (> 4.9 ppm) and H2 signals at 3.8 ppm. The Fuc residues were also identified by their H5 and H6 (CH3) signals (Figure 1A) and their H5/H6 cross-peaks clearly visible in the 2D spectra (Figure 2A and Table III). H1/H2 cross-peaks from ß-linked Gal confirmed the presence of several fucosylated determinants (Figures 1, 2A, 2B and Table III). Other possible {alpha}-linked monosaccharides (Gal, GalNAc, and GlcNAc) were not detected. Thus, Ley [(Fuc{alpha}1->2)Galß1->4(Fuc{alpha}1->3)GlcNAc)] was the major fucosylated determinant on MG1. The Leb determinant [(Fuc{alpha}1->2)Galß1->3 (Fuc{alpha}1->4)GlcNAc)], and all possible combinations of a single Fuc linked to either C-2 of Gal (H type 1 and 2; Fuc{alpha}1->2Galß1-> 3/4GlcNAc), C-3 of GlcNAc [Lex; Galß1->4(Fuc{alpha}1->3)GlcNAc], or C-4 of GlcNAc [Lea; Galß1->3(Fuc{alpha}1->4) GlcNAc], were also found.



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Fig. 1. 1D NMR spectrum of the total mixture of MG1 oligosaccharides recorded at 25°C. O-linked oligosaccharides were released from purified MG1 by reductive ß-elimination in 0.05 M NaOH/1 M NaBH4, desalted on Dowex 50WX8, and transferred to D20 (see Materials and methods). (A) The entire 1D spectrum; assignments of the major resolved signals are indicated. (B) Expansion of the Fuc H1 region with annotations for type 1 chain (Galß1->3GlcNAc)–based structures (top) and type 2 chain (Galß1->4GlcNAc)–based structures (bottom).

 


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Fig. 2. 2D DQF-COSY and TOCSY NMR spectra of the total mixture of MG1 oligosaccharides. (A) The entire 2D DQF-COSY spectrum; assignments of the major resolved signals are indicated. (B) A region of DQF-COSY spectrum showing cross-peaks between Fuc H1 and H2; annotations for type 1 chain (Galß1->3GlcNAc)–based structures and type 2 chain (Galß1->4GlcNAc)–based structures are labeled. (C) A region of the DQF-COSY spectrum showing cross-peaks between NeuAc H3ax and H3eq. (D) A region of the TOSCY spectrum showing cross-peaks between Gal H1 and H3 or H4.

 


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Table III. Assignments of signals (ppm) in 3H-NMR analysis of alditol mixture released from MG1a

 
NeuAc residues were less abundant than Fuc, but easily identified in the NMR spectra by their H3ax and H3eq signals. About two-thirds were found in NeuAc{alpha}2->3Gal (H3ax/H3eq at about 1.8/2.76 ppm) and one-third in NeuAc{alpha}2->6GalNAcol (H3ax/H3eq at about 1.7/2.73 ppm), with small amounts in NeuAc{alpha}2->6Gal (H3ax/H3eq at about 1.72/2.67 ppm) (Figures 1A, 2A, 2C and Table III). The cross-peaks from Gal H1/H3 and Gal H1/H4 (Figure 2D) were observed in the TOCSY spectrum in an area that did not contain other major peaks. When substituted with NeuAc at C-3, the Gal H3 signals shifted downfield to > 4.11 ppm. Three partially resolved groups of signals could be assigned to Gal H1/H3 in the TOCSY spectrum and hence gave positive confirmation of the presence of NeuAc{alpha}2->3Gal. All had typical H1 signals at about 4.52–4.54 ppm. For the top cluster, H3 was at 4.08 ppm, indicative of Gal in the sLex determinant [NeuAc{alpha}2->3Galß1->4(Fuc{alpha}1->3)GlcNAc] and for Gal in NeuAc{alpha}2->3Gal ß1->3GlcNAc. An H1/H2 cross-peak from Gal in sLex at 4.52/3.52 was also positively identified. The other two signals with H3 around 4.11 and 4.12 ppm are typical of the NeuAc{alpha}2->3Galß1->3GalNAcol and NeuAc{alpha}2->3Galß1->4GlcNAc structures, respectively. In addition, evidence for NeuAc{alpha}2->3, as in blood group Sda/Cad determinants, was detected by a weak NeuAc H3ax/H3eq cross-peak in the COSY spectrum at 2.68/1.87 ppm and an H1/H3 Gal cross-peak in the TOCSY spectrum at about 4.53/4.15 ppm (Figures 2C and D, respectively). Because H3 of Gal in sLea, usually found at about 4.04 ppm, was missing, this epitope was either absent or scarce in the MG1 sample.

The cross-peak in the TOCSY spectrum at 4.45/4.10 is typical of the H1/H4 signals of branched Gal residues, substituted with GlcNAc at both C-3 and C-6 (Figure 2D). The same signal from Gal substituted with GlcNAc only at C-3, which is found above 4.12 ppm (often at about 4.15), was missing. Hence, the Gal-GlcNAc backbones of the MG1 glycans were predominantly extended by branching, although smaller amounts of linear extension may contribute to the bottom part of the cross-peak.

The region of the 1D NMR spectra between 4 and 4.8 ppm was very complex and could not be fully interpreted (Figure 1A). Nevertheless, a great deal of information about core structure was deduced. Among others were signals from H1 of ß-Gal and ß-GlcNAc, as well as various signals from GalNAcol. A strong cluster of signals from H2 of GalNAcol was identified at about 4.39 ppm based on the position and fine structure of the cross-peak with the two H1 signals. This chemical shift is typical of core structures containing Galß1->3GalNAcol (core 1 and 2). Because the H2 GalNAcol signal from core 1 and 2 was clearly visible in the 1D spectrum and no other signal was found in the 4.40 ppm region, we could use these as a reference to compare the amount of GalNAcol with other saccharides. The relative size of the NMR signals agreed with the molar ratios of constituent saccharides estimated in the composition analysis (Table I). The H1/H2 and H3/H4 cross-peaks of GlcNAcß1->3GalNAcol (as in core 3– and 4–containing saccharides) were weak (Table III) and overlapped with other signals. Cross-peaks from H5 and H6, although typically weaker, permitted identification of both ß-GlcNAc and NeuAc at C-6 of GalNAcol and unsubstituted C-6 of GalNAcol (Table III). GalNAcol saccharides, free or substituted at C-6 with NeuAc, were identified by their H3/H4 cross-peak (Table III).

Monosaccharide composition of neutral, sialylated, and sulfated MG1-derived glycans
The released oligosaccharides where then separated into neutral, sialylated, and sulfated fractions and analyzed for monosaccharide composition (Table IV). For comparison purposes, the table also includes the composition of the corresponding glycan fractions purified from MG2, information that we previously published (Prakobphol et al., 1998Go). The molar ratios of the monosaccharides were calculated relative to GalNAcol, the reduced product of the GalNAc that had been linked to the mucin protein core during the release reaction. The molar distribution of neutral, sialylated, and sulfated MG1 oligosaccharides was 56, 26, and 19 mole%, respectively. MG2 contained a similar percentage of neutral species (49 mole%), but a much higher percentage of sialylated oligosaccharides (40 mole%) and a lower percentage of sulfated species (11 mole%). In this way calculated average MG1 oligosaccharide chain length was substantially longer than that of MG2: 13 versus 4 in the neutral fraction, 17 versus 10 in the sialylated fraction, and, most strikingly, 41 versus 7 in the sulfated group. These oligosaccharide lengths are longer than when estimated by MS (see below). This discrepancy is always found and largely due to MS favoring low masses, but it can also be attributed to a possible underestimate of the GalNAcol during monosaccharide analyses. However, the relative differences between MG1 and MG2 are valid, as are the general trends in the length of the oligosaccharides.


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Table IV. Monosaccharide composition analysis of neutral, sialylated, and sulfated oligosaccharides from MG1 and MG2
 
MS analyses of the neutral and sialylated fractions
The neutral and sialylated oligosaccharide fractions were permethylated and analyzed by MALDI-TOF MS (Figure 3). Fifty-five molecular ions of neutral oligosaccharides of <= 20 monosaccharide residues were detected. The deduced monosaccharide composition of the individual components showed the composition seen for polylactosamine structures, approximately equal amounts of Gal and GlcNAc (Table V). The composition of 15 of the annotated ions had masses suggesting two more Hex than HexNAc residues. This is unusual, and no structures or epitopes supporting this unusual composition were detected by GC-MS or NMR. However, further exploration of the nature of the ions at m/z 1171.6 (labeled N5.3) and m/z 1345.6 (N6.3) from both MG1 and MG2 by nano-electrospray ionization tandem mass spectrometry (ESI-MS/MS) generated product ions corresponding to the saccharide structures given (data not shown). No definitive structures could be predicted, but were suggestive of -Hex-HexNAcol-Hex core structures.



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Fig. 3. MALDI-TOF MS spectra of permethylated neutral and sialylated oligosaccharides from MG1. Molecular ions occur as lithium adducts [M+Li]+. Contaminants are marked with an asterisk. The deduced oligosaccharide compositions are listed in Table V. Only masses above m/z 1000 are shown.

 

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Table V. Permethylated neutral oligosaccharides from MG1 and MG2 detected by MALDI-TOF MS
 
In the sialylated oligosaccharide fraction, 24 molecular ions consisting of up to 19 residues were detected (Table VI). Only five of the sialylated oligosaccharides contained a second NeuAc, and most compounds were also fucosylated.


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Table VI. Permethylated sialic acid–containing oligosaccharides from MG1 analyzed by MALDI-TOF MS and GC-MS
 
Permethylated neutral and sialylated oligosaccharides were fractionated by gel filtration, the smaller oligosaccharides were collected and analyzed by GC and GC-MS (Figure 4). Sixteen neutral and eight sialylated compounds were possible to characterize by GC-MS (Tables VII and VIII). The interpretation was based on the mass spectra obtained from each peak, where most fragment ions detected were oxonium and inductive ions (Prakobphol et al., 1998Go; Carlstedt et al., 1993Go; Karlsson et al., 1989Go). The majority of the oligosaccharides had core 1 (Hex1->3HexNAc) or core 2 [Hex1->3(HexNAc1->6)HexNAc] type sequences. The neutral oligosaccharides were nearly all fucosylated, with Fuc linked to Gal, or to GlcNAc in H, Leb and/or Ley-like epitopes. NeuAc was found linked either to Gal residues or to C-6 of GalNAcol.



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Fig. 4. High-temperature GC of permethylated neutral and sialylated oligosaccharides. The peaks were labeled with numbers and letters. These labels were then used to construct the deduced oligosaccharide sequences that are listed in Tables VII and VIII.

 

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Table VII. Comparison of neutral oligosaccharides from MG1 and MG2 analyzed with GC-MS
 

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Table VIII. Comparison of sialylated oligosaccharides from MG1 and MG2 analyzed with GC-MS
 
Considered together, the results from MS and NMR were consistent and indicated that the structures of the MG1 oligosaccharides were much more diverse than those of the MG2 (Prakobphol et al., 1998Go). Core 1 and 2 structures were shown to be prominent by both MS and NMR. Three core 3 sequences, N3.3, N4.3, and N5.4 (Table VII, Figure 3) were detected as minor components by GC-MS as well as in the NMR spectra by weak signals. Small amounts of core 4 sequences were only detected by NMR. The Gal-GlcNAc backbone was predominantly extended by branching. The neutral oligosaccharides were complex and highly fucosylated. The sialylated compounds were less complex and contained both the NeuAc{alpha}2->3Gal and the NeuAc{alpha}2->6GalNAcol sequences. In contrast to MG2, which carried fewer fucosylated determinants, MG1 carried several fucosylated determinants, including H type 1 and 2 (Fuc{alpha}1->2Galß1->3/4GlcNAc), Lea [Galß1->3(Fuc{alpha}1->4)GlcNAc], Leb [(Fuc{alpha}1->2)Galß1 ->3(Fuc{alpha}1->4)GlcNAc)], Lex [Galß1->4(Fuc{alpha}1->3) GlcNAc], sLex [NeuAc{alpha}2->3Galß1->4 (Fuc{alpha}1->3)GlcNAc], and Ley [(Fuc{alpha}1->2)Galß1->4 (Fuc{alpha}1->3)GlcNAc)]. Finally, free GalNAcol or GalNAcol carrying a single NeuAc substitution at C-6 were detected by both GC/MS, NMR spectroscopy.

Size fractionation of sulfated MG1-derived glycans
Detailed structural analysis of the sulfated oligosaccharides, the least abundant fraction with the most complex oligosaccharides, awaits purification of larger amounts of MG1. Here we can only report the results of preliminary analyses (data not shown). We estimated the sizes of the oligosaccharide by gel filtration on two serially coupled Superdex Peptide columns that have a fractionation range of 100–9000 Da for peptides. Aliquots of individual fractions were analyzed for monosaccharide composition. The oligosaccharides eluted as a single broad peak between 15 and 22 ml, that is, close to the void volume (15.5 ml). For comparison and calibration purposes, oligosaccharides released from porcine small intestinal mucins, with an average chain length of five residues, eluted between 25 and 35 ml; salt eluted at 38 ml. These results suggested that the average oligosaccharide is very large and might have a mass of up to 8 kDa. This could suggest a chain length of about 40 sugar residues, lengths also suggested from the results of the monosaccharide compositional analysis (Table IV).


    Discussion
 Top
 Abstract
 Introduction
 Results
 Discussion
 Material and methods
 Acknowledgments
 Abbreviations
 References
 
Molecular cloning techniques have recently revealed information about the MG1 protein core (apomucin) (Desseyn et al., 1997aGo, 1998). The gene contains 48 exons, encodes ~ 6000 amino acids, and produces a polypeptide of ~ Mr 600,000. The MUC5B central exon, the largest ever reported for a vertebrate gene, encodes four super-repeats of 528 amino acids. In turn, each super-repeat contains a region of imperfectly conserved 29-residue tandem repeats, a conserved region without repeats, and a Cys-rich subdomain.

Very little is known about MG1 glycosylation, which accounts for approximately 80% of the mucin’s molecular mass (Levine et al., 1987Go; Loomis et al., 1987Go). To our knowledge this is the first report on the structure of the O-glycans of purified MG1. The results from MALDI-TOF MS, GC-MS, and NMR spectroscopy showed the presence of highly fucosylated oligosaccharides. Fuc was present in a large variety of terminal linkages that included blood group H epitopes (types 1 and 2), as well as Lea, Leb, Lex, and Ley determinants. A portion of these results is in agreement with the data of Klein et al. (1992)Go. These investigators characterized, by using NMR, the high-molecular-weight oligosaccharides that were present in a pronase digest of a pooled sample of whole saliva obtained from 20 blood group O donors. The Fuc-containing epitopes we detected in association with purified MG1 were among those previously reported, an expected result because MG1 was undoubtedly a component of the pooled sample. However, we also noted significant differences, a result we also expected because, in addition to MG1, saliva contains many other highly glycosylated components. For example, we primarily found core 1 and 2 structures, wherease Klein et al. (1992)Go report a substantial number of cores types 3 and 4.

One of our more interesting observations were that the sulfated fraction contained oligosaccharides of an extraordinary length—the average size was approximately 40 monosaccharide residues, accounting for 19 mole% of the glycans expressed on the protein. Because there is very little information about such structures, it is difficult to know whether they are unique to MG1 or found on other mucin cores. Our groups have not detected these unusual structures on any other highly glycosylated salivary components (Prakobphol et al., 1998Go; Gillece-Castro et al., 1991Go). However, long sulfated oligosaccharides, with up to 200 residues, have been suggested on mucins isolated from the sputum of a cystic fibrosis patient (Sangadala et al., 1993Go).

Previously we characterized the glycans of MG2, the other major mucin found in human saliva (Prakobphol et al., 1998Go). The experimental strategy included all the basic elements that were applied in the present study. Briefly, we purified (to homogeneity) MG2 from the SM/SL saliva of a single individual. The O-glycans were chemically released and their structures determined by using a combination of NMR and MS techniques. Accordingly, we were able to compare the O-glycosylation patterns of the two mucins. Here we report major differences in the glycosylation of these two mucins. These differences included the much longer chain length of the MG1 sulfated species. The chain length of the neutral and sialylated glycans was also longer, evident from both the monosaccharide composition analyses (Table IV) and the spectra of the oligosaccharides obtained by MALDI-TOF MS (Figure 3). These subsets of MG1 oligosaccharides carried up to 20 residues, whereas components of the neutral and sialylated MG2 fractions had a maximum chain length of 12 monosaccharides.

The differences in glycosylation between the two salivary mucins affected not only length but also terminal epitopes. For example, MG1 carried a wide variety of the fucosylated terminal epitopes, which included blood group H, Lea, Lea, Leb, Lex, and Ley, whereas MG2 carried fewer fucosylated epitopes. Finally, our previous study indicated that MG1, but not MG2, carried the ABO blood group structures (Prakobphol et al., 1993Go). Since, the MG1 sample that we analyzed in this study was obtained from an individual with blood type O, we were unable to determine which species carried these additional modifications.

With regard to oligosaccharides sequences GC-MS showed that MG1 and MG2 shared only 6 out of 22 neutral species (Table VII) and 4 out of 13 sialylated oligosaccharides (Table VIII). The data from our study also indicated that MG1 carries a much more diverse repertoire of oligosaccharide structures than MG2. MALDI-TOF MS revealed 79 molecular ions, as compared to 38 for MG2. If the GC-MS results are added, then MG1 carries 87 different glycan structures versus the 43 isolated from MG2. These numbers are underestimates because MALDI-TOF MS only shows molecular ions and does not distinguish between isomeric oligosaccharides. This is important because the complexity and number of potential isomers grows quickly as a function of the number of sugar residues. For example, Table VII shows six different structures with identical masses that are built from the same five sugar residues. Taking into account the number of isomers and the large sulfated oligosaccharides that have not been characterized in detail, it is likely that the MG1 mucin carries several hundred different oligosaccharide structures.

The functions of salivary mucins have been reviewed (Tabak, 1995Go). In addition to well-recognized roles in alimentation, they form the pellicle that coats the resident hard (Fisher et al., 1987Go; Al-Hashimi and Levine, 1989Go) and soft (Slomiany et al., 1989Go) oral tissues. Here, mucins interact with cells that traffic into and out of the oral cavity. It has been appreciated for some time that microorganisms attach to host glycans (Karlsson, 1989Go). Together, the results of our recent work show a high degree of diversity in expression of glycans by MG1, few of which are shared with MG2. The end result is an enormous repertoire of potential binding sites for microorganisms in the oral cavity. A few of these interactions have been described. For example, the observation that MG1 interacts with the gastrointestinal pathogen H. pylori (Veerman et al., 1997Go) is consistent, first, with reports that this organism adheres via saccharides with Leb epitopes (Boren et al., 1993Go), and second, with the evidence presented here that MG1 carries these substituents. With regard to oral bacterial species, Gibbons and Qureshi (1978)Go found that several strains of Streptococcus mutans bound to a blood group-reactive mucin in whole saliva, presumably MG1. Finally, there are also reports that Haemophilus influenza strains bind to MG1 (Veerman et al., 1995Go).

Our previous work also showed that the salivary mucins (e.g., MG1 and MG2) are L-selectin ligands (Prakobphol et al., 1998Go), a possibility that was suggested by our detection of Lex and sLex epitopes among the low-molecular-weight salivary mucin carbohydrate structures. Subsequently, we proved the functional significance of this interaction, as additional experiments showed that MG2 can mediate leukocyte rolling and tethering in a parallel plate flow chamber that assays adhesion as a function of shear stress (Prakobphol et al., 1999Go). Because MG1 oligosaccharides also carry Lex epitopes, as well as complicated sulfate-containing glycans, it is possible that the high-molecular-mass salivary mucin, like MG2, can mediate leukocyte adhesion under flow. Although the significance of the leukocyte interactions with mucins is not yet known, it is interesting to note that bacteria can adhere via sugar sequences other than Lex and sLex, suggesting that mucins may coordinate adhesion of these different cell types, and thereby play a role in the immunological processes modulating the oral ecology (Prakobphol et al., 1999Go).

Finally, information about salivary mucin structure will allow us to determine whether alterations in the oligosaccharide repertoire are associated with particular disease states. Although the absence of saliva has devastating consequences for oral health, little is known about the role of individual salivary components, including the mucins. Content and structure would appear to be particularly relevant. Mucin content of human saliva obtained from older patients (65–85 years) is significantly lower than that of younger individuals (18–35 years; Denny et al., 1991Go), raising the interesting possibility that when levels of these molecules fall below a critical threshold, certain age-related pathologies may ensue. With regard to structure, several variations are possible. A preliminary report suggests that MUC5B, the gene that encodes MG1, may show little polymorphism (Debailleul et al., 1998Go). However, glycoforms of this mucin have been identified (Prakobphol et al., 1993Go; Bolscher et al., 1995Go; Thornton et al., 1997Go), suggesting that glycosylation may be an important variable. The results reported here give us the necessary background information to begin studies if individuals who express certain oligosaccharide epitopes on specific mucins are predisposed to either oral health or disease.


    Material and methods
 Top
 Abstract
 Introduction
 Results
 Discussion
 Material and methods
 Acknowledgments
 Abbreviations
 References
 
Isolation of MG1
Human MG1 was purified from SM/SL saliva, collected as the ductal secretion, from a single donor with blood type O, using the method described by Ramasubbu et al. (1991)Go. The purified mucin had the expected electrophoretic characteristics, that is, appeared as a heterodisperse band with an Mr > 1,000,000.

Immunostaining of MG1 with monoclonal antibody PAN H2
Serial dilutions of purified MG1 (0.16 mg/300 µl) and, as a negative control, guanidinium chloride insoluble porcine small intestine mucin (solubilized by reduction), were immobilized on polyvinylidene difluoride membrane strips. The samples were partially deglycosylated by treatment with a mixture of trifluoromethanesulfonic acid (5 ml) and toluene (300 µl) for 5 h at –20°C, followed by rinsing with 25 mM Tris–HCl, pH 8, and distilled water. Then the membranes were incubated with 2% bovine serum albumin (BSA) overnight at room temperature before exposure to the monoclonal antibody PAN H2 (Nielsen et al., 1997Go) diluted (1:100 v/v) in 2% BSA/phosphate buffered saline for 2 h at room temperature. After washing, the bound IgG was detected by incubation with goat anti-mouse IgG conjugated to alkaline phosphatase (Dako, Denmark) and visualized by developing with nitro-blue tetrazolium and bromo-chloro-indolyl-phosphate (p-toluidine salt).

Amino acid analysis
Amino acid analysis was performed as described elsewhere (Spackman et al., 1958Go) on an Alpha Plus amino acid analyzer (Pharmacia Biotech, Uppsala, Sweden).

Release of oligosaccharides
The O-linked oligosaccharides were released from MG1 (4.5 mg) by reductive ß-elimination in 0.05 M potassium hydroxide and 1 M sodium borohydride (Carlstedt et al., 1993Go). The total mixture of saccharides was desalted on Dowex 50WX8, followed by co-distillation of borate methyl ester with MeOH/1% acetic acid to remove borate.

Proton NMR spectroscopy
The released and desalted oligosaccharide mixture was analyzed by 1D and 2D DQF-COSY and TOCSY proton NMR. Deuterium exchange was performed twice in 0.5 ml 99.95% D2O (30 min) followed by 0.5 ml 99.98% D2O (30 min). The sample was lyophilized between each exchange. The oligosaccharides were transferred to a 5-mm NMR tube and analyzed with a Varian Innova 600 MHz instrument [5 mm 1H (13C,X) triple resonance pulsed field gradient probe] at 25°C, using acetone as an internal standard. The NMR spectra were interpreted by comparison with spectral data published for a large number of O-linked glycans and other relevant saccharides. Overviews are given by Kamerling and Vliegenthart (1992)Go and in Sugabase, a database available on the Web (www.boc.chem.ruu.nl/sugabase/sugabase.html).

Fractionation of oligosaccharides
After NMR analyses, the oligosaccharides were separated into neutral, sialic acid–containing, and sulfate-containing fractions (Karlsson et al., 1995Go). Briefly, the released and desalted oligosaccharides were applied to an anion exchange column, and the neutral oligosaccharides were eluted with MeOH. After an on-column derivatization of the carboxyl groups to methyl esters, the sialylated species were eluted with MeOH and the sulfated species were eluted with pyridinium acetate. The methyl esters of the sialylated oligosaccharides were converted to methyl amides (Karlsson et al., 1995Go).

Monosaccharide composition analyses
Monosaccharides were analyzed as described (Karlsson and Hansson, 1995Go), by using a Dionex GP40 pump with an ED40 pulsed amperometric detector (Dionex, Sunnyvale, CA). NeuAc was quantified after reversion of the methyl esterified residues to carboxyl groups by saponification in 0.1 M sodium hydroxide (50°C, 2 h). After neutralization, the samples were hydrolyzed in 100 µl of 0.1 M HCl at 80°C or 1 h and analyzed by high pH anion exchange chromatography, using pulsed amperometric detection with muraminic acid as an internal standard. The column (CarboPac PA1, 4 x 250 mm; Dionex) with a guard column (CarboPac PA1, 4 x 50 mm) was eluted isocratically at 1 ml/min with 0.1 M sodium acetate and 0.05 M sodium hydroxide (from a 50% solution, J.T. Baker, The Netherlands).

MS
The neutral and sialylated oligosaccharides from MG1 and MG2 were permethylated and analyzed with MALDI-TOF MS as previously described (Prakobphol et al., 1998Go). Prior to analysis by high-temperature GC and GC–electron impact MS (Karlsson et al., 1995Go), the permethylated oligosaccharides of MG1 were fractionated into low- (1–7 monosaccharide residues) and high-molecular-mass species (>= 7 residues) by gel chromatography on a Sephadex LH20 column eluted with MeOH (Thomsson et al., 1998Go).

Gel chromatography of sulfated oligosaccharides
The high-performance liquid chromatographysystem consisted of a Pharmacia-LKB 2150 pump and a Pharmacia 2212 Helirac fraction collector. The sulfated oligosaccharides were dissolved in 200 µl H2O before injection. The sample was applied to two serially connected Superdex peptide columns HR 30/10 (1 cm x 30 cm; Amersham Pharmacia Biotech) and eluted with 25 mM ammonium bicarbonate at 0.2 ml/min. Fractions (0.5 ml) were collected and pooled, and their monosaccharide composition was determined.


    Acknowledgments
 Top
 Abstract
 Introduction
 Results
 Discussion
 Material and methods
 Acknowledgments
 Abbreviations
 References
 
This work was supported by the Swedish Medical Research Council (7461), the Swedish NMR center, the Glycoconjugates in Biological Systems program sponsored by the Swedish Foundation for Strategic Research, the IngaBritt and Arne Lundbergs Foundation, and the National Institutes of Health (DE07244). The mass spectrometers were obtained by grants from F.R.N. and the Knut and Alice Wallenberg Foundation. Charlotta Damberg, Tomas Larsson, and Hasse Karlsson are acknowledged for technical assistance. We also thank Nancy J. Phillips and Karoline Scheffler for helpful discussion.


    Abbreviations
 Top
 Abstract
 Introduction
 Results
 Discussion
 Material and methods
 Acknowledgments
 Abbreviations
 References
 
BSA, bovine serum albumin; DQF-COSY, double quantum-filtered correlated spectroscopy; ESI-MS/MS, nano-electrospray ionization tandem mass spectrometry; GC, gas chromatography; MALDI-TOF MS, matrix-assisted laser desorption/ionization time-of-flight mass spectrometry; NMR, nuclear magnetic resonance; MS, mass spectrometry; SM/SL, submandibular/sublingual; TOCSY, total correlated spectroscopy.


    Footnotes
 
1 To whom correspondence should be addressed Back


    References
 Top
 Abstract
 Introduction
 Results
 Discussion
 Material and methods
 Acknowledgments
 Abbreviations
 References
 
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