Glycobiology Advance Access originally published online on January 3, 2003
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Glycobiology, 2003, Vol. 13, No. 5 363-366
© 2003 Oxford University Press
Distinct localization of MUC5B glycoforms in the human salivary glands
3 Department of Dental Basic Sciences, Section of Oral Biochemistry, Academic Centre for Dentistry Amsterdam (acta), Vrije Universiteit, Van Der Boechorststraat 7, 1081 Bt Amsterdam, the Netherlands
4 Department of Pathology, VUMC, De Boelelaan 1107, 1084 HV Amsterdam, The Netherlands
Received on September 25, 2002; revised on November 19, 2002; accepted on December 4, 2002
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Abstract |
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Salivary mucins, encoded by the MUC5B gene, make up a heterogeneous family of molecules, which are secreted by several glands, including the submandibular, sublingual, and palatine glands. Previous studies have shown that heterogeneity in the salivary mucin population is related to its multiglandular origin. In the present study we address the question to what extent the mucin (MUC5B) population from a single human salivary gland is made up of different glycoforms. Using monoclonal antibodies to defined protein and sulfated carbohydrate epitopes specific to MUC5B, we conduct an immunohistochemical study of different salivary gland types, including submandibular, sublingual, and labial glands. In all tissues studied we found a mosaic expression pattern of sulfo-Lewis a antigen, recognized by mAb F2, which in salivary glands is exclusively present on MUC5B. On the other hand, mucous acini were uniformly labeled by mAb EU-MUC5Bb, evoked against a peptide-stretch of the tandem repeat region of MUC5B. Double staining with both antibodies confirmed the presence of MUC5B-positive/sulfo-Lewis a-positive cells, as well as MUC5B-positive/sulfo-Lewis a-negative cells within one glandular unit. These results indicate that one and the same salivary gland synthesizes different MUC5B glycoforms.
Key words: glycoforms / immunohistology / MUC5B / salivary glands / sulfo-Lewis a
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Introduction |
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Saliva is a protective secretion produced largely by three major exocrine glands—the parotid, submandibular, and sublingual—and additionally by several minor glands in the mouth. An important role in the protection of the oral tissues is played by salivary components, including secretory IgA, lactoferrin, lysozyme, proline-rich proteins, histatins, and mucins (Iontcheva et al., 1997
). These latter macromolecules are large glycoproteins that are abundantly decorated with O-linked glycans. At least two structurally and functionally distinct populations of mucins are present in human saliva: the high-molecular-mass (Mr>106 Da) oligomeric, gel-forming MG1 population and the lower-molecular-mass (Mr 1.2–1.5x105 Da) monomeric MG2 mucins (Loomis et al., 1987; Ramasubbu et al., 1991; Wu et al., 1994; Nieuw Amerongen et al., 1995), a product of the MUC7 gene (Bobek et al., 1993). The protein core of MG1 is encoded by MUC5B (Nielsen et al., 1997; Thornton et al., 1999, Wickstrøm et al., 1998). Characterization of the MUC5B carbohydrates revealed that these make a tremendously heterogeneous set of neutral, sialylated, and sulfated oligosaccharides, varying in length between 2 and more than 40 monosaccharide residues (Thomson et al., 2002). Furthermore, it has been demonstrated that different types of salivary glands secrete differently glycosylated MUC5B species (Veerman et al., 1992; Bolscher et al., 1995; Thornton et al., 1999; Wickstrøm et al., 1998). The goal of the present study was to examine to what extent the MUC5B population secreted by a single salivary gland is made up of a homogeneous set of molecules. We approached this by studying the colocalization of a MUC5B-polypeptide epitope and a specific sulfated carbohydrate epitope in human salivary glands. Using monoclonal antibodies directed to well-characterized protein and carbohydrate epitopes on MUC5B, we found that within one salivary gland differently glycosylated MUC5B species are expressed.
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Results |
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To verify the specificity of the antisera used in the immunohistological study, a number of saliva samples from different individuals were analyzed by western blotting (Figure 1). In all saliva samples tested (so far from more than 50 individuals), mAb F2 and mAb EU-MUC5Bb labeled exclusively high Mr material, corroborating previous findings that the sulfo-Lewis a antigen is a common epitope of high Mr salivary mucins. Strikingly, the material recognized by mAb EU-MUC5Bb remained in the stacking gel, whereas F2, in addition, recognized species that entered the separation gel. Anti-MUC7 antibody CpMG2, which is directed to a sequence located in the C-terminus of MUC7, bound only to MUC7, which exhibited intraindividual variation in apparent molecular size, confirming previous reports (Bolscher et al., 1999
; Kirkbride et al., 2001). Control incubations with nonrelevant mAbs of the same subclasses or with control rabbit antiserum were negative.
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With these sera sections from formalin-fixed, paraffin-embedded submandibular, sublingual, and labial glands were stained (Figures 2 and 3). MAb EU-MUC5Bb exhibited a uniform staining pattern, labeling virtually all mucous acini in the submandibular gland, sublingual gland, and labial gland (e.g., Figures 2A, 2E, 2 J), whereas mAb F2 stained focally (e.g., Figures 2D, 2H, 2L). Staining with this antibody showed the presence of positive as well as negative mucous acini within one glandular tissue. Even within one acinus unit, both F2-positive and F2-negative mucous cells adjacent to each other were observed (Figures 2H, 2L). In all glands, only serous cells were labeled by anti-MUC7 antibody CpMG2, in a uniform pattern. This is illustrated in Figure 2M for a labial gland tissue section, showing that serous demilune cells, capping mucous acini, were labeled by this antibody. Essentially the same localization pattern was found in sublingual gland tissue sections (not shown). In the submandibular gland, besides the demilune cells, also serous acini were labeled by this antiserum (not shown). Slides incubated with control antisera were all negative.
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To further confirm the disparity in staining produced by mAbs EU-MUC5Bb and F2 and to rule out the possibility that the observed difference was due to technical artifacts, we stained one and the same lip biopsy section simultaneously with EU-MUC5Bb and F2, followed by detection using isotype-specific, fluorescently tagged secondary antibodies (Figure 3). These double-labeled sections (Figure 3C) confirmed that the F2 and the EU-MUC5Bb signals did only partially overlap; EU-MUC5Bb-positive/F2-positive as well as EU-MUC5Bb-positive/F2-negative patches were present. No EU-MUC5Bb-negative/F2-positive sections were found, supporting the conclusion that F2 is specific to a subpopulation of salivary MUC5B. Altogether this experiment strongly suggests that both F2-positive and F2-negative MUC5B molecules are expressed by one and the same salivary gland.
Control experiments involved probing with a single antibody-secondary antibody and probing with the mixture of primary antibodies (mAbF2/mAb EU-MUC5Bb), followed by incubation with either tetramethyl rhodamine isothiocyanate (TRITC)-labeled anti-mouse IgG1 (to detect EU-MUC5Bb) or fluorescein isothiocyanate (FITC)-labeled anti-mouse IgM (to detect F2). In this case the same staining patterns were obtained as when tissues were probed with a single primary-secondary antibody combination (not shown). In another control experiment sections were incubated with F2 or EU-MUC5Bb alone and then probed with the nonmatching secondary antibody (TRITC-labeled goat anti-mouse IgG1 and FITC-labeled goat anti-mouse IgM, respectively). In these cases tissues were completely negative, demonstrating the absence of cross-reactivity between secondary antibodies and nonmatching Ig isotypes (not shown).
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Discussion |
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A number of biochemical studies have revealed that in saliva differently glycosylated MUC5B species are present. This heterogeneity is partly explained by the multiglandular origin of salivary mucins (Veerman et al., 1992
; Bolscher et al., 1995). However, it has remained unknown whether the MUC5B population from one and the same gland type is immunochemically homogeneous. In a previous study heterogeneous expression of the F2 epitope has been demonstrated in lung tissue (Wickstrøm et al., 1998). However, besides MUC5B, the respiratory tract expresses other MUC genes, including MUC5AC (Roger et al., 2000, 2001). Thus it cannot be excluded that the heterogeneity in the F2 labeling was due to the fact that some cells or acini expressed another MUC type (e.g., MUC5AC instead of MUC5B) with a different pattern of glycosylation. This possibility was obviated in the present study by labeling the same tissue section simultaneously with two antibodies (against MUC5B-peptide and the F2 epitope). In this way, also artifacts due to, say, incomplete fixation are avoided. The present study, therefore, is the first to prove unambiguously that only one salivary gland expresses differently glycosylated MUC5B mucins. Mosaic patterns of expression have been demonstrated in the endometrium for carbohydrates present on the membrane-bound MUC1. Ultrastructural analysis revealed intercellular variation in glycosylation, even between neighboring cells (Campbell et al., 2000).
In the present study we used mAb F2 to detect a specific sulfoglycosylation motif. In saliva, the F2 epitope is specific for the large mucins, MUC5B (Figure 1), facilitating the interpretation of immunohistochemical data. Other "mucin-specific" antisera, for example, directed against Lewis antigens or ABO blood group antigens, may recognize (besides MUC5B) MUC7 and gp340 (Ligtenberg et al., 2000; Bikker et al., 2002), which makes it difficult to unequivocally assign a positive signal to a specific mucin or glycoprotein species.
Nevertheless, with only one antibody against a MUC5B-specific glycosylation motif, different MUC5B (sulfo)glycoforms could be demonstrated in the present study. In view of the tremendous variation in MUC5B oligosaccharides (Thomson et al., 2002), it can be speculated that similar nonuniform patterns of expression can be found for a number of other carbohydrate epitopes as well. Thus, groups of cells, or individual cells within one gland, may secrete a MUC5B molecule carrying a unique carbohydrate signature. Previous biochemical and biophysical analysis already pointed to the existence of differently glycosylated MUC5B subpopulations in single glandular secretions (Bolscher et al., 1995; Thornton et al., 1999). The finding that within one secretory acinus different glycoforms are expressed suggests that the diversity in mucin molecules may be much larger than so far assumed. However, sophisticated imaging techniques enabling (immuno)chemical analysis of individual mucin molecules (McMaster et al., 1999) are needed to prove this unequivocally.
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Material and methods |
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Western blotting and electrophoresis
Saliva was collected from human volunteers by drooling in an ice-chilled vessel and clarified by centrifugation at 10,000xg for 5 min. The study was approved by the Institutional Ethical Board of the Academic Hospital of the Vrije Universiteit at Amsterdam (protocol OWBR010), and informed consent was obtained from all donors. Western blot analysis was conducted essentially as previously described (Veerman et al., 1996
). In short, saliva samples were analyzed on 15% polyacrylamide gel, using the PhastSystem (Pharmacia, Uppsala, Sweden). After electrophoresis, proteins were transferred onto nitrocellulose membranes by diffusion blotting. Nitrocellulose filters were probed with mAbs (
5 µg/ml), dissolved in phosphate buffered saline supplemented with 0.5% Tween 20. Bound antibodies were detected by alkaline phosphate conjugated to anti-mouse immunoglobulins, using nitro-blue-tetrazolium and 5-bromo-4-chloro-indolyl-phosphate as substrates. Controls, incubated with nonrelated (monoclonal or polyclonal) antibodies, were negative.
Antibodies
MAb F2 (IgM subclass) recognizes the SO3Gal[beta]1-3GlcNAc moiety of the sulfo-Lewis a antigen (Veerman et al., 1997). MAb EU-MUC5Bb (IgG1 subclass) was evoked against the amino acid sequence RNREQVGKFKM, located in four of the cysteine-rich domains of the tandem repeat region of MUC5B, and was obtained from the European Consortium (Concerted Action contract number BMH4-CT98-3222). CpMG2, a rabbit polyclonal antibody, is directed to the amino acid sequence LLNRIIDDMVEQ, located in the C-terminus of MUC7 (Bolscher et al., 1999).
Immunohistochemistry on human tissue specimens
Sections were prepared from paraffin-embedded specimens of human salivary gland tissues that had been removed for therapeutic or diagnostic purposes. The material was made up of two sublingual and submandibular glands resections (male, 69 years; female, 65 years), and three labial biopsies (male, 55 years; female, 65 years). The study was approved by the Institution Ethical Board of the Academic Hospital Vrije Universiteit at Amsterdam, and informed consent was obtained from all tissue donors. Neutral-buffered formaldehyde fixed, paraffin-embedded tissue sections (4 µm) were mounted on ChemMate Capillary Gap Slides (Dako, Glostrup, Denmark), dried at 60°C, deparaffinized, and incubated with 0.3% H2O2 in methanol for 30 min to remove endogenous peroxidase activity. Slides were incubated with antisera (mAbs F2 and EU-MUC5Bb, 1:5 diluted, and polyclonal antibody CpMG2, 1:100 diluted) for 25 min at room temperature and, after rinsing, incubated with rabbit anti-mouse horseradish peroxidase, or goat anti-rabbit horseradish peroxidase. Immunostaining was followed by brief nuclear counterstaining in Mayers hematoxylin. Finally, coverslips were mounted with AquaTex (Merck, Darmstadt, Germany). Controls were performed by replacement of the primary antibody with an unrelated antibody of the same subclass.
Double labeling of tissues was conducted as follows: sections from lip biopsies were deparaffinized and blocked with normal goat serum (1:50) for 10 min. The slides were incubated with a 1:1 mixture of mAb F2 and EU-MUC5Bb for 60 min. After rinsing, sections were incubated with a mixture of TRITC-labeled goat anti-mouse IgG1 and FITC-labeled goat anti-mouse IgM, 1:100 diluted in phosphate buffered saline supplemented with 10% normal human serum/normal goat serum. Immunostaining was followed by brief nuclear counterstaining with 4',6'-diamidino-2-phenylindole hydrochloride (1:10).
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Footnotes |
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2 Present address: Department of Pathology, University Hospital Groningen, P.O. Box 30.001, 9700 RB Groningen, The Netherlands
1 To whom correspondence should be addressed; e-mail: eci.veerman.obc.acta{at}med.vu.nl
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Abbreviations |
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FITC, fluorescein isothiocyanate; mAb, monoclonal antibody; TRITC, tetramethyl rhodamine isothiocyanate.
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