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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Key words: glycoforms / immunohistology / MUC5B / salivary glands / sulfo-Lewis a
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Introduction |
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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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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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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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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
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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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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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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, 2056320569.
Bolscher, J., Veerman, E., Nieuw Amerongen, A.V., 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, 801806.[ISI][Medline]
Bolscher, J.G.M., Groenink, J., van der Kwaak, J.S., van den Keijbus, P.A.M., van't Hof, W., Veerman, E.C.I., and Nieuw Amerongen, A.V. (1999) Detection and quantification of MUC7 in submandibular, sublingual, palatine, and labial saliva by anti-peptide antiserum. J. Dent. Res., 78, 13621369.[Abstract]
Campbell, S., Larsen, J., Seif, M.W., Allen, T.D., Knox, F., Jones, C.J.P., and Aplin, J.D. (2000) Mosaic characteristics of human endometrial epithelium in vitro: analysis of secretory markers and cell surface ultrastructure. Mol. Hum. Reprod., 6, 4149.
Iontcheva, I., Oppenheim, F.G., and Troxler, R.F. (1997) Human salivary mucin MG1 selectively forms heterotypic complexes with amylase, proline-rich proteins, statherin, and histatins. J. Dent. Res., 76, 734743.[Abstract]
Kirkbride, H.J., Bolscher, J.G.M., Nazmi K., Vinall, L.E., Nash, M.W., Moss, F.M., Mitchell, D.M., and Swallow, D.M. (2001) Genetic polymorphism of MUC7: allele frequencies and association with asthma. Eur. J. Hum. Gen., 9, 347354.[CrossRef][ISI][Medline]
Ligtenberg, A.J.M., Veerman, E.C.I., and Nieuw Amerongen, A.V. (2000) A role for Lewis a antigens on salivary agglutinin in binding to Streptococcus mutans. Leeuwenhoek, 77, 2130.[CrossRef]
Loomis, R.E., Prakobphol, A., Levine, M.J., Reddy, M.S., and Jones, P.C. (1987) Biochemical and biophysical comparison of two mucins from human submandibular-sublingual saliva. Arch. Biochem. Biophys., 258, 452464.[ISI][Medline]
McMaster, T.J., Berry, M., Corfield, A.P., and Miles, M.J. (1999) Atomic force microscopy of the submolecular architecture of hydrated ocular mucins. Biophys. J., 77, 533541.
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, 413419.[Abstract]
Nieuw Amerongen, A.V., Bolscher, J.G.M., and Veerman, E.C.I. (1995) Salivary mucins: protective functions in relation to their diversity. Glycobiology, 5, 733740.[ISI][Medline]
Ramasubbu, N., Reddy, M.S., Bergey, E.J., Haraszthy, G.G., Soni, S.D., and Levine, M.J. (1991) Large-scale purification and characterisation of the major phosphoproteins and mucins of human submandibular-sublingual saliva. Biochem. J., 280, 341352.[ISI][Medline]
Roger, P., Gascard, J.P., Bara, J., de Montpreville, V.T., Yeadon, M., and Brink, C. (2000) ATP induced MUC5AC release from human airways in vitro. Mediat. Inflamm., 9, 277284.[CrossRef][ISI]
Roger, P., Gascard, J.P., Bara, J., Dulmet, E., and Brink, C. (2001) EGTA treatment of human airways in vitro unmasks M1/MUC5AC mucin in submucosal glands. Eur. Respir. J., 18, 176183.
Thomson, K.A., Prakobphol, A., Leffler, H., Reddy, M.S., Levine, M.J., Fisher, S.J., and Hansson, G.C. (2002) The salivary mucin MG1 (MUC5B) carries a repertoire of unique oligosaccharides that is large and divcrse. Glycobiology, 12, 114.
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, 293302.
Veerman, E.C.I., Van den Keybus, P.A.M., Valentijn Benz, M., and Nieuw Amerongen, A.V. (1992) Isolation of different high-Mr mucin species from human whole saliva. Biochem. J., 283, 807811.[ISI][Medline]
Veerman, E.C.I., Ligtenberg, A. J. M., Schenkels, L. C. P. M., Walgreen-Weterings, E., and Nieuw Amerongen, A.V. (1995) Binding of human high-molecular weight salivary mucins (MG1) to Hemophilus parainfluenzae. J. Dent. Res., 74, 351357.[Abstract]
Veerman, E.C.I., van den Keybus, P.A.M., Vissink, A., and Nieuw Amerongen, A.V. (1996) Human glandular salivas: their separate collection and analysis. Eur. J. Oral Sci., 104, 346352.[ISI][Medline]
Veerman, E.C.I., Bolscher, J.G.M., Appelmelk, B.J., Bloemena, E., van den Berg, T.K., and Nieuw Amerongen, A.V. (1997) A monoclonal antibody directed against high Mr salivary mucins recognizes the SO3Gal[beta]1-3GlcNAc moiety of sulfo-Lewis a. A histochemical survey of human and rat tissue. Glycobiology, 7, 3744.[Abstract]
Wickstrøm, 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, 685693.[ISI][Medline]
Wu, A.M., Csako, G., and Herp, A. (1994) Structure, biosynthesis, and function of salivary mucins. Mol. Cell. Biochem., 137, 3955.[ISI][Medline]
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