N-Glycan Modifications with Negative Charge in a Natural Polymer Mucin from Bovine Submaxillary Glands, and Their Structural Role

Bovine submaxillary mucin (BSM) is a natural polymer used in biomaterial applications for its viscoelasticity, lubricity, biocompatibility, and biodegradability. N-glycans are important for mucin stability and function, but their structures have not been fully characterized, unlike that of O-glycans. In this study, BSM N-glycans were investigated using liquid chromatography-tandem mass spectrometry. The microheterogeneous structures of 32 N-glycans were identified, and the quantities (%) of each N-glycan relative to total N-glycans (100%) were obtained. The terminal N-acetylgalactosamines in 12 N-glycans (sum of relative quantities; 27.9%) were modified with mono- (10 glycans) and disulfations (2 glycans). Total concentration of all sulfated N-glycans was 6.1 pmol in BSM (20 µg), corresponding to 25.3% of all negatively charged glycans (sum of present N-glycans and reported O-glycans). No N-glycans with sialylated or phosphorylated forms were identified, and sulfate modification ions were the only negative charges in BSM N-glycans. Mucin structures, including sulfated N-glycans located in the hydrophobic terminal regions, were indicated. This is the first study to identify the structures and quantities of 12 sulfated N-glycans in natural mucins. These sulfations play important structural roles in hydration, viscoelasticity control, protection from bacterial sialidases, and polymer stabilization to support the functionality of BSM via electrostatic interactions.

Karakterisasi Biokimia Lektin Makroalga Sargassum polycystum dan Turbinaria ornata/Biochemical Characterisation of Lectin Derived from Sargassum polycystum and Turbinaria ornata Macroalgae

Kemampuan lektin untuk mengikat karbohidrat secara spesifik dan reversible dapat dikembangkan dalam berbagai aplikasi, misalnya sebagai reagen histokimia. Penelitian ini bertujuan untuk mempelajari spesifisitas pengikatan lektin makroalga Sargassum polycystum dan Turbinaria ornata pada berbagai jenis karbohidrat, stabilitas aktivitas hemaglutinasi lektin pada berbagai rentang suhu dan pH, serta pengaruh kation divalen pada aktivitasnya. Uji penghambatan hemaglutinasi secara kualitatif dan kuantitatif dilakukan untuk mempelajari spesifisitas pengikatan lektin terhadap 20 jenis gula dan glikoprotein. Untuk melihat stabilitas aktivitasnya, lektin S. polycystum dan T. ornata diberi perlakuan pemanasan pada rentang suhu 30-100 oC, perlakuan pH 3-10 dan perlakuan kation divalen MgCl2 dan CaCl2 kemudian diuji aktivtitas hemagglutinasinya. Ekstrak kaya lektin S. polycystum dan T. ornata mampu mengenali dan mengikat 8 glikoprotein secara kualititatif, yaitu fetuin (Fe), asialo Fe (aFe), thyroglobulin from bovine (BTG), asialo BTG, thyroglobulin from porcine (PTG), asialo PTG (aPTG), asialo mucin from bovine submaxillary glands (aBSM), dan asialo transferrin (aTf), namun tidak mempunyai afinitas terhadap gula sederhana. Lektin S. polycystum memiliki spesifisitas pengikatan terbaik terhadap aFe dan transferrin (Minimum Inhibitory Concentration/MIC 250 µg/ml), sementara Lektin T. ornata memiliki spesifisitas pengikatan terbaik terhadap aPTG (MIC 31.25 µg/ml), PTG (MIC 125 µg/ml), dan BTG (MIC 250 µg/ml). Aktivitas hemaglutinasi lektin S. polycystum stabil pada suhu 30-80 oC dan suasana netral hingga basa (pH 7-10), namun kurang stabil pada suasana asam (pH 3-6). Aktivitas lektin T. ornata relatif tidak stabil pada suhu 40-100 oC, sedikit menurun pada pH sangat asam, namun stabil pada rentang pH 5-10. Keberadaan kation divalent Ca2+ dan Mg2+ sedikit menurunkan aktivitas lektin S. polycystum dan T. ornata.