Enzymatic sulphation of mucus glycoprotein in rat sublingual salivary gland

Sulphotransferase activity catalysing the transfer of sulphate ester group from 3′-phosphoadenosine-5′-phosphosulphate to salivary mucus glycoprotein was located in detergent extracts of the Golgi-rich membrane fraction of rat sublingual salivary glands. Optimum enzyme activity was obtained with 0.5 per cent Triton X-100, 20 mM NaF and 2 mM MgCl₂, at pH 6.8, using desulphated sublingual salivary mucus; glycoprotein. The enzyme was equally capable of sulphation of the proteolytically degraded and desulphated glycoprotein, whereas the acceptor capacity of intact salivary mucus glycoprotein was about four times lower. The Golgi enzyme preparation also catalysed the sulphation of galactosylceramide. However, the sulphation of mucus glycoprotein was not affected by the presence of this glycolipid, suggesting that the Sulphotransferase involved in mucin sulphation is different from that responsible for the synthesis of galactosylceramide sulphate. The apparent K_m of the sublingual-gland mucus glycoprotein Sulphotransferase for salivary mucin was 7.7 μM. The ³⁵S-labelled glycoprotein product of the enzyme reaction gave, in CsCl density gradient, a band in which the ³⁵S label coincided with the glycoprotein. Alkaline borohydride reductive cleavage of this glycoprotein released the label into the reduced acidic oligosaccharide fraction. Upon thin-layer chromatography, two [³⁵S]-oligosaccharides were detected. These were identified as penta- and heptasaccharides, each bearing a labelled sulphate ester group on the terminal N-acetylglucosamine residue. Based on the results of chemical and enzymatic analyses of the intact and desulphated compounds the following structures for these oligosaccharides are suggested: SO₃ → GlcNAcβ → Galβ → GlcNAcβ → Galβ → GlcNAcβ → (NeuAc → )GalNAc-ol and SO₃ → GlcNAcβ → Galβ → GlcNAcβ → (NeuAc → )GalNAc-ol.