Alterations of mucin O-glycosylation in response to intestinal infection. Importance of specific glycosyltransferases

Sammanfattning: Mucins are large glycoproteins constituting the major protein component of the mucus layer covering the epithelial surfaces. The mucins are characterised by a dense and heterogeneous O-glycosylation, important for their chemical and physiological properties. The mucus layer forms a selective physical barrier serving to protect the epithelium from physical and chemical stress. With their heterogeneous glycosylation, the mucins probably have a role in the interplay with microbes dwelling at the epithelia, by constituting the targets for microbial adhesins. An increased understanding of mucin O-glycosylation and its biosynthesis would aid in exploring the mechanisms and occurrence of such an interplay.We have studied the O-glycosylation of rat small intestinal mucins, mainly Muc2, during an infection with the parasitic nematode Nippostrongylus brasiliensis. Mucins were collected from the rats at different stages of the infection, and oligosaccharides were released from the protein backbone and separated into neutral, sialylated and sulphated species. The oligosaccharides were structurally analysed, using gas chromatography-mass spectrometry, NMR and high pH anion exchange chromatography. The comparison of the oligosaccharides from normal and infected rats revealed three specific alterations appearing transiently during the infection, indicating a dynamic regulation of mucin O-glycosylation. The alterations were 1) an increased amount of N-acetylneuraminic acid relative to N-glycolylneuraminic acid, 2) the appearance of blood group A terminal epitopes and 3) the appearance of blood group Sda/Cad terminal epitopes. The enzymes responsible for the two first alterations were by northern blotting identified as the CMP-NeuAc hydroxylase and the blood group A GalNAc-transferase, respectively. The rat blood group A glycosyltransferase was cloned and sequenced at the genomic and cDNA levels. The gene was mapped to chromosome 3q11-12 by fluorescence in-situ hybridisation and radiation hybrid mapping. During the cDNA cloning, two similar sequences with 95 percent similarity were identified, both encoding active transferases. By PCR studies on inbred, outbred and interbred rats, the two sequences were shown to be allelic. The unusually large difference between the alleles might indicate an evolutionary pressure in favour of a high mutation frequency for this gene.An aspect of mucin glycosylation that has been difficult to address is the glycosylation of specific O-glycan sites, partly due to the large number of such sites in the mucins. As a step towards such an analysis, a short recombinant reporter protein was constructed, based on sequence from the glycosylated domain of MUC1 and containing only eight O-glycan sites. When this protein was produced in CHO K1 cells, the major oligosaccharides present were mono- and disialylated core 1 glycans, as determined by liquid chromatography-electrospray ionisation-mass spectrometry (LC-ESI-MS) and LC-ESI-MS/MS on released oligosaccharides. N-terminal peptide sequencing showed that all individual O-glycan sites were glycosylated, to an extent of 75-95 percent at the different sites. The occupancy of the O-glycan sites could be increased by co-expression with polypeptide-GalNAc-T4, which in vitro has been shown to add GalNAc to the polypeptide backbone of MUC1 glycopeptides. Our results confirmed the proposed activity of GalNAc-T4 in vivo.