ESI-MS-MS Characterisation of N-linkedGlycosylation in Native CauliflowerXyloglucan Endotransglycosylase (XET)

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Campuzano, I[1];Richie, M[1];Langridge, J[1];Brumer, H[2];Henriksson, H[2];Teeri, T[2].
BMSS; 9th-12th September; 2001
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Xyloglucan endotransglycosylases (XETs) are a class of enzymes closely related to the well-studied glycosyl hydrolases (GH) which have been implicated in playing a role in plant cell wall expansion during growth and development. XET catalyses the cleavage and re-ligation of high molecular weight xyloglucan, which acts as a glue to hold cellulose microfibrils together in a composite matrix thorough hydrogen bonding interactions [1]. On the basis of sequence similarity, all known xyloglucan endotransglycosylases (XETs) have been classified as members of glycosyl hydrolase family 16 [//]. analogy, the beta1-3, beta1-4 endoglucanases, utilise a two-steph configuration retaining mechanism like that shown in Scheme 1. Early studies have indicated that protein glycosylation may be important for the catalytic function of heterologously expressed Arabidopsis thaliana XET[2], although this was not explored in any mechnistic detail. Interestingly, a conserved N-glycosylation site is found in Family 16 XETs proximal to the active site glutamic acid residues. The ability to purify cauliflower XET from the native source as a single glycoform has promted us to re-examine the role of protein glycosylation regarding the ability of XET to favour transglycosylation over hydrolysis. We report here that characterisation of this glycosylation by a combined LC-MS and MS/MS appraoch on native and enxymatically deglycosylated samples.

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