Sequence-structure-function relationships of glycosyltransferases in families GT43, GT47, and GT64

Extracellular carbohydrates are an essential aspect of biology, playing central roles in cell-cell interaction, cell shape, and infectious disease. In eukaryotes, extracellular glycans and glyco-conjugates are synthesised or matured by glycosyltransferase enzymes in the Golgi apparatus. Most glycosyltransferases catalyse the formation of unique glycosidic bonds; thus, the activities of these enzymes are responsible for controlling the diversity of glycan structures. The study of glycosyltransferases can therefore grant insight into the biology of the Golgi apparatus and the ætiology of disease, as well as providing a means to engineer carbohydrate structures for therapeutic or material applications. GT43 members IRX9 and IRX14 are involved in the synthesis of xylan in Arabidopsis, and are thought to form a multimeric complex with GT47 member IRX10. In this work, I expressed transmembrane fragments of these proteins in E. coli and used a reporter assay to demonstrate the sequence-dependent oligomerisation of the IRX9 transmembrane domain. Furthermore, I showed that an IRX9 mutant containing a transmembrane G28I point mutation was unable to complement the phenotype of the irx9 mutant and appeared to be mislocalised when transiently expressed in tobacco leaves. In animals, exostosin glycosyltransferases synthesise the backbone of heparan sulphate, and contain both a GT47 and a GT64 domain. Using cryo-EM, I solved the structure of EXTL3, the largest exostosin, in complex with UDP. The structure revealed that the EXTL3 GT47 domain adopts a GT-B fold that has been inactivated by structural changes to the active site. The structure also demonstrated that the GT47 and GT64 active sites are likely separated by a surprising distance in these bi-domain enzymes. Members of GT47 clade A have particularly diverse activities in plants. I used the structure of EXTL3 to rationalise differences in substrate specificity between these enzymes. Although I was unable to explain the difference in activity between a recently characterised xylan arabinopyranosyltransferase and a homologous xylan galactosyltransferase, I was nevertheless successful in identifying a novel xyloglucan-specific enzyme from Coffea canephora on the basis of its protein sequence. As a whole, the results in this thesis provide insight into the relationship between amino acid sequence, atomic structure, and function in glycosyltransferases. The findings hint at a potential nano-scale organisation of the Golgi apparatus that will be exciting to investigate further.

Name dc:type.qualificationname

Doctor of Philosophy (PhD)

Level dc:type.qualificationlevel

Doctoral

Grantor dc:publisher.institution

University of Cambridge

Year dc:date.issued

2021

Author dc:creator

• Wilson, Louis

Advisor dc:contributor.advisor

• Dupree, Paul

• biochemistry
• glycosyltransferase
• glycobiology
• Golgi
• polysaccharide
• cryo-EM
• Arabidopsis
• cell wall

Licence

• https://www.repository.cam.ac.uk/bitstreams/b18a1b8b-2b25-4150-acdf-d5485063acea/download
• https://www.rioxx.net/licenses/all-rights-reserved/

Language dc:language

eng

DOI dc:identifier.doi

https://doi.org/10.17863/CAM.72489

OAI identifier oai:identifier

oai:www.repository.cam.ac.uk:1810/325034