Keusch, Jeremy Joel Hugo; (1997) Immunodetection and regulation of beta1,4-galactosyltransferase in B lymphocytes. Doctoral thesis (Ph.D), UCL (University College London).

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Abstract

β1,4-galactosyltransferase (β1,4-GalTase) is a glycosyltransferase localized in the trans Golgi of most mammalian cells where it is involved in the galactosylation of glycoconjugates, transferring galactose from UDP-galactose to the non-reducing end of exposed GlcNAc residues. This project examined β1,4-GalTase expression in various B cells and its ability to galactosylate different acceptor substrates in vitro and in situ. B cells, isolated from the peripheral blood of patients with rheumatoid arthritis (RA) and controls, were assayed for β1,4-GalTase activity in a newly developed ELISA- based assay which utilized the neoglycoprotein, GlcNAc-pITC-BSA, as the acceptor substrate. The previously reported decrease in B cell β1,4-GalTase activity from patients with RA was not apparent with the GlcNAc-pITC-BSA substrate, but could be detected using ovalbumin as an acceptor. A number of anti-β1,4-GalTase monoclonal antibodies (mAbs) were produced against soluble human milk β1,4-GalTase. These mAbs were purified, conjugated and extensively characterized enabling the immunodetection of β1,4-GalTase in different assay systems. The binding affinities of the anti-β1,4-GalTase mAbs were measured against several different types of purified β1,4-GalTase using surface plasmon resonance and were all found to be of moderately high binding affinity (KB approximately 108 M-1 against human milk β1,4-GalTase). All the anti-β1,4-GalTase mAbs cross-reacted with bovine milk β1,4-GalTase although to different extents. Two anti-β1,4-GalTase mAbs against non-competing epitopes were used to develop a sensitive (>1 ng/ml) β1,4-GalTase protein quantification ELISA which could measure β1,4-GalTase protein from cell lysates and extracellular sources. Preliminary data suggested that there was no apparent difference between the β1,4-GalTase protein levels in the B cells from controls and patients with RA. B cells which differed in their levels of β1,4-GalTase activity were produced following the stable transfection of IgG-secreting B cells, from the same cell line, with human β1,4-GalTase cDNA in the sense and antisense orientations. The β1,4-GalTase sense transfected B cells had higher levels of β1,4-GalTase expression and secreted IgG with more galactosylated structures than those B cells transfected with the antisense. However, factors other than β1,4-GalTase expression levels influenced the galactosylation of IgG. Experiments and results presented in this thesis were discussed with particular reference to the under-galactosylated IgG observed in patients with RA.

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