Neoglycoproteins: in vitro introduction of glycosyl units at glutamines in beta-casein using transglutaminase.
Exploring different methods for preparing neoglycoproteins with a specific number of oligosaccharides in specific positions, we have used guinea pig liver transglutaminase to incorporate glycosyl units into glutamine residues in beta-casein. In order to prevent epsilon-(gamma-glutamyl)lysine cross-link formation, the lysine residues of beta-casein were first blocked either by amidination with ethyl acetimidate or by acylation with succinic anhydride. The glycosyl donor substrates prepared for this work were maltotriose reductively aminated with cadaverine, N-(Glc-Glc-glucitol-1)-cadaverine, and an asparaginyl nonasaccharide from ovalbumin modified with a 6-aminohexanoyl group at the alpha-amino group. The transglutaminase-catalyzed incorporation of these two donors into the beta-casein derivatives was monitored in comparison to the incorporation of the commonly used transglutaminase substrate dansylcadaverine under conditions of optimal incorporation (multiple additions of enzyme, large excess of donor, and long incubation time). For both dansylcadaverine and Glc-Glc-Glc(OH)-cadaverine, 5 and 8 mol of donor were incorporated per mol of amidinated and succinylated beta-casein, respectively. Competition experiments showed that the two donor substrates are incorporated into the same glutamine sites. Partial sequencing of the glycosylated beta-casein permitted the identification of glutamine residues 56, 79, 167, 175, and 194 as the primary sites of incorporation in amidinated casein with residues 54 and 182 as possible sites for partial glycosylation. The results are consistent with a specific glycosylation of only selected glutamines in this transglutaminase-catalyzed process.(ABSTRACT TRUNCATED AT 250 WORDS)[1]References
- Neoglycoproteins: in vitro introduction of glycosyl units at glutamines in beta-casein using transglutaminase. Yan, S.B., Wold, F. Biochemistry (1984) [Pubmed]
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