O-glycosylation of EGF repeats: identification and initial characterization of a UDP-glucose: protein O-glucosyltransferase.
O-Glucose is an unusual form of posttranslational modification consisting of glucose directly attached to protein through O-linkage. Several serum proteins (factor VII, factor IX, protein Z, and thrombospondin) contain this unique modification on their epidermal growth factor (EGF)-like repeats. Comparison of the glycosylation sites on these proteins revealed a putative consensus sequence for O-glucose modification: C(1)XSXPC(2), where C(1) and C(2) are the first and second conserved cysteines of the EGF repeat. We identify and characterize an enzymatic activity capable of adding glucose to EGF repeats: UDP-glucose: protein O-glucosyltransferase. Using extracts of Chinese hamster ovary cells as the enzyme source, recombinant factor VII EGF repeat as the acceptor, and UDP-[(3)H]glucose as the donor, we show that the activity is linearly dependent on time, enzyme amount, and substrate concentration. As with most glycosyltransferases, metal ions (such as manganese) are required for activity. Analysis demonstrated that the glucose is added in O-linkage to the EGF repeat. Mutation of the serine to alanine in the predicted glycosylation site abrogates glycosylation, as does reduction and alkylation of the EGF repeat, suggesting that the enzyme recognizes not only the consensus sequence but also the 3D structure of the EGF repeat. Detection of O-glucosyltransferase activity in extracts of cell lines from insects to humans and a variety of rat tissues suggests that O-glucose modification is widespread in biology. These studies lay the foundation for future work on the biological role of the O-glucose modification.
Shao
Li
L
Department of Biochemistry and Cell Biology, Institute for Cell and Developmental Biology, State University of New York at Stony Brook, Stony Brook, NY 11794-5215, USA.
Luo
Yi
Y
Moloney
Daniel J
DJ
Haltiwanger
Robert
R
D020816:Amino Acid Motifs; D000818:Animals; D016466:CHO Cells; D002460:Cell Line; D016384:Consensus Sequence; D006224:Cricetinae; D004815:Epidermal Growth Factor / Q000737:chemistry*; D005260:Female; D020869:Gene Expression Profiling; D005964:Glucosyltransferases / Q000737:chemistry* / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism*; D006031:Glycosylation; D006801:Humans; D006863:Hydrogen-Ion Concentration; D051381:Rats; D013379:Substrate Specificity; D013696:Temperature; D014532:Uridine Diphosphate Glucose / Q000378:metabolism*
D004815:Epidermal Growth Factor; D005964:Glucosyltransferases; D014532:Uridine Diphosphate Glucose