Abstract
Protein O-glucosyltransferase 1/Rumi-mediated glucosylation of Notch epidermal growth factor-like (EGF-like) domains plays an important role in Notch signaling. Protein O-glucosyltransferase 1 shows specificity for folded EGF-like domains, it can only glycosylate serine residues in the C1XSXPC2 motif, and it possesses an uncommon dual donor substrate specificity. Using several EGF-like domains and donor substrate analogs, we have determined the structures of human Protein O-glucosyltransferase 1 substrate/product complexes that provide mechanistic insight into the basis for these properties. Notably, we show that Protein O-glucosyltransferase 1’s requirement for folded EGF-like domains also leads to its serine specificity and that two distinct local conformational states are likely responsible for its ability to transfer both glucose and xylose. We also show that Protein O-glucosyltransferase 1 possesses the potential to xylosylate a much broader range of EGF-like domain substrates than was previously thought. Finally, we show that Protein O-glucosyltransferase 1 has co-evolved with EGF-like domains of the type found in Notch.
Highlights
Protein O-glucosyltransferase 1 (POGLUT1) glucosylates the epidermal growth factor-like (EGF-like) domains found in diverse substrates including Notch and its ligands[1,2,3] (Fig. 1a)
The X-ray crystal structure of hPOGLUT1 was determined in complex with human factor VII EGF-like domain 1 and human Notch[1] EGF-like domain 12, as well as a synthetic POGLUT1 substrate, EGF(+), the consensus sequence for human EGF-like domains that are POFUT1 substrates[17] (Supplementary Table 1, Supplementary Fig. 4)
POGLUT1/Rumi recognizes a diverse array of EGF-like domain substrates and can only glucosylate/xylosylate those that are properly folded, properties shared by POFUT1
Summary
Protein O-glucosyltransferase 1 (POGLUT1) glucosylates the epidermal growth factor-like (EGF-like) domains found in diverse substrates including Notch and its ligands[1,2,3] (Fig. 1a). POGLUT1 displays dual donor substrate specificity as it can utilize both UDP-glucose and UDP-xylose, a a. Structures of Drosophila Rumi (dRumi) in complex with human Factor IX EGF-like domain 1 (hF9EGF1) and UDP have provided insight into EGFlike domain recognition and the possible effects of disease causing mutations[16]. We show that POGLUT1’s serine specificity stems from the backbone conformation of the glycosylation motif and, as such, its requirement for a folded EGF-like domain substrate. We show that POGLUT1 does not require a diserine-containing glycosylation motif to efficiently xylosylate its substrates, an observation suggesting that it can xylosylate a much wider range of EGF-like domains than previously thought. We show that the POGLUT1 glycosylation motif is found predominantly among EGF-like domains of the type found in Notch and that POGLUT1 has co-evolved with these domains through animal evolution
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