Rumi functions as both a protein O -glucosyltransferase and a protein O -xylosyltransferase

Abstract

Mutations in rumi result in a temperature-sensitive loss of Notch signaling in Drosophila . Drosophila Rumi is a soluble, endoplasmic reticulum-retained protein with a CAP10 domain that functions as a protein O -glucosyltransferase. In human and mouse genomes, three potential Rumi homologues exist: one with a high degree of identity to Drosophila Rumi (52%), and two others with lower degrees of identity but including a CAP10 domain (KDELC1 and KDELC2). Here we show that both mouse and human Rumi, but not KDELC1 or KDELC2, catalyze transfer of glucose from UDP-glucose to an EGF repeat from human factor VII. Similarly, human Rumi, but not KDELC1 or KDELC2, rescues the Notch phenotypes in Drosophila rumi clones. During characterization of the Rumi enzymes, we noted that, in addition to protein O -glucosyltransferase activity, both mammalian and Drosophila Rumi also showed significant protein O -xylosyltransferase activity. Rumi transfers Xyl or glucose to serine 52 in the O -glucose consensus sequence ( ) of factor VII EGF repeat. Surprisingly, the second serine (S53) facilitates transfer of Xyl, but not glucose, to the EGF repeat by Rumi. EGF16 of mouse Notch2, which has a diserine motif in the consensus sequence ( ), is also modified with either O -Xyl or O -glucose glycans in cells. Mutation of the second serine (S590A) causes a loss of O -Xyl but not O -glucose at this site. Altogether, our data establish dual substrate specificity for the glycosyltransferase Rumi and provide evidence that amino acid sequences of the recipient EGF repeat significantly influence which donor substrate (UDP-glucose or UDP-Xyl) is used.

Site-specific O-Glucosylation of the Epidermal Growth Factor-like (EGF) Repeats of Notch

“…2B). Using the radioactive assay, mN2 EGF16 was an excellent substrate for both Poglut and Poxylt activity of Poglut/Rumi, consistent with our prior mass spectral results (15). The FA9 N54S mutant showed similar Poglut activity compared with wild type but significantly higher Poxylt activity (Fig.…”

“…EGF27 from mN1 (26) and EGF16 from mouse Notch2 (mN2) (14)). We also found an O-xylose modification at the O-glucose consensus site of EGF16 from mN2 and demonstrated that Poglut/Rumi utilizes UDP-xylose as well as UDP-glucose as donor substrate (15). The presence of a di-serine motif within the O-glucose consensus sequence, as found in mN2 EGF16, appears to enhance use of UDP-xylose by Poglut/Rumi (15).…”

“…Our recent site-mapping and biochemical analyses showed that mN2 EGF16 is modified with O-xylose glycans as well as O-glucose glycans due to the presence of a di-serine motif inside the O-glucose consensus sequence (15). To examine whether mN2 EGF16 is a better substrate for O-xylose transfer in vitro than other EGF repeats, we expressed it in bacteria and incubated overnight with Poglut/Rumi and UDP-glucose or UDP-xylose (Fig.…”

Dual Roles of O-Glucose Glycans Redundant with Monosaccharide O-Fucose on Notch in Notch Trafficking

“…At least five different glycan modifications have been found on the Notch EGF-like repeats: N-linked glycan modifications (8) and four O-linked glycan modifications: O-fucose (9 -11), O-glucose (12,13), O-GlcNAc (14), and O-GalNAc (15). A potential O-xylosylation of the EGF-like repeats has also been observed in vitro (16). The individual modifications have been analyzed to determine their specific functions and generate a model for how glycosylation regulates receptor functions (8 -15).…”

O-Glycosylation modulates the stability of epidermal growth factor-like repeats and thereby regulates Notch trafficking

“…The plasmids used for secretion assays were pSecTag2c-Notch1 EGF(1-36)-Myc-His 6 (44), pcDNA4-POGLUT1-Myc-His 6 (45), and pSecTag2c-POFUT1-Myc-His 6 (7). A plasmid encoding human IgG (3) was used as secretion control.…”