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.

Fig. 1.

Human Rumi is the functional and biochemical homologue of Drosophila Rumi. (A) Molecular structure of human proteins containing a CAP10 domain. SP, signal peptide; KDEL, KDEL ER-retention signal; FLMN, filamin-like domain. (B) Percent identity of amino acid sequences of human Rumi, KDELC1, and KDELC2 with dRumi. (C–F) Thoraces of adult flies harboring randomly located rumi^Δ26 mutant clones. The lower panels show close-ups of the boxes in the upper panels. The rumi^Δ26 mutant clones result in loss of sensory organs on thorax (C, dotted lines in the close-up). Simultaneous overexpression of a FLAG-tagged version of hRumi restores the lost sensory organs in rumi^Δ26 mutant clones (D). Note that the rescued sensory organs—four of which are marked by asterisks—lose the y⁺ transgene on the reciprocal 3R chromosome and are therefore yellow, as opposed to rumi^+/- heterozygous and rumi^+/+ sensory organs, which look darker. Human KDELC1–FLAG (E) or hKDELC2–FLAG (F) were not able to rescue the phenotype (dotted lines in the close-ups). (G–J) Wings of adult flies harboring randomly located rumi⁴⁴ mutant clones. Loss of rumi results in the thickening of wing veins (brackets) and “Notches” of tissue loss in the wing margin (arrowhead) (G). Only hRumi–FLAG is able to rescue these phenotypes (compare H with I and J). All animals were raised at 30 °C after the second instar larval stage. (K) Protein expression detected by Western blot analysis using anti-Myc antibody in the culture media from 293T cells transfected transiently with an empty vector control (lane 1), human Rumi (lane 2), KDELC1 (lane 3), or KDELC2 (lane 4), all with a C-terminal Myc-His₆ tag. (L) The Poglut activity in the culture media from the same cells as Fig. 1K. The values indicate the mean ± SEM.

Fig. 2.

Rumi has Poxylt activity as well as Poglut activity. (A) Donor substrate specificity of N-terminal His-tagged mouse Rumi (His-mRumi). A representative dataset from two independent assays is shown. (B) Structure of UDP-glucose and UDP-Xyl. (C) Enzyme dependence of Poglut (blue) or Poxylt (red) activities of His-mRumi toward hFVII EGF repeats (5 μM). (D) EGF dependence of Poglut (blue) or Poxylt (red) activities of His-mRumi in the presence of 10 μM UDP-sugars. Note, kinetic parameters were calculated using the data points below 5 μM of EGF repeats by Lineweaver–Burk plot because the decreased activities at higher range may be due to substrate inhibition. (E) UDP-sugar dependence of Poglut (blue) or Poxylt (red) activities of His-mRumi toward hFVII EGF repeats (5 μM). (F) Poglut (blue) or Poxylt (red) activities of FLAG-tagged versions of wild-type or G169E mutant hRumi toward hFVII EGF repeats (5 μM). The values indicate mean ± SEM.

Fig. 3.

The diserine motif in the O-glucose consensus sequence is an indicator for O-Xyl transfer by Rumi. (A) Poglut (Top) or Poxylt activity (Middle) of His-mRumi, or Pofut1 activity of human Pofut1 toward hFVII EGF repeats modified with O-fucose (Fuc-EGF), O-glucose (Glc-EGF), O-xylose (Xyl-EGF), or unmodified hFVII EGF repeats (5 μM). Data are representative of two independent assays. (B) EGF dependence of Poglut (Upper) or Poxylt (Lower) activity of hRumi–FLAG toward hFVII EGF repeat (WT, blue), S52A (red), or S53A (green) mutants, and hFIX EGF repeat (purple). The values indicate mean ± SEM. (C) HCD analysis after trypsin digestion of the hFVII EGF repeats unmodified or modified with O-glucose or O-Xyl by hRumi. Collision-induced dissociation fragmentation confirmed the identity of the peptide and the modifications (Fig. S6). (i) HCD fragmentation of the doubly charged form of the peptide MDGDQCASSPCQDGGSCK (m/z 980.3) from unmodified hFVII EGF repeats. Numerous sequence fragment y ions are observed that confirm the identity of the peptide. (ii) HCD fragmentation of the doubly charged form of the O-glucosylated peptide (m/z 1,061.3) from O-glucosylated hFVII EGF repeats. Numerous sequence fragment y ions are observed that not only confirm the identity of the peptide, but also the attachment of a hexose (glucose) to the first serine within the diserine motif. Although y10 + Hex (m/z 1,257.4) was not detected, y11 + Hex (m/z 1,344.4), y12 + Hex (m/z 1,415.4), and y13 + Hex (m/z 1,575.5) ions were detected, confirming the attachment of hexose to the first serine. (iii) HCD fragmentation of the doubly charged form of the O-xylosylated peptide (m/z 1,046.3) from O-xylosylated hFVII EGF repeats. Numerous sequence fragment y ions are observed that not only confirm the identity of the peptide, but also the attachment of a pentose (xylose) to the first serine within the diserine motif. Although y10 + Pen (m/z 1,227.4) was not detected, y11 + Pen (m/z 1,314.4), y12 + Pen (m/z 1,385.5), and y13 + Pen (m/z 1,545.5) ions were detected.

Fig. 4.

O-Xyl trisaccharide on EGF16 of mNotch2 is dependent on the diserine motif. Extracted ion chromatograms (EICs) of the ions corresponding to (glyco)peptides containing the O-glucose consensus sequence from EGF16 of mNotch2 derived from Asp-N digests of wild-type (A) or the mutated (B) mNotch2 proteins (see Fig. S8 for mass spectra confirming the identity of these ions). (A) EICs of the ions corresponding to the unglycosylated (Top), O-glucosylated (Middle), and O-xylosylated (Bottom) forms of from wild-type mNotch2 EGF16. The MS data were searched for the doubly charged form of these (glyco)peptides: m/z 801.0 for the unglycosylated, m/z 1,014.0 for the O-glucosylated, and m/z 999.0 for the O-xylosylated forms. (B) EICs of the ions corresponding to the unglycosylated (Top), O-glucosylated (Middle), and O-xylosylated (Bottom) forms of from the mutated mNotch2 EGF16. The MS data were searched for the doubly charged form of these (glyco)peptides: m/z 793.0 for the unglycosylated (Top), m/z 1,006.0 for the O-glucosylated (Middle), and m/z 991.0 for the O-xylosylated (Bottom) forms.