Rumi functions as both a protein O-glucosyltransferase and a protein O-xylosyltransferase - PubMed (original) (raw)

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.