POMGNT1 Is Glycosylated by Mucin-Type <i>O</i>-Glycans
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Protein <i>O</i>-linked mannose β1,2-<i>N</i>-acetylglucosaminyltransferase 1 (POMGNT1) is a Golgi glycosyltransferase that catalyzes the formation of the <i>N</i>-acetylglucosamine (GlcNAc) β1→2Man linkage of <i>O</i>-mannosyl glycan. POMGNT1 is not modified by <i>N</i>-glycans because there are no potential <i>N</i>-glycosylation sites; however, it is not clear whether POMGNT1 is modified by <i>O</i>-glycans. To determine whether POMGNT1 is <i>O</i>-glycosylated, we prepared recombinant human POMGNT1 from HEK293T cells. The recombinant POMGNT1 was recognized by <i>Sambucus sieboldiana</i> lectin (SSA), and sialidase digestion of POMGNT1 decreased SSA reactivity and enhanced the reactivity of <i>Arachis hypogaea</i> lectin (PNA). These results suggest that POMGNT1 is modified by a sialylated core-1 <i>O</i>-glycan. Next, we analyzed the structures of the <i>O</i>-glycans on POMGNT1 by β-elimination and pyrazolone-labeling methods in combination with mass spectrometry. We identified several mucin-type <i>O</i>-glycans containing (NeuAc)<sub>1</sub>(Hex)<sub>1</sub>(HexNAc)<sub>1</sub>, (NeuAc)<sub>2</sub>(Hex)<sub>1</sub>(HexNAc)<sub>1</sub>, and (NeuAc)<sub>2</sub>(Hex)<sub>2</sub>(HexNAc)<sub>2</sub>. To examine whether the <i>O</i>-glycans affect the functions and properties of POMGNT1, we compared glycosylated and non-glycosylated forms of recombinant sPOMGNT1 for their activity and surface hydrophobicity using the hydrophobic probe 1-anilino-8-naphthalene sulfonate (ANS). POMGNT1 activity and surface hydrophobicity were not affected by the presence or absence of <i>O</i>-glycans.
- Biological and Pharmaceutical Bulletin
Biological and Pharmaceutical Bulletin 38(9), 1389-1394, 2015
The Pharmaceutical Society of Japan