POMGNT1 Is Glycosylated by Mucin-Type O-Glycans.
Protein O-linked mannose β1,2-N-acetylglucosaminyltransferase 1 (POMGNT1) is a Golgi glycosyltransferase that catalyzes the formation of the N-acetylglucosamine (GlcNAc) β1→2Man linkage of O-mannosyl glycan. POMGNT1 is not modified by N-glycans because there are no potential N-glycosylation sites; however, it is not clear whether POMGNT1 is modified by O-glycans. To determine whether POMGNT1 is O-glycosylated, we prepared recombinant human POMGNT1 from HEK293T cells. The recombinant POMGNT1 was recognized by Sambucus sieboldiana lectin (SSA), and sialidase digestion of POMGNT1 decreased SSA reactivity and enhanced the reactivity of Arachis hypogaea lectin (PNA). These results suggest that POMGNT1 is modified by a sialylated core-1 O-glycan. Next, we analyzed the structures of the O-glycans on POMGNT1 by β-elimination and pyrazolone-labeling methods in combination with mass spectrometry. We identified several mucin-type O-glycans containing (NeuAc)1(Hex)1(HexNAc)1, (NeuAc)2(Hex)1(HexNAc)1, and (NeuAc)2(Hex)2(HexNAc)2. To examine whether the O-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 O-glycans.
Molecular Glycobiology, Research Team for Mechanism of Aging, Tokyo Metropolitan Geriatric Hospital and Institute of Gerontology.
Xin
X
Xin
Keiko
K
Akasaka-Manya
Hiroshi
H
Manya
Jun-ichi
J
Furukawa
Naoyuki
N
Kuwahara
Kazue
K
Okada
Hiroki
H
Tsumoto
Nobuaki
N
Higashi
Ryuichi
R
Kato
Yasuro
Y
Shinohara
Tatsuro
T
Irimura
Tamao
T
Endo
D006031:Glycosylation; D057809:HEK293 Cells; D006801:Humans; D017351:N-Acetylglucosaminyltransferases / Q000378:metabolism*; D037121:Plant Lectins / Q000378:metabolism; D011134:Polysaccharides / Q000378:metabolism*; D011994:Recombinant Proteins / Q000378:metabolism; D054788:Ribosome Inactivating Proteins / Q000378:metabolism
D037121:Plant Lectins; D011134:Polysaccharides; D011994:Recombinant Proteins; C050600:Sambucus nigra lectins; D017351:N-Acetylglucosaminyltransferases; C443106:protein O-mannose beta-1,2-N-acetylglucosaminyltransferase; D054788:Ribosome Inactivating Proteins