An essential oligomannosidic glycan chain in the catalytic domain of autotaxin, a secreted lysophospholipase-D.
Autotaxin/NPP2, a secreted lysophospholipase-D, promotes cell proliferation, survival, and motility by generating the signaling molecule lysophosphatidic acid. Here we show that ectonucleotide pyrophosphatase/phosphodiesterase 2 (NPP2) is N-glycosylated on Asn-53, Asn-410, and Asn-524. Mutagenesis and deglycosylation experiments revealed that only the glycosylation of Asn-524 is essential for the expression of the catalytic and motility-stimulating activities of NPP2. The N-glycan on Asn-524 was identified as Man8/9GlcNAc2, which is rarely present on mature eukaryotic glycoproteins. Additional studies show that this Asn-524-linked glycan is not accessible to alpha-1,2-mannosidase, suggesting that its non-reducing termini are buried inside the folded protein. Consistent with a structural role for the Asn-524-linked glycan, only the mutation of Asn-524 augmented the sensitivity of NPP2 to proteolysis and increased its mobility during Blue Native PAGE. Asn-524 is phylogenetically conserved and maps to the catalytic domain of NPP2, but a structural model of this domain suggests that Asn-524 is remote from the catalytic site. Our study defines an essential role for the Asn-524-linked glycan chain of NPP2.
Jansen
Silvia
S
Laboratory of Biosignaling and Therapeutics, Department of Molecular Cell Biology, Faculty of Medicine, Catholic University of Leuven, B-3000 Leuven, Belgium.
Callewaert
Nico
N
Dewerte
Isabelle
I
Andries
Maria
M
Ceulemans
Hugo
H
Bollen
Mathieu
M
D000595:Amino Acid Sequence; D000818:Animals; D001216:Asparagine / Q000235:genetics / Q000378:metabolism; D002236:Carbohydrate Conformation; D020134:Catalytic Domain; D002460:Cell Line; D017124:Conserved Sequence; D004789:Enzyme Activation; D006026:Glycoside Hydrolases / Q000378:metabolism; D006031:Glycosylation; D006801:Humans; D008958:Models, Molecular; D008969:Molecular Sequence Data; D009154:Mutation / Q000235:genetics; D009844:Oligosaccharides / Q000737:chemistry* / Q000378:metabolism*; D010727:Phosphoric Diester Hydrolases / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*; D011755:Pyrophosphatases / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*; D051381:Rats; D016415:Sequence Alignment; D000099189:Lysophospholipase D
D001216:Asparagine; D006026:Glycoside Hydrolases; D009844:Oligosaccharides; D010727:Phosphoric Diester Hydrolases; D011755:Pyrophosphatases; D000099189:Lysophospholipase D; C028569:oligomannoside