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J. Biol. Chem., Vol. 282, Issue 15, 11084-11091, April 13, 2007

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An Essential Oligomannosidic Glycan Chain in the Catalytic Domain of Autotaxin, a Secreted Lysophospholipase-D^*

Silvia Jansen¹, Nico Callewaert², Isabelle Dewerte, Maria Andries, Hugo Ceulemans³, and Mathieu Bollen⁴

From the Laboratory of Biosignaling and Therapeutics, Department of Molecular Cell Biology, Faculty of Medicine, Catholic University of Leuven, B-3000 Leuven, Belgium and Unit for Molecular Glycobiology, DMBRVIB and L-ProBE, Ghent University, B-9052 Ghent, Belgium

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 Man_8/9GlcNAc₂, which is rarely present on mature eukaryotic glycoproteins. Additional studies show that this Asn-524-linked glycan is not accessible to -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.

Received for publication, December 15, 2006 , and in revised form, February 16, 2007.

Note Added in Proof—Following the acceptance of our manuscript we have found that the removal of the essential glycan chain at Asn-524 of NPP2 by N-glycosidase F and endoglycosidase H requires the presence of EDTA, which contaminates these enzyme preparations. We noted that EDTA also increases the mobility of NPP2 during Blue-Native PAGE, indicating that it causes a major conformational change. This mobility shift was reversed by the addition of metals. These data add further support to our proposal that the Asn-524 linked glycan chain is buried inside the polypeptide chain and is only accessible to deglycosylating enzymes following denaturation or treatments, such as an incubation with EDTA, that induce major conformational changes.

^* This work was supported by the Fund for Scientific Research-Flanders (Grant G.0524.06), a Flemish Concerted Research Action, and the Prime Minister's office (IAP/V-05). The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

¹ Recipient of a Research Foundation-Flanders (FWO) Ph.D. fellowship.

² Recipient of a Marie Curie Excellence Grant (EU-FP6).

³ Recipient of an FWO postdoctoral fellowship.

⁴ To whom correspondence should be addressed: Afdeling Biochemie, Campus Gasthuisberg, KULeuven, Herestraat 49, PB901, B-3000 Leuven, Belgium. Tel.: 32-16-34-57-01; Fax: 32-16-34-59-95; E-mail: Mathieu.Bollen{at}med.kuleuven.be.

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