br Materials and methods br Results br Discussion

Materials and methods
Results
Discussion
Author contributions JH purified protein, performed vanadate inhibition experiments, crystallized, collected data, determined and refined the ATX-VO5 structure, analysed all the structures, prepared the displayed items and supervised ALHE; WJK contributed the ATX-LPA structure; ALHE was involved in ATX-VO5 crystallization; LvZ performed initial vanadate inhibition experiments; RPJ finalized the refinement of all presented structures; AP supervised research and participated in structure determination, refinement and deposition; JH and AP wrote the paper with assistance from WJK and RPJ. JH and AP contributed equally to this publication.
Author information
Acknowledgements Crystallographic experiments were performed at the PXI and PXIII beamlines at the Swiss Light Source, Paul Scherrer Institut, Villigen, Switzerland. AP and JH would like to thank the beamline scientists for excellent technical support. JH would also like to thank Drs. Mariano Stornaiuolo, Prakash Rucktooa and Wouter H. Moolenaar for helpful discussion and technical advice, and the NKI Protein Facility for access to infrastructure. This work was supported by a KWF grant to AP (NKI-2010-4781), an NWO-TOP grant to AP (700.10.354) and an NWO-Vidi grant to RPJ (723.013.003).
Introduction Serum autotaxin (ATX) is a lysophospholipase D (lysoPLD) and the major source of extracellular lysophophatidic IOX-2 (LPA) [1], [2]. ATX and LPA serum levels tightly correlate in humans and mice [3], [4], [5]. LPA mediates diverse (patho)physiological processes through 6 different G-protein coupled LPA-receptors (LPAR1–6) [6]. Numerous cell types express ATX [7], [8], [9], [10]. Increased ATX and/or LPAR expression levels are described in a yet increasing variety of tissues during embryonic development as well as disease, reviewed in [11]. Moreover, increased activity of ATX secreted in the systemic circulation was recently proposed to contribute to the pathophysiology of pruritus in various cholestatic liver diseases [12], [13] and in atopic dermatitis (AD) [14], [15], [16]. Increased serum ATX activity is a feature of uncomplicated pregnancy, but a marked elevation is diagnostic for intrahepatic cholestasis of pregnancy (ICP) [17], a cholestatic condition during pregnancy by definition accompanied by pruritus. The source of increased serum ATX activity in pruritus of cholestasis remains to be unraveled. A possible mechanism might be an increase of the actual serum ATX protein concentration, which is only supported by scarce data from cholestatic patients and pregnant women (Western blot) [12] as well as patients with atopic dermatitis (ELISA) [14]. Whereas adipose tissue is known to be a significant contributor to serum ATX levels in mice [18], the only (easily accessible) human tissue in which ATX expression was studied as a potential source of elevated serum levels is placenta. No difference in placental Atx mRNA levels between patients with ICP and pregnant controls was observed although serum ATX activity was markedly higher in ICP patients [17], virtually excluding the placenta as a major source of elevated serum ATX activity in ICP. As an alternative explanation for elevated ATX activity in cholestasis-associated pruritus such as in ICP, serum factors inhibiting or inducing the enzymatic activity of ATX might be altered during disease, as was proposed for atopic dermatitis [16]. A third optional explanation might be decreased clearance of serum ATX under cholestatic conditions, which was proposed to be mediated by sinusoidal cells in the liver [19].
Materials and methods
Results
Discussion The lysophospholipase D, autotaxin (ATX), provides targeted LPA release and signaling in a broad range of (patho)physiological processes. A physiological role of the abundant ATX in the systemic circulation has not yet been identified. In cholestasis and in atopic dermatitis serum ATX and LPA levels correlate with the intensity of pruritus (itch) [12], [13], [14], [16]. LPA is a pruritogen, as intradermal injection elicits scratching in mice [12], [15], [28], [29]. We therefore hypothesized that ATX-derived LPA plays a key role in systemic pruritus of patients with cholestatic liver disease. Till date, the origin of increased serum ATX activity during cholestasis and pregnancy was unknown. We hypothesized that increased activity could be the result of an increase in protein concentration (due to either increased production or reduced clearance) or increased enzymatic activity.