Autotaxin: structure-function and signaling

      Autotaxin (ATX), or ecto-nucleotide pyrophosphatase/phosphodiesterase-2, is a secreted lysophospholipase D (lysoPLD) that hydrolyzes extracellular lysophospholipids into the lipid mediator lysophosphatidic acid (LPA), a ligand for specific G protein-coupled receptors. ATX-LPA signaling is essential for development and has been implicated in a great diversity of (patho)physiological processes, ranging from lymphocyte homing to tumor progression. Structural and functional studies have revealed what makes ATX a unique lysoPLD, and how secreted ATX binds to its target cells. The ATX catalytic domain shows a characteristic bimetallic active site followed by a shallow binding groove that can accommodate nucleotides as well as the glycerol moiety of lysophospholipids, and by a deep lipid-binding pocket. In addition, the catalytic domain has an open tunnel of unknown function adjacent to the active site. Here, we discuss our current understanding of ATX structure-function relationships and signaling mechanisms, and how ATX isoforms use distinct mechanisms to target LPA production to the plasma membrane, notably binding to integrins and heparan sulfate proteoglycans. We also briefly discuss the development of drug-like inhibitors of ATX.
      Autotaxin (ATX), also known as ecto-nucleotide pyrophosphatase/phosphodiesterase (ENPP)2, is a secreted lysophospholipase D (lysoPLD) that belongs to the ENPP family. The ENPP family consists of seven members with structurally related catalytic domains that hydrolyze phosphodiester bonds in various substrates, including nucleoside triphosphates, lysophospholipids, and choline phosphate esters (
      • Bollen M.
      • Gijsbers R.
      • Ceulemans H.
      • Stalmans W.
      • Stefan C.
      Nucleotide pyrophosphatases/phosphodiesterases on the move.
      ,
      • Stefan C.
      • Jansen S.
      • Bollen M.
      NPP-type ectophosphodiesterases: unity in diversity.
      ,
      • Zimmermann H.
      • Zebisch M.
      • Strater N.
      Cellular function and molecular structure of ecto-nucleotidases.
      ). ATX produces the lipid mediator lysophosphatidic acid (LPA; mono-acyl-glycerol-3-phosphate) from extracellular lysophosphatidylcholine (LPC) (
      • Tokumura A.
      • Majima E.
      • Kariya Y.
      • Tominaga K.
      • Kogure K.
      • Yasuda K.
      • Fukuzawa K.
      Identification of human plasma lysophospholipase D, a lysophosphatidic acid-producing enzyme, as autotaxin, a multifunctional phosphodiesterase.
      ,
      • Umezu-Goto M.
      • Kishi Y.
      • Taira A.
      • Hama K.
      • Dohmae N.
      • Takio K.
      • Yamori T.
      • Mills G.B.
      • Inoue K.
      • Aoki J.
      • et al.
      Autotaxin has lysophospholipase D activity leading to tumor cell growth and motility by lysophosphatidic acid production.
      ,
      • van Meeteren L.A.
      • Moolenaar W.H.
      Regulation and biological activities of the autotaxin-LPA axis.
      ) and other (non-choline) lysophospholipids such as lysophosphatidylethanolamine and lysophosphatidylserine (
      • Aoki J.
      • Inoue A.
      • Okudaira S.
      Two pathways for lysophosphatidic acid production.
      ,
      • Bolen A.L.
      • Naren A.P.
      • Yarlagadda S.
      • Beranova-Giorgianni S.
      • Chen L.
      • Norman D.
      • Baker D.L.
      • Rowland M.M.
      • Best M.D.
      • Sano T.
      • et al.
      The phospholipase A1 activity of lysophospholipase A-I links platelet activation to LPA production during blood coagulation.
      ). ATX is widely expressed, with the highest mRNA levels detected in the brain, spinal cord, ovary, lung, intestine, and kidney. Relatively high ATX expression is also found in lymph nodes, specifically at the high endothelial venules that control lymphocyte entry (
      • Bai Z.
      • Cai L.
      • Umemoto E.
      • Takeda A.
      • Tohya K.
      • Komai Y.
      • Veeraveedu P.T.
      • Hata E.
      • Sugiura Y.
      • Kubo A.
      • et al.
      Constitutive lymphocyte transmigration across the basal lamina of high endothelial venules is regulated by the autotaxin/lysophosphatidic acid axis.
      ,
      • Kanda H.
      • Newton R.
      • Klein R.
      • Morita Y.
      • Gunn M.D.
      • Rosen S.D.
      Autotaxin, an ectoenzyme that produces lysophosphatidic acid, promotes the entry of lymphocytes into secondary lymphoid organs.
      ,
      • Umemoto E.
      • Hayasaka H.
      • Bai Z.
      • Cai L.
      • Yonekura S.
      • Peng X.
      • Takeda A.
      • Tohya K.
      • Miyasaka M.
      Novel regulators of lymphocyte trafficking across high endothelial venules.
      ).
      The bioactive product of ATX, LPA, acts on specific G protein-coupled receptors (GPCRs) that activate multiple signaling pathways (
      • van Meeteren L.A.
      • Moolenaar W.H.
      Regulation and biological activities of the autotaxin-LPA axis.
      ,
      • Choi J.W.
      • Herr D.R.
      • Noguchi K.
      • Yung Y.C.
      • Lee C.W.
      • Mutoh T.
      • Lin M.E.
      • Teo S.T.
      • Park K.E.
      • Mosley A.N.
      • et al.
      LPA receptors: subtypes and biological actions.
      ). The biological outcome of LPA signaling depends on LPA receptor (co-)expression patterns and tissue context. The best known cellular responses to LPA include the stimulation of cell migration, proliferation, and survival, but inhibitory responses have also been documented (
      • Jongsma M.
      • Matas-Rico E.
      • Rzadkowski A.
      • Jalink K.
      • Moolenaar W.H.
      LPA is a chemorepellent for B16 melanoma cells: action through the cAMP-elevating LPA5 receptor.
      ,
      • Oda S.K.
      • Strauch P.
      • Fujiwara Y.
      • Al Shami A.
      • Oravecz T.
      • Tigyi G.
      • Pelanda R.
      • Torres R.M.
      Lysophosphatidic acid inhibits CD8 T cell activation and control of tumor progression.
      ). However, the list of biological responses to LPA receptor stimulation is remarkably diverse, as it ranges from mitogenic and chemotactic activities to neurite remodeling and ion channel activation (
      • van Meeteren L.A.
      • Moolenaar W.H.
      Regulation and biological activities of the autotaxin-LPA axis.
      ,
      • Choi J.W.
      • Herr D.R.
      • Noguchi K.
      • Yung Y.C.
      • Lee C.W.
      • Mutoh T.
      • Lin M.E.
      • Teo S.T.
      • Park K.E.
      • Mosley A.N.
      • et al.
      LPA receptors: subtypes and biological actions.
      ). The ATX-LPA signaling axis is essential for vascular and neural development [reviewed in (
      • Moolenaar W.H.
      • Houben A.J.
      • Lee S.J.
      • van Meeteren L.A.
      Autotaxin in embryonic development.
      )] and has been implicated in a great variety of physiological and pathological processes, including lymphocyte homing (
      • Bai Z.
      • Cai L.
      • Umemoto E.
      • Takeda A.
      • Tohya K.
      • Komai Y.
      • Veeraveedu P.T.
      • Hata E.
      • Sugiura Y.
      • Kubo A.
      • et al.
      Constitutive lymphocyte transmigration across the basal lamina of high endothelial venules is regulated by the autotaxin/lysophosphatidic acid axis.
      ,
      • Kanda H.
      • Newton R.
      • Klein R.
      • Morita Y.
      • Gunn M.D.
      • Rosen S.D.
      Autotaxin, an ectoenzyme that produces lysophosphatidic acid, promotes the entry of lymphocytes into secondary lymphoid organs.
      ,
      • Zhang Y.
      • Chen Y.C.
      • Krummel M.F.
      • Rosen S.D.
      Autotaxin through lysophosphatidic acid stimulates polarization, motility, and transendothelial migration of naive T cells.
      ), pulmonary fibrosis (
      • Tager A.M.
      • LaCamera P.
      • Shea B.S.
      • Campanella G.S.
      • Selman M.
      • Zhao Z.
      • Polosukhin V.
      • Wain J.
      • Karimi-Shah B.A.
      • Kim N.D.
      • et al.
      The lysophosphatidic acid receptor LPA1 links pulmonary fibrosis to lung injury by mediating fibroblast recruitment and vascular leak.
      ,
      • Oikonomou N.
      • Mouratis M.A.
      • Tzouvelekis A.
      • Kaffe E.
      • Valavanis C.
      • Vilaras G.
      • Karameris A.
      • Prestwich G.D.
      • Bouros D.
      • Aidinis V.
      Pulmonary autotaxin expression contributes to the pathogenesis of pulmonary fibrosis.
      ), neuropathic pain (
      • Inoue M.
      • Rashid M.H.
      • Fujita R.
      • Contos J.J.
      • Chun J.
      • Ueda H.
      Initiation of neuropathic pain requires lysophosphatidic acid receptor signaling.
      ), cardiovascular disease (
      • Smyth S.S.
      • Mueller P.
      • Yang F.
      • Brandon J.A.
      • Morris A.J.
      Arguing the case for the autotaxin-lysophosphatidic acid-lipid phosphate phosphatase 3-signaling nexus in the development and complications of atherosclerosis.
      ), cholestatic pruritus (
      • Kremer A.E.
      • Martens J.J.
      • Kulik W.
      • Rueff F.
      • Kuiper E.M.
      • van Buuren H.R.
      • van Erpecum K.J.
      • Kondrackiene J.
      • Prieto J.
      • Rust C.
      • et al.
      Lysophosphatidic acid is a potential mediator of cholestatic pruritus.
      ), and tumor progression (
      • Houben A.J.
      • Moolenaar W.H.
      Autotaxin and LPA receptor signaling in cancer.
      ,
      • Liu S.
      • Umezu-Goto M.
      • Murph M.
      • Lu Y.
      • Liu W.
      • Zhang F.
      • Yu S.
      • Stephens L.C.
      • Cui X.
      • Murrow G.
      • et al.
      Expression of autotaxin and lysophosphatidic acid receptors increases mammary tumorigenesis, invasion, and metastases.
      ,
      • Marshall J.C.
      • Collins J.W.
      • Nakayama J.
      • Horak C.E.
      • Liewehr D.J.
      • Steinberg S.M.
      • Albaugh M.
      • Vidal-Vanaclocha F.
      • Palmieri D.
      • Barbier M.
      • et al.
      Effect of inhibition of the lysophosphatidic acid receptor 1 on metastasis and metastatic dormancy in breast cancer.
      ,
      • Peyruchaud O.
      • Leblanc R.
      • David M.
      Pleiotropic activity of lysophosphatidic acid in bone metastasis.
      ).
      ATX, named after its first discovered activity as an “autocrine motility factor” (
      • Stracke M.L.
      • Krutzsch H.C.
      • Unsworth E.J.
      • Arestad A.
      • Cioce V.
      • Schiffmann E.
      • Liotta L.A.
      Identification, purification, and partial sequence analysis of autotaxin, a novel motility-stimulating protein.
      ), is synthesized as a prepro-enzyme and, after N-glycosylation and proteolytic maturation, secreted as an active lysoPLD along the classical secretory route (
      • Jansen S.
      • Stefan C.
      • Creemers J.W.
      • Waelkens E.
      • Van Eynde A.
      • Stalmans W.
      • Bollen M.
      Proteolytic maturation and activation of autotaxin (NPP2), a secreted metastasis-enhancing lysophospholipase D.
      ,
      • Koike S.
      • Keino-Masu K.
      • Ohto T.
      • Masu M.
      The N-terminal hydrophobic sequence of autotaxin (ENPP2) functions as a signal peptide.
      ). ATX acts locally rather than systemically, although ATX is also present in the circulation where it accounts for maintaining plasma LPA levels. Circulating ATX has as a short half-life, however, as it is rapidly cleared by the liver (
      • Jansen S.
      • Andries M.
      • Vekemans K.
      • Vanbilloen H.
      • Verbruggen A.
      • Bollen M.
      Rapid clearance of the circulating metastatic factor autotaxin by the scavenger receptors of liver sinusoidal endothelial cells.
      ). Likely sources of plasma ATX are the lymphatic high endothelial venules and adipose tissue, which express and secrete ATX at high levels (
      • Kanda H.
      • Newton R.
      • Klein R.
      • Morita Y.
      • Gunn M.D.
      • Rosen S.D.
      Autotaxin, an ectoenzyme that produces lysophosphatidic acid, promotes the entry of lymphocytes into secondary lymphoid organs.
      ,
      • Dusaulcy R.
      • Rancoule C.
      • Gres S.
      • Wanecq E.
      • Colom A.
      • Guigne C.
      • van Meeteren L.A.
      • Moolenaar W.H.
      • Valet P.
      • Saulnier-Blache J.S.
      Adipose-specific disruption of autotaxin enhances nutritional fattening and reduces plasma lysophosphatidic acid.
      ). Despite much progress made in understanding ATX-LPA receptor signaling, the inner workings of ATX have long remained elusive. Recent structural studies have changed the situation and provide new insights into ATX functioning, and what makes ATX a unique lysoPLD.
      In this review, we discuss our current understanding of ATX structure-function relationships, isoforms and signaling mechanisms, and how ATX binds to its targets cells. We also briefly discuss the development of small-molecule inhibitors of ATX and their binding modes.

      LPA AND ITS RECEPTORS

      LPA acts on six distinct GPCRs (termed LPA1–6 or LPAR1–6), which are differentially expressed and show both overlapping and distinct signaling properties (
      • Choi J.W.
      • Herr D.R.
      • Noguchi K.
      • Yung Y.C.
      • Lee C.W.
      • Mutoh T.
      • Lin M.E.
      • Teo S.T.
      • Park K.E.
      • Mosley A.N.
      • et al.
      LPA receptors: subtypes and biological actions.
      ). LPA receptors can be divided into two subfamilies. The classical LPA1–3 receptors belong to the so-called “endothelial differentiation gene” (Edg) family, which includes five GPCRs for the lipid mediator sphingosine 1-phosphate (S1P) (
      • Chun J.
      • Hla T.
      • Lynch K.R.
      • Spiegel S.
      • Moolenaar W.H.
      International Union of Basic and Clinical Pharmacology. LXXVIII. Lysophospholipid Receptor Nomenclature.
      ). Three additional LPA receptors (LPA4–6) are more closely related to the purinergic receptor (P2Y) family of GPCRs (
      • Yanagida K.
      • Kurikawa Y.
      • Shimizu T.
      • Ishii S.
      Current progress in non-Edg family LPA receptor research.
      ).
      LPA and its major precursor LPC comprises various molecular species that vary in the length and degree of saturation of their fatty acid chain, which is esterified at the sn-1 (or, less common, sn-2) position of the glycerol backbone. Ether-linked 1-alkyl-LPA and 1-alkenyl-LPA species also exist, but are much less abundant. All six LPA receptors can be stimulated by 1-acyl-LPA, albeit with different potencies. Of note, some LPA receptors (LPA3 and LPA6) prefer unsaturated 2-acyl-LPA as a ligand, while LPA5 exhibits a strong preference for ether-linked 1-alkyl-LPA species (
      • Jongsma M.
      • Matas-Rico E.
      • Rzadkowski A.
      • Jalink K.
      • Moolenaar W.H.
      LPA is a chemorepellent for B16 melanoma cells: action through the cAMP-elevating LPA5 receptor.
      ,
      • Williams J.R.
      • Khandoga A.L.
      • Goyal P.
      • Fells J.I.
      • Perygin D.H.
      • Siess W.
      • Parrill A.L.
      • Tigyi G.
      • Fujiwara Y.
      Unique ligand selectivity of the GPR92/LPA5 lysophosphatidate receptor indicates role in human platelet activation.
      ). Detailed accounts of LPA receptor signaling pathways, their impact on gene expression, and biological outcomes can be found elsewhere (
      • van Meeteren L.A.
      • Moolenaar W.H.
      Regulation and biological activities of the autotaxin-LPA axis.
      ,
      • Choi J.W.
      • Herr D.R.
      • Noguchi K.
      • Yung Y.C.
      • Lee C.W.
      • Mutoh T.
      • Lin M.E.
      • Teo S.T.
      • Park K.E.
      • Mosley A.N.
      • et al.
      LPA receptors: subtypes and biological actions.
      ,
      • Stortelers C.
      • Kerkhoven R.
      • Moolenaar W.H.
      Multiple actions of LPA on fibroblasts revealed by transcriptional profiling.
      ).

      LPA PRODUCTION AND BIOAVAILABILITY

      All LPA species examined can be produced by ATX-mediated choline release from the corresponding LPC substrate. LPC is by far the most abundant lysophospholipid in plasma and serum (concentration 100–200 μM), where it is bound mainly to albumin, but also to lipoproteins and α-1-acid glycoprotein (
      • Tokumura A.
      • Nishioka Y.
      • Yoshimoto O.
      • Shinomiya J.
      • Fukuzawa K.
      Substrate specificity of lysophospholipase D which produces bioactive lysophosphatidic acids in rat plasma.
      ,
      • Ojala P.J.
      • Hermansson M.
      • Tolvanen M.
      • Polvinen K.
      • Hirvonen T.
      • Impola U.
      • Jauhiainen M.
      • Somerharju P.
      • Parkkinen J.
      Identification of alpha-1 acid glycoprotein as a lysophospholipid binding protein: a complementary role to albumin in the scavenging of lysophosphatidylcholine.
      ,
      • Croset M.
      • Brossard N.
      • Polette A.
      • Lagarde M.
      Characterization of plasma unsaturated lysophosphatidylcholines in human and rat.
      ). Lysophospholipids such as LPC and lysophosphatidylethanolamine can exist as free monomers in aqueous solutions, with critical micelle concentrations in the low micromolar range, while they bind to albumin with relatively low affinity [Kd ∼2–5 μM (
      • Thumser A.E.
      • Voysey J.E.
      • Wilton D.C.
      The binding of lysophospholipids to rat liver fatty acid-binding protein and albumin.
      ,
      • Guo S.
      • Shi X.
      • Yang F.
      • Chen L.
      • Meehan E.J.
      • Bian C.
      • Huang M.
      Structural basis of transport of lysophospholipids by human serum albumin.
      )]. Therefore, free monomeric LPC exists in dynamic equilibrium with the large carrier-bound pool(s) and most likely serves as the physiological substrate for ATX. Consistent with this, a few micromoles of free LPC is sufficient for ATX to mediate LPA receptor activation (
      • Umezu-Goto M.
      • Kishi Y.
      • Taira A.
      • Hama K.
      • Dohmae N.
      • Takio K.
      • Yamori T.
      • Mills G.B.
      • Inoue K.
      • Aoki J.
      • et al.
      Autotaxin has lysophospholipase D activity leading to tumor cell growth and motility by lysophosphatidic acid production.
      ,
      • Jongsma M.
      • Matas-Rico E.
      • Rzadkowski A.
      • Jalink K.
      • Moolenaar W.H.
      LPA is a chemorepellent for B16 melanoma cells: action through the cAMP-elevating LPA5 receptor.
      ).
      While ATX is the primary LPA-producing phospholipase, a second but less common route of LPA production involves the hydrolysis of phosphatidic acid (PA) in the outer leaflet of the plasma membrane by membrane-associated phospholipase A1 (PA-PLA1) (
      • Aoki J.
      • Inoue A.
      • Okudaira S.
      Two pathways for lysophosphatidic acid production.
      ,
      • Inoue A.
      • Arima N.
      • Ishiguro J.
      • Prestwich G.D.
      • Arai H.
      • Aoki J.
      LPA-producing enzyme PA-PLAalpha regulates hair follicle development by modulating EGFR signalling.
      ). The resulting unsaturated 2-acyl-LPA acts preferentially on the LPA6 receptor. Studies in mice show that the PA-PLA1-LPA6 signaling axis regulates hair follicle development (
      • Inoue A.
      • Arima N.
      • Ishiguro J.
      • Prestwich G.D.
      • Arai H.
      • Aoki J.
      LPA-producing enzyme PA-PLAalpha regulates hair follicle development by modulating EGFR signalling.
      ), and that ATX is dispensable for this process (
      • Grisanti L.
      • Rezza A.
      • Clavel C.
      • Sennett R.
      • Rendl M.
      Enpp2/Autotaxin in dermal papilla precursors is dispensable for hair follicle morphogenesis.
      ). It is further of note that bioactive LPA can also be produced by exogenous PLDs, notably those from Streptomyces chromofuscus (
      • van Dijk M.C.
      • Postma F.
      • Hilkmann H.
      • Jalink K.
      • van Blitterswijk W.J.
      • Moolenaar W.H.
      Exogenous phospholipase D generates lysophosphatidic acid and activates Ras, Rho and Ca2+ signaling pathways.
      ) and the toxic PLDs from spider (Loxosceles) and certain pathogenic corynebacteria (
      • van Meeteren L.A.
      • Frederiks F.
      • Giepmans B.N.
      • Pedrosa M.F.
      • Billington S.J.
      • Jost B.H.
      • Tambourgi D.V.
      • Moolenaar W.H.
      Spider and bacterial sphingomyelinases D target cellular lysophosphatidic acid receptors by hydrolyzing lysophosphatidylcholine.
      ,
      • Lee S.
      • Lynch K.R.
      Brown recluse spider (Loxosceles reclusa) venom phospholipase D (PLD) generates lysophosphatidic acid (LPA).
      ). The latter PLDs hydrolyze both sphingomyelin and various lysophospholipids to produce ceramide-1-phosphate and bioactive LPA, respectively. It is currently unclear to what extent excessive LPA production contributes to the toxicity of these exogenous PLDs.
      In a cellular context, newly produced LPA is rapidly degraded by membrane-associated lipid phosphate phosphatases that dephosphorylate LPA into biologically inactive mono-acylglycerol (
      • van der Bend R.L.
      • de Widt J.
      • van Corven E.J.
      • Moolenaar W.H.
      • van Blitterswijk W.J.
      Metabolic conversion of the biologically active phospholipid, lysophosphatidic acid, in fibroblasts.
      ,
      • Sciorra V.A.
      • Morris A.J.
      Roles for lipid phosphate phosphatases in regulation of cellular signaling.
      ,
      • Ren H.
      • Panchatcharam M.
      • Mueller P.
      • Escalante-Alcalde D.
      • Morris A.J.
      • Smyth S.S.
      Lipid phosphate phosphatase (LPP3) and vascular development.
      ,
      • Brindley D.N.
      • Pilquil C.
      Lipid phosphate phosphatases and signaling.
      ). In cell-free seminal plasma, on the other hand, LPA is degraded by soluble prostatic acid phosphatase (
      • Tanaka M.
      • Kishi Y.
      • Takanezawa Y.
      • Kakehi Y.
      • Aoki J.
      • Arai H.
      Prostatic acid phosphatase degrades lysophosphatidic acid in seminal plasma.
      ). When injected intravenously, LPA is rapidly cleared from the circulation by hepatic uptake, most likely in the liver sinusoidal endothelial cells (
      • Salous A.K.
      • Panchatcharam M.
      • Sunkara M.
      • Mueller P.
      • Dong A.
      • Wang Y.
      • Graf G.A.
      • Smyth S.S.
      • Morris A.J.
      Mechanism of rapid elimination of lysophosphatidic acid and related lipids from the circulation of mice.
      ), as is radio-labeled ATX (
      • Jansen S.
      • Andries M.
      • Vekemans K.
      • Vanbilloen H.
      • Verbruggen A.
      • Bollen M.
      Rapid clearance of the circulating metastatic factor autotaxin by the scavenger receptors of liver sinusoidal endothelial cells.
      ). The bioavailability of LPA may also be regulated by LPA-binding proteins. For example, in the presence of albumin, LPA activates LPA1 and LPA2 receptors, but not LPA3 receptors (
      • Hama K.
      • Bandoh K.
      • Kakehi Y.
      • Aoki J.
      • Arai H.
      Lysophosphatidic acid (LPA) receptors are activated differentially by biological fluids: possible role of LPA-binding proteins in activation of LPA receptors.
      ). High-affinity binding to plasma gelsolin may also influence LPA's bioavailability (
      • Goetzl E.J.
      • Lee H.
      • Azuma T.
      • Stossel T.P.
      • Turck C.W.
      • Karliner J.S.
      Gelsolin binding and cellular presentation of lysophosphatidic acid.
      ,
      • Osborn T.M.
      • Dahlgren C.
      • Hartwig J.H.
      • Stossel T.P.
      Modifications of cellular responses to lysophosphatidic acid and platelet-activating factor by plasma gelsolin.
      ). In summary then, the local level of receptor-active LPA is determined by a complex interplay between ATX, free lysophospholipid substrates, lipid phosphatases, and LPA-binding proteins.

      ATX AND ITS ISOFORMS

      ATX and its closest relatives, ENPP1 and ENPP3, are multi-domain proteins consisting of two N-terminal Cys-rich somatomedin B-like (SMB) domains, a central catalytic phosphodiesterase (PDE) domain (approximately 400 amino acids), and a C-terminal nuclease-like (NUC) domain that is catalytically inactive (Fig. 1). Despite their structural similarities, the three ENPPs have very different physiological and catalytic activities. ATX functions as a lysoPLD, ENPP1 is a membrane-bound nucleotide pyrophosphatase (
      • Stefan C.
      • Jansen S.
      • Bollen M.
      NPP-type ectophosphodiesterases: unity in diversity.
      ), and ENPP3 may preferentially hydrolyze nucleotide sugars (
      • Korekane H.
      • Park J.Y.
      • Matsumoto A.
      • Nakajima K.
      • Takamatsu S.
      • Ohtsubo K.
      • Miyamoto Y.
      • Hanashima S.
      • Kanekiyo K.
      • Kitazume S.
      • et al.
      Identification of ectonucleotide pyrophosphatase/phosphodiesterase 3 (ENPP3) as a regulator of N-acetylglucosaminyltransferase GnT-IX (GnT-Vb).
      ). Although ATX is capable of hydrolyzing nucleotides in vitro, the apparent affinity of ATX for LPC is some 10-fold higher than for nucleotides and, furthermore, extracellular nucleotide levels are normally very low. In fact, all known biological effects of ATX are attributable to LPA production and subsequent receptor stimulation. However, additional noncatalytic functions of ATX cannot be excluded, as will be discussed below.
      Figure thumbnail gr1
      Fig. 1Domain structure of the major ATX isoforms (α, β, γ, δ) compared with that of ENPP1, an ecto-nucleotide pyrophosphatase. The α, β, and γ splice variants of ATX/ENPP2 differ by the presence or absence of sequences encoded by exons 12 and 21. ATXδ lacks 12 bp at the 3′ end of exon 19, encoding four amino acids in the lasso loop or L2 linker region (
      • Hashimoto T.
      • Okudaira S.
      • Igarashi K.
      • Hama K.
      • Yatomi Y.
      • Aoki J.
      Identification and biochemical characterization of a novel autotaxin isoform, ATXδ, with a four-amino acid deletion.
      ). ATXα is the original melanoma-derived isoform, ATXβ is the canonical isoform, and ATXγ is the “brain-specific” form. The ATX-specific deletion (18 aa) in the catalytic PDE domain is key to formation of the lipid-binding pocket and open tunnel. The polybasic insertion (blue) in ATXα contains a consensus furin cleavage site as indicated (
      • Houben A.J.
      • van Wijk X.M.
      • van Meeteren L.A.
      • van Zeijl L.
      • van de Westerlo E.M.
      • Hausmann J.
      • Fish A.
      • Perrakis A.
      • van Kuppevelt T.H.
      • Moolenaar W.H.
      The polybasic insertion in autotaxin alpha confers specific binding to heparin and cell surface heparan sulfate proteoglycans.
      ). The indicated deletion (six amino acids) in the catalytic domain of ENPP1 may promote nucleotide binding (
      • Jansen S.
      • Perrakis A.
      • Ulens C.
      • Winkler C.
      • Andries M.
      • Joosten R.P.
      • Van Acker M.
      • Luyten F.P.
      • Moolenaar W.H.
      • Bollen M.
      Structure of NPP1, an ectonucleotide pyrophosphatase/phosphodiesterase involved in tissue calcification.
      ). SP, signal peptide; TM, transmembrane domain. For further details see text.
      The intricacy of ATX-LPA signaling is further enhanced by alternative splicing of ATX mRNA, which gives rise to distinct isoforms (Fig. 1). The three best known splice variants of ATX, termed α, β, and γ, differ by the presence or absence of sequences encoded by exons 12 and 21 (
      • van Meeteren L.A.
      • Moolenaar W.H.
      Regulation and biological activities of the autotaxin-LPA axis.
      ,
      • Giganti A.
      • Rodriguez M.
      • Fould B.
      • Moulharat N.
      • Coge F.
      • Chomarat P.
      • Galizzi J.P.
      • Valet P.
      • Saulnier-Blache J.S.
      • Boutin J.A.
      • et al.
      Murine and human autotaxin alpha, beta, and gamma isoforms: gene organization, tissue distribution, and biochemical characterization.
      ). A recently identified fourth isoform (ATXδ) is nearly identical to ATXβ, as it lacks four residues in the “lasso loop” or L2 linker region that connects the PDE and NUC domains (
      • Hashimoto T.
      • Okudaira S.
      • Igarashi K.
      • Hama K.
      • Yatomi Y.
      • Aoki J.
      Identification and biochemical characterization of a novel autotaxin isoform, ATXδ, with a four-amino acid deletion.
      ) (Fig. 1). However, isoform-specific functions of ATX have long remained uncharacterized. The canonical and most predominant isoform, termed ATXβ, was originally cloned from teratocarcinoma cells and later found to be identical to plasma lysoPLD (
      • Tokumura A.
      • Majima E.
      • Kariya Y.
      • Tominaga K.
      • Kogure K.
      • Yasuda K.
      • Fukuzawa K.
      Identification of human plasma lysophospholipase D, a lysophosphatidic acid-producing enzyme, as autotaxin, a multifunctional phosphodiesterase.
      ,
      • Umezu-Goto M.
      • Kishi Y.
      • Taira A.
      • Hama K.
      • Dohmae N.
      • Takio K.
      • Yamori T.
      • Mills G.B.
      • Inoue K.
      • Aoki J.
      • et al.
      Autotaxin has lysophospholipase D activity leading to tumor cell growth and motility by lysophosphatidic acid production.
      ), accounting for LPA production in the circulation, although ATXδ may contribute as well. Virtually all of our current understanding of ATX is derived from studies on ATXβ.
      The “long” isoform, termed ATXα, is the original melanoma-derived “autocrine motility factor” (
      • Stracke M.L.
      • Krutzsch H.C.
      • Unsworth E.J.
      • Arestad A.
      • Cioce V.
      • Schiffmann E.
      • Liotta L.A.
      Identification, purification, and partial sequence analysis of autotaxin, a novel motility-stimulating protein.
      ). ATXα is less widely expressed and is characterized by a 52-residue polybasic insertion (encoded by exon 12) in the heart of the catalytic domain. The “brain-specific” ATXγ isoform contains a 25-aa insertion (encoded by exon 21) close to the NUC domain (
      • Giganti A.
      • Rodriguez M.
      • Fould B.
      • Moulharat N.
      • Coge F.
      • Chomarat P.
      • Galizzi J.P.
      • Valet P.
      • Saulnier-Blache J.S.
      • Boutin J.A.
      • et al.
      Murine and human autotaxin alpha, beta, and gamma isoforms: gene organization, tissue distribution, and biochemical characterization.
      ,
      • Kawagoe H.
      • Soma O.
      • Goji J.
      • Nishimura N.
      • Narita M.
      • Inazawa J.
      • Nakamura H.
      • Sano K.
      Molecular cloning and chromosomal assignment of the human brain-type phosphodiesterase I/nucleotide pyrophosphatase gene (PDNP2).
      ,
      • Fuss B.
      • Baba H.
      • Phan T.
      • Tuohy V.K.
      • Macklin W.B.
      Phosphodiesterase I, a novel adhesion molecule and/or cytokine involved in oligodendrocyte function.
      ). It is currently unclear whether any of the ATX isoforms are specifically associated with a distinct (patho)physiological condition.
      All ATX isoforms display similar lysoPLD activities and substrate preferences (
      • Giganti A.
      • Rodriguez M.
      • Fould B.
      • Moulharat N.
      • Coge F.
      • Chomarat P.
      • Galizzi J.P.
      • Valet P.
      • Saulnier-Blache J.S.
      • Boutin J.A.
      • et al.
      Murine and human autotaxin alpha, beta, and gamma isoforms: gene organization, tissue distribution, and biochemical characterization.
      ,
      • Hashimoto T.
      • Okudaira S.
      • Igarashi K.
      • Hama K.
      • Yatomi Y.
      • Aoki J.
      Identification and biochemical characterization of a novel autotaxin isoform, ATXδ, with a four-amino acid deletion.
      ,
      • Houben A.J.
      • van Wijk X.M.
      • van Meeteren L.A.
      • van Zeijl L.
      • van de Westerlo E.M.
      • Hausmann J.
      • Fish A.
      • Perrakis A.
      • van Kuppevelt T.H.
      • Moolenaar W.H.
      The polybasic insertion in autotaxin alpha confers specific binding to heparin and cell surface heparan sulfate proteoglycans.
      ). ATX-catalyzed substrate hydrolysis follows simple Michaelis-Menten kinetics, with no evidence for an interfacial activation mechanism and consistent with the notion that ATX acts on free lysophospholipids in the aqueous phase. However, there are significant differences in kinetic parameters between natural and artificial substrates, such as LPC versus fluorescent analogs (
      • Ferguson C.G.
      • Bigman C.S.
      • Richardson R.D.
      • van Meeteren L.A.
      • Moolenaar W.H.
      • Prestwich G.D.
      Fluorogenic phospholipid substrate to detect lysophospholipase D/autotaxin activity.
      ,
      • Saunders L.P.
      • Cao W.
      • Chang W.C.
      • Albright R.A.
      • Braddock D.T.
      • De La Cruz E.M.
      Kinetic analysis of autotaxin reveals substrate-specific catalytic pathways and a mechanism for lysophosphatidic acid distribution.
      ).
      Because the insertions in ATXα and ATXγ appear not to affect catalytic activity, they could serve to confer distinct cellular localization, processing, or binding partner preference. On the basis of the ATXβ structure (see below), it is safe to conclude that those insertions do not perturb the structure of the mature ATXα and ATXγ proteins. We recently showed that ATXα binds specifically to negatively charged heparin with high affinity (Kd ∼10−8 M), most likely through the Arg/Lys-rich clusters in the insertion, whereas ATXβ does not (
      • Houben A.J.
      • van Wijk X.M.
      • van Meeteren L.A.
      • van Zeijl L.
      • van de Westerlo E.M.
      • Hausmann J.
      • Fish A.
      • Perrakis A.
      • van Kuppevelt T.H.
      • Moolenaar W.H.
      The polybasic insertion in autotaxin alpha confers specific binding to heparin and cell surface heparan sulfate proteoglycans.
      ). Heparin enhanced the lysoPLD activity of ATXα toward LPC up to 2-fold, but it had no detectable effect on the activity of ATXβ (
      • Houben A.J.
      • van Wijk X.M.
      • van Meeteren L.A.
      • van Zeijl L.
      • van de Westerlo E.M.
      • Hausmann J.
      • Fish A.
      • Perrakis A.
      • van Kuppevelt T.H.
      • Moolenaar W.H.
      The polybasic insertion in autotaxin alpha confers specific binding to heparin and cell surface heparan sulfate proteoglycans.
      ).
      Heparin is structurally very similar to heparan sulfate (HS), which is present as HS proteoglycans (HSPGs) at the cell surface and in extracellular matrices, where it recruits growth factors, chemokines, and other molecules to fine tune signaling events (
      • Kreuger J.
      • Spillmann D.
      • Li J.P.
      • Lindahl U.
      Interactions between heparan sulfate and proteins: the concept of specificity.
      ). Indeed, ATXα, but not ATXβ, was found to bind abundantly to cultured mammalian cells in a manner strictly dependent on HS (
      • Houben A.J.
      • van Wijk X.M.
      • van Meeteren L.A.
      • van Zeijl L.
      • van de Westerlo E.M.
      • Hausmann J.
      • Fish A.
      • Perrakis A.
      • van Kuppevelt T.H.
      • Moolenaar W.H.
      The polybasic insertion in autotaxin alpha confers specific binding to heparin and cell surface heparan sulfate proteoglycans.
      ). Thus, by mediating bindings to HSPGs, the ATXα insertion loop likely serves to target LPA production close to the LPA receptors. The insertion loop also contains a conserved furin cleavage site, but the functional significance of ATXα cleavage at this site is unclear (
      • Houben A.J.
      • van Wijk X.M.
      • van Meeteren L.A.
      • van Zeijl L.
      • van de Westerlo E.M.
      • Hausmann J.
      • Fish A.
      • Perrakis A.
      • van Kuppevelt T.H.
      • Moolenaar W.H.
      The polybasic insertion in autotaxin alpha confers specific binding to heparin and cell surface heparan sulfate proteoglycans.
      ). As heparin stimulates ATXα activity in vitro, binding of ATXα to HSPGs may not only target LPA production to the plasma membrane, but also increase the catalytic efficiency of this particular isoform locally. Cocrystallization studies using defined heparin fragments should help determine the precise heparin/HS-binding mode of ATXα.

      ATX STRUCTURE-FUNCTION

      Structural studies have revealed how the different domains of ATX are organized and interact, and what makes ATX a unique lysoPLD (
      • Hausmann J.
      • Kamtekar S.
      • Christodoulou E.
      • Day J.E.
      • Wu T.
      • Fulkerson Z.
      • Albers H.M.
      • van Meeteren L.A.
      • Houben A.J.
      • van Zeijl L.
      • et al.
      Structural basis of substrate discrimination and integrin binding by autotaxin.
      ,
      • Nishimasu H.
      • Okudaira S.
      • Hama K.
      • Mihara E.
      • Dohmae N.
      • Inoue A.
      • Ishitani R.
      • Takagi J.
      • Aoki J.
      • Nureki O.
      Crystal structure of autotaxin and insight into GPCR activation by lipid mediators.
      ). The crystal structure of ATXβ (Fig. 2) shows that the central catalytic PDE domain interacts extensively with the SMB domains on one side and with the NUC domain on the other. This interaction is strengthened by an N-linked glycan between both domains and by an inter-domain disulfide bridge. In addition, a long “lasso” loop, starting at the end of the PDE domain, wraps tightly around the NUC domain and enters the NUC fold from the opposite side. All these features serve to maintain the structural rigidity of the ATX catalytic domain (
      • Hausmann J.
      • Kamtekar S.
      • Christodoulou E.
      • Day J.E.
      • Wu T.
      • Fulkerson Z.
      • Albers H.M.
      • van Meeteren L.A.
      • Houben A.J.
      • van Zeijl L.
      • et al.
      Structural basis of substrate discrimination and integrin binding by autotaxin.
      ,
      • Nishimasu H.
      • Okudaira S.
      • Hama K.
      • Mihara E.
      • Dohmae N.
      • Inoue A.
      • Ishitani R.
      • Takagi J.
      • Aoki J.
      • Nureki O.
      Crystal structure of autotaxin and insight into GPCR activation by lipid mediators.
      ). The NUC domain tightly binds Ca2 via an EF hand-like motif, the precise function of which remains to be determined (
      • Hausmann J.
      • Kamtekar S.
      • Christodoulou E.
      • Day J.E.
      • Wu T.
      • Fulkerson Z.
      • Albers H.M.
      • van Meeteren L.A.
      • Houben A.J.
      • van Zeijl L.
      • et al.
      Structural basis of substrate discrimination and integrin binding by autotaxin.
      ).
      Figure thumbnail gr2
      Fig. 2The ATX structure shown as a semi-transparent surface. The two N-terminal SMB domains are shown in dark and light magenta, the PDE domain in green, and the NUC domain in light blue. The approximate volumes of the shallow groove, the open tunnel, and the hydrophobic pocket are represented as cyan, dark blue, and orange solid surfaces, respectively. A phosphate ion trapped in the active site is shown as a stick model. The volumes of the groove, pocket, and tunnel were traced manually in Chimera (
      • Pettersen E.F.
      • Goddard T.D.
      • Huang C.C.
      • Couch G.S.
      • Greenblatt D.M.
      • Meng E.C.
      • Ferrin T.E.
      UCSF Chimera–a visualization system for exploratory research and analysis.
      ), using pseudo-atoms placed on the protein surface to approximate the volume.

       A shallow groove and a deep hydrophobic pocket

      The catalytic domain of ATX and that of ENPP1, whose structure was also recently determined (
      • Jansen S.
      • Perrakis A.
      • Ulens C.
      • Winkler C.
      • Andries M.
      • Joosten R.P.
      • Van Acker M.
      • Luyten F.P.
      • Moolenaar W.H.
      • Bollen M.
      Structure of NPP1, an ectonucleotide pyrophosphatase/phosphodiesterase involved in tissue calcification.
      ,
      • Kato K.
      • Nishimasu H.
      • Okudaira S.
      • Mihara E.
      • Ishitani R.
      • Takagi J.
      • Aoki J.
      • Nureki O.
      Crystal structure of Enpp1, an extracellular glycoprotein involved in bone mineralization and insulin signaling.
      ), is structurally similar to that of the prototypic nucleotide pyrophosphatase/phosphodiesterase (NPP) from Xanthomonas axonopodis (XaNPP), an evolutionary relative of alkaline phosphatases (
      • Zalatan J.G.
      • Fenn T.D.
      • Brunger A.T.
      • Herschlag D.
      Structural and functional comparisons of nucleotide pyrophosphatase/phosphodiesterase and alkaline phosphatase: implications for mechanism and evolution.
      ). The catalytic sites in ATX, ENPP1, and XaNPP are almost identical, with the nucleophile Thr in proximity with two zinc ions, coordinated by conserved His and Asp residues. All three enzymes share a shallow groove, capable of binding the substrate nucleotides and directing the phosphodiester bond for catalysis, as revealed by the structures of XaNPP and ENPP1 in complex with various nucleotides. The same shallow groove also accommodates the glycerol moiety of lysophospholipid substrates (
      • Nishimasu H.
      • Okudaira S.
      • Hama K.
      • Mihara E.
      • Dohmae N.
      • Inoue A.
      • Ishitani R.
      • Takagi J.
      • Aoki J.
      • Nureki O.
      Crystal structure of autotaxin and insight into GPCR activation by lipid mediators.
      ). Yet the glycerol moiety is not critical for recognition by ATX because a nonglycero-lysophospholipid, notably sphingosylphosphorylcholine, also can serve as a good substrate for ATX (
      • Clair T.
      • Aoki J.
      • Koh E.
      • Bandle R.W.
      • Nam S.W.
      • Ptaszynska M.M.
      • Mills G.B.
      • Schiffmann E.
      • Liotta L.A.
      • Stracke M.L.
      Autotaxin hydrolyzes sphingosylphosphorylcholine to produce the regulator of migration, sphingosine-1-phosphate.
      ).
      The ATX catalytic domain is unique in that it has evolved a deep hydrophobic pocket that is not found in ENPP1 (
      • Jansen S.
      • Perrakis A.
      • Ulens C.
      • Winkler C.
      • Andries M.
      • Joosten R.P.
      • Van Acker M.
      • Luyten F.P.
      • Moolenaar W.H.
      • Bollen M.
      Structure of NPP1, an ectonucleotide pyrophosphatase/phosphodiesterase involved in tissue calcification.
      ,
      • Kato K.
      • Nishimasu H.
      • Okudaira S.
      • Mihara E.
      • Ishitani R.
      • Takagi J.
      • Aoki J.
      • Nureki O.
      Crystal structure of Enpp1, an extracellular glycoprotein involved in bone mineralization and insulin signaling.
      ) or, to the best of our knowledge, in any other phospholipase. This pocket has an estimated volume of about 800 Å3, is about 15 Å deep, and is located down from the shallow groove, sculpted inside the hydrophobic core of the catalytic domain (Fig. 2 and Fig. 3). Formation of this pocket has been made possible by the deletion of a 18-aa stretch, unique to ATX. This was apparently the crucial evolutionary event that allowed ATX to function specifically as a lysoPLD. The pocket can readily accommodate lysophospholipids as shown by cocrystal structures of ATX and various LPA species, and confirmed by mutational and biochemical studies (
      • Nishimasu H.
      • Okudaira S.
      • Hama K.
      • Mihara E.
      • Dohmae N.
      • Inoue A.
      • Ishitani R.
      • Takagi J.
      • Aoki J.
      • Nureki O.
      Crystal structure of autotaxin and insight into GPCR activation by lipid mediators.
      ). It appears that the acyl chains of LPA adopt distinct conformations in the pocket, while the pocket itself shows some structural plasticity (
      • Nishimasu H.
      • Okudaira S.
      • Hama K.
      • Mihara E.
      • Dohmae N.
      • Inoue A.
      • Ishitani R.
      • Takagi J.
      • Aoki J.
      • Nureki O.
      Crystal structure of autotaxin and insight into GPCR activation by lipid mediators.
      ). The short-chain, saturated species, 14:0 and 16:0, are readily accommodated in the pocket, whereas 18:0 does not seem to fit. In contrast, 18:1 and 18:3 bend at the unsaturated bonds, while unsaturated 22:6 adopts a U-shaped conformation in the pocket (
      • Nishimasu H.
      • Okudaira S.
      • Hama K.
      • Mihara E.
      • Dohmae N.
      • Inoue A.
      • Ishitani R.
      • Takagi J.
      • Aoki J.
      • Nureki O.
      Crystal structure of autotaxin and insight into GPCR activation by lipid mediators.
      ). It remains to be seen if the observed conformations of bound LPA are identical to those adopted by the respective LPC substrates, or whether they represent an intermediate conformation prior to LPA release.
      Figure thumbnail gr3
      Fig. 3A close-up of the lipid-binding pocket and open tunnel in ATX, shown as solid surfaces colored according to hydrophobicity (blue, hydrophilic; red, hydrophobic). The left panel emphasizes the deep hydrophobic pocket for lipid binding in contrast with the hydrophilic shallow groove. The right panel focuses on the open tunnel, showing both hydrophobic and hydrophilic features. The picture was generated using Chimera software (
      • Pettersen E.F.
      • Goddard T.D.
      • Huang C.C.
      • Couch G.S.
      • Greenblatt D.M.
      • Meng E.C.
      • Ferrin T.E.
      UCSF Chimera–a visualization system for exploratory research and analysis.
      ).
      The lipid-binding pocket and active site in ATX appear freely accessible to solvent and lipid substrates (
      • Hausmann J.
      • Kamtekar S.
      • Christodoulou E.
      • Day J.E.
      • Wu T.
      • Fulkerson Z.
      • Albers H.M.
      • van Meeteren L.A.
      • Houben A.J.
      • van Zeijl L.
      • et al.
      Structural basis of substrate discrimination and integrin binding by autotaxin.
      ,
      • Nishimasu H.
      • Okudaira S.
      • Hama K.
      • Mihara E.
      • Dohmae N.
      • Inoue A.
      • Ishitani R.
      • Takagi J.
      • Aoki J.
      • Nureki O.
      Crystal structure of autotaxin and insight into GPCR activation by lipid mediators.
      ). This contrasts to the canonical phospholipases that function as interfacial enzymes, where the active site is occluded by a lid or an auto-inhibitory loop that is removed upon interaction with membranes, thereby allowing the lipid substrate to bind. Typical examples of interfacial enzymes are the secreted PLA2s (
      • Lambeau G.
      • Gelb M.H.
      Biochemistry and physiology of mammalian secreted phospholipases A2.
      ), where catalysis involves facilitated phospholipid diffusion from the membrane bilayer directly into the catalytic site (
      • Scott D.L.
      • White S.P.
      • Otwinowski Z.
      • Yuan W.
      • Gelb M.H.
      • Sigler P.B.
      Interfacial catalysis: the mechanism of phospholipase A2.
      ). But ATX would not need a deep lipid-binding pocket if catalysis would take place at the membrane-water interface (or carrier surface, for that matter). Once free LPC is bound in the ATX pocket, catalysis may be driven through “substrate destabilization.” In this process, the choline head group in close proximity to the zinc ions destabilizes the complex and lowers the activation energy barrier of the PDE reaction (
      • North E.J.
      • Osborne D.A.
      • Bridson P.K.
      • Baker D.L.
      • Parrill A.L.
      Autotaxin structure-activity relationships revealed through lysophosphatidylcholine analogs.
      ).

       An open tunnel

      An unexpected and intriguing feature of the ATX catalytic domain is the presence of an open tunnel (or “channel”), which forms a sort of “T-junction” with the shallow groove (
      • Hausmann J.
      • Kamtekar S.
      • Christodoulou E.
      • Day J.E.
      • Wu T.
      • Fulkerson Z.
      • Albers H.M.
      • van Meeteren L.A.
      • Houben A.J.
      • van Zeijl L.
      • et al.
      Structural basis of substrate discrimination and integrin binding by autotaxin.
      ,
      • Nishimasu H.
      • Okudaira S.
      • Hama K.
      • Mihara E.
      • Dohmae N.
      • Inoue A.
      • Ishitani R.
      • Takagi J.
      • Aoki J.
      • Nureki O.
      Crystal structure of autotaxin and insight into GPCR activation by lipid mediators.
      ). This tunnel is absent in ENPP1 and the bacterial NPP. The walls of this tunnel are formed partially by the catalytic domain, and partially by the first SMB domain. The deletion of the 18-aa stretch in ATX, responsible for the formation of the lipid-binding pocket, may also have facilitated the formation of this tunnel, allowing the interaction with the SMB1 domain. The ATX tunnel is partially hydrophobic in nature, but at least one of its walls is hydrophilic (Fig. 3). It has been suggested that the tunnel functions as an LPA exit channel, as inferred from the observed electron density maps that allowed modeling of LPA acyl chains (
      • Nishimasu H.
      • Okudaira S.
      • Hama K.
      • Mihara E.
      • Dohmae N.
      • Inoue A.
      • Ishitani R.
      • Takagi J.
      • Aoki J.
      • Nureki O.
      Crystal structure of autotaxin and insight into GPCR activation by lipid mediators.
      ,
      • Moolenaar W.H.
      • Perrakis A.
      Insights into autotaxin: how to produce and present a lipid mediator.
      ). This could allow delivery of LPA to its cognate GPCRs. Although attractive, this model remains to be validated experimentally. Delivery of LPA from the pocket into the tunnel involves a relatively long path, in which case significant structural rearrangements must occur. Given the nature and the size of the tunnel, a variety of molecules may also be accommodated, at least transiently. Its proximity to the active site suggests implications for catalysis and/or product uptake and delivery.

       The SMB domains and integrin binding

      SMB domains are known to mediate protein-protein interactions. The two N-terminal SMB domains in ATX are of special interest as they interact extensively with the catalytic domain. Both SMB domains are structurally similar, but have differential intra- and inter-molecular inter­actions. While SMB1 is involved in the formation of the tunnel, the SMB2 surface is in the best position to engage with other binding partners (
      • Hausmann J.
      • Kamtekar S.
      • Christodoulou E.
      • Day J.E.
      • Wu T.
      • Fulkerson Z.
      • Albers H.M.
      • van Meeteren L.A.
      • Houben A.J.
      • van Zeijl L.
      • et al.
      Structural basis of substrate discrimination and integrin binding by autotaxin.
      ,
      • Fulkerson Z.
      • Wu T.
      • Sunkara M.
      • Kooi C.V.
      • Morris A.J.
      • Smyth S.S.
      Binding of autotaxin to integrins localizes lysophosphatidic acid production to platelets and mammalian cells.
      ). Indeed, ATX binds to activated platelets and other cells through β1 and β3 integrins via its N-terminal SMB2 domain (
      • Fulkerson Z.
      • Wu T.
      • Sunkara M.
      • Kooi C.V.
      • Morris A.J.
      • Smyth S.S.
      Binding of autotaxin to integrins localizes lysophosphatidic acid production to platelets and mammalian cells.
      ,
      • Pamuklar Z.
      • Federico L.
      • Liu S.
      • Umezu-Goto M.
      • Dong A.
      • Panchatcharam M.
      • Fulerson Z.
      • Berdyshev E.
      • Natarajan V.
      • Fang X.
      • et al.
      Autotaxin/lysopholipase D and lysophosphatidic acid regulate murine hemostasis and thrombosis.
      ). ATX-integrin binding provides one mechanism to localize LPA production to the cell surface. The SMB2 domain contains a canonical RGD integrin-binding motif, but mutagenesis studies revealed that ATX-integrin interaction is RGD-independent (
      • Hausmann J.
      • Kamtekar S.
      • Christodoulou E.
      • Day J.E.
      • Wu T.
      • Fulkerson Z.
      • Albers H.M.
      • van Meeteren L.A.
      • Houben A.J.
      • van Zeijl L.
      • et al.
      Structural basis of substrate discrimination and integrin binding by autotaxin.
      ). In fact, the structural context of the RGD motif lacks the required flexibility to bind integrins. Precisely how SMB2 interacts with integrin β subunits remains to be explored.
      ATX also binds to activated lymphocytes via integrins, an interaction that leads to enhanced lymphocyte motility in response to localized LPA production (
      • Bai Z.
      • Cai L.
      • Umemoto E.
      • Takeda A.
      • Tohya K.
      • Komai Y.
      • Veeraveedu P.T.
      • Hata E.
      • Sugiura Y.
      • Kubo A.
      • et al.
      Constitutive lymphocyte transmigration across the basal lamina of high endothelial venules is regulated by the autotaxin/lysophosphatidic acid axis.
      ,
      • Kanda H.
      • Newton R.
      • Klein R.
      • Morita Y.
      • Gunn M.D.
      • Rosen S.D.
      Autotaxin, an ectoenzyme that produces lysophosphatidic acid, promotes the entry of lymphocytes into secondary lymphoid organs.
      ,
      • Zhang Y.
      • Chen Y.C.
      • Krummel M.F.
      • Rosen S.D.
      Autotaxin through lysophosphatidic acid stimulates polarization, motility, and transendothelial migration of naive T cells.
      ). The binding site on ATX is unknown, but it is of note that a known integrin-binding motif (LDV) on the surface of the PDE domain is exposed and probably accessible for integrin interaction (
      • Moolenaar W.H.
      • Perrakis A.
      Insights into autotaxin: how to produce and present a lipid mediator.
      ). In conclusion, the available evidence supports a model in which distinct domains mediate ATX binding to integrins, thus serving to localize ATX to the plasma membrane (
      • Fulkerson Z.
      • Wu T.
      • Sunkara M.
      • Kooi C.V.
      • Morris A.J.
      • Smyth S.S.
      Binding of autotaxin to integrins localizes lysophosphatidic acid production to platelets and mammalian cells.
      ).
      Integrins not only function as cell-matrix adhesion sites, but also mediate outside-in signaling events. Recent evidence suggests that ATX may exert noncatalytic signaling functions via cell-surface integrins. Specifically, ATX-mediated directional migration of MDA-MB-231 carcinoma cells was not completely abrogated upon full inhibition of lysoPLD activity (
      • Wu T.
      • Kooi C.V.
      • Shah P.
      • Charnigo R.
      • Huang C.
      • Smyth S.S.
      • Morris A.J.
      Integrin-mediated cell surface recruitment of autotaxin promotes persistent directional cell migration.
      ). Furthermore, the isolated SMB domains of ATX were still capable of stimulating cell migration, although the effect was rather modest (
      • Wu T.
      • Kooi C.V.
      • Shah P.
      • Charnigo R.
      • Huang C.
      • Smyth S.S.
      • Morris A.J.
      Integrin-mediated cell surface recruitment of autotaxin promotes persistent directional cell migration.
      ). Collectively, the data support the notion that ATX may exert LPA-independent functions in an integrin-dependent manner, a scenario that warrants further investigation.

      ATX AS A DRUG TARGET: SMALL-MOLECULE INHIBITORS

      Given the involvement of ATX-LPA receptor signaling in several pathologies, it is logical that much effort has been spent in developing specific ATX inhibitors both in academia and in industry (
      • Barbayianni E.
      • Magrioti V.
      • Moutevelis-Minakakis P.
      • Kokotos G.
      Autotaxin inhibitors: a patent review.
      ). As an extracelullar PDE, ATX is an attractive and highly druggable target. Some first-generation ATX inhibitors have been based on the finding that LPA and S1P can inhibit ATX activity against very low LPC concentrations, nucleotides, and artificial substrates (Ki about 100 nM) (
      • van Meeteren L.A.
      • Ruurs P.
      • Christodoulou E.
      • Goding J.W.
      • Takakusa H.
      • Kikuchi K.
      • Perrakis A.
      • Nagano T.
      • Moolenaar W.H.
      Inhibition of autotaxin by lysophosphatidic acid and sphingosine 1-phosphate.
      ). However, as we discussed previously, there is no evidence that ATX is subject to product inhibition by LPA under physiological conditions (
      • Moolenaar W.H.
      • Perrakis A.
      Insights into autotaxin: how to produce and present a lipid mediator.
      ). Nevertheless, a number of nonhydrolyzable LPA analogs have been developed as ATX inhibitors, but their potency is very poor when tested in ATX-mediated LPC hydrolysis assays. It therefore seems unlikely that the reported in vivo effects of those LPA analogs are attributable to ATX inhibition. In fact, LPA analogs are rapidly cleared from the circulation and have as yet not been shown to lower plasma LPA levels (
      • Salous A.K.
      • Panchatcharam M.
      • Sunkara M.
      • Mueller P.
      • Dong A.
      • Wang Y.
      • Graf G.A.
      • Smyth S.S.
      • Morris A.J.
      Mechanism of rapid elimination of lysophosphatidic acid and related lipids from the circulation of mice.
      ).
      Non-lipid small-molecule inhibitors obviously hold more promise. High-throughput screening has identified thiazolidinedione-based compounds as a new class of ATX inhibitors (
      • Albers H.M.
      • Dong A.
      • van Meeteren L.A.
      • Egan D.A.
      • Sunkara M.
      • van Tilburg E.W.
      • Schuurman K.
      • van Tellingen O.
      • Morris A.J.
      • Smyth S.S.
      • et al.
      Boronic acid-based inhibitor of autotaxin reveals rapid turnover of LPA in the circulation.
      ). Their potency was increased dramatically by introduction of a boronic acid moiety, designed to target the catalytic Thr residue in ATX. Similar results were obtained with compounds from an independent screen (
      • Kawaguchi M.
      • Okabe T.
      • Okudaira S.
      • Nishimasu H.
      • Ishitani R.
      • Kojima H.
      • Nureki O.
      • Aoki J.
      • Nagano T.
      Screening and X-ray crystal structure-based optimization of autotaxin (ENPP2) inhibitors, using a newly developed fluorescence probe.
      ). Injection of a boronic acid-based ATX inhibitor into mice results in a rapid fall in plasma LPA levels (
      • Albers H.M.
      • Dong A.
      • van Meeteren L.A.
      • Egan D.A.
      • Sunkara M.
      • van Tilburg E.W.
      • Schuurman K.
      • van Tellingen O.
      • Morris A.J.
      • Smyth S.S.
      • et al.
      Boronic acid-based inhibitor of autotaxin reveals rapid turnover of LPA in the circulation.
      ), consistent with LPA being rapidly cleared from the circulation (
      • Salous A.K.
      • Panchatcharam M.
      • Sunkara M.
      • Mueller P.
      • Dong A.
      • Wang Y.
      • Graf G.A.
      • Smyth S.S.
      • Morris A.J.
      Mechanism of rapid elimination of lysophosphatidic acid and related lipids from the circulation of mice.
      ). The crystal structure of ATX in complex with a boronic acid-based inhibitor (HA155) revealed that it forms a reversibly covalent bond with the Thr nucleophile in the lipid-binding pocket of ATX (
      • Hausmann J.
      • Kamtekar S.
      • Christodoulou E.
      • Day J.E.
      • Wu T.
      • Fulkerson Z.
      • Albers H.M.
      • van Meeteren L.A.
      • Houben A.J.
      • van Zeijl L.
      • et al.
      Structural basis of substrate discrimination and integrin binding by autotaxin.
      ). Another small-molecule inhibitor of ATX, termed PF-8380, shows adequate oral bioavailability and potency in reducing LPA levels in plasma and at sites of inflammation (
      • Gierse J.
      • Thorarensen A.
      • Beltey K.
      • Bradshaw-Pierce E.
      • Cortes-Burgos L.
      • Hall T.
      • Johnston A.
      • Murphy M.
      • Nemirovskiy O.
      • Ogawa S.
      • et al.
      A novel autotaxin inhibitor reduces lysophosphatidic acid levels in plasma and the site of inflammation.
      ), but precisely how the compound binds to ATX is unknown.
      The tripartite ATX binding site, featuring the groove, pocket, and surface, offers much potential for selective and specific inhibitors. While both the groove and pocket of ATX are well characterized for their binding of substrates and inhibitors, the open tunnel may be an attractive target for inhibitor design as well. Figure 4 shows characteristic bindings poses for lipids and inhibitors in ATX, and for nucleotides in ENPP1. The lipid and nucleotide products bound to ATX and ENPP1 suggest that many additional cores can be envisaged in the further development of inhibitors [see also (
      • Fells J.I.
      • Lee S.C.
      • Fujiwara Y.
      • Norman D.D.
      • Lim K.G.
      • Tsukahara R.
      • Liu J.
      • Patil R.
      • Miller D.D.
      • Kirby R.J.
      • et al.
      Hits of a high-throughput screen identify the hydrophobic pocket of autotaxin/lysophospholipase D as an inhibitory surface.
      )]. The ATX inhibitors structurally characterized until now share common characteristics and explore the hydrophobic pocket with fluoro-benzene and chloro-benzene moieties. The reversible covalent bond of the boron atom with the Thr nucleophile is a special case that has not been explored in drug development, with the notable exception of the proteasome inhibitor Bortezomib (
      • Groll M.
      • Berkers C.R.
      • Ploegh H.L.
      • Ovaa H.
      Crystal structure of the boronic acid-based proteasome inhibitor bortezomib in complex with the yeast 20S proteasome.
      ). As ATX is an extracellular target, boron-based chemistry might be well tolerated in lead compounds and drug candidates in vivo. Novel small-molecule inhibitors with adequate pharmacokinetics should serve as useful tools for elucidating the role of ATX in normal physiology and pathophysiology in mice, and hopefully may be applied in future clinical studies.
      Figure thumbnail gr4
      Fig. 4Structures of various substrates and inhibitors bound to ATX/ENPP2 and ENPP1. Top row, structures of distinct LPA species bound to ATX (
      • Nishimasu H.
      • Okudaira S.
      • Hama K.
      • Mihara E.
      • Dohmae N.
      • Inoue A.
      • Ishitani R.
      • Takagi J.
      • Aoki J.
      • Nureki O.
      Crystal structure of autotaxin and insight into GPCR activation by lipid mediators.
      ). Middle row, structures of small-molecule inhibitors bound to ATX (
      • Albers H.M.
      • Dong A.
      • van Meeteren L.A.
      • Egan D.A.
      • Sunkara M.
      • van Tilburg E.W.
      • Schuurman K.
      • van Tellingen O.
      • Morris A.J.
      • Smyth S.S.
      • et al.
      Boronic acid-based inhibitor of autotaxin reveals rapid turnover of LPA in the circulation.
      ,
      • Kawaguchi M.
      • Okabe T.
      • Okudaira S.
      • Nishimasu H.
      • Ishitani R.
      • Kojima H.
      • Nureki O.
      • Aoki J.
      • Nagano T.
      Screening and X-ray crystal structure-based optimization of autotaxin (ENPP2) inhibitors, using a newly developed fluorescence probe.
      ). Bottom row, structures of nucleotides bound to ENPP1 (
      • Kato K.
      • Nishimasu H.
      • Okudaira S.
      • Mihara E.
      • Ishitani R.
      • Takagi J.
      • Aoki J.
      • Nureki O.
      Crystal structure of Enpp1, an extracellular glycoprotein involved in bone mineralization and insulin signaling.
      ). The structures shown are available in the PDB. The structure of 3BoA (
      • Kawaguchi M.
      • Okabe T.
      • Okudaira S.
      • Nishimasu H.
      • Ishitani R.
      • Kojima H.
      • Nureki O.
      • Aoki J.
      • Nagano T.
      Screening and X-ray crystal structure-based optimization of autotaxin (ENPP2) inhibitors, using a newly developed fluorescence probe.
      ) has been re-refined and manually adjusted to impose the correct geometry of the ligand (
      • Joosten R.P.
      • Joosten K.
      • Murshudov G.N.
      • Perrakis A.
      PDB_REDO: constructive validation, more than just looking for errors.
      ) (A. Perrakis, unpublished observations communicated to the authors of the original publication).

      CONCLUDING REMARKS

      ATX is arguably the most fascinating member of the ENPP family, as it is the major LPA-producing enzyme being involved in a great diversity of physiological and pathological processes. Crystal structures of ATX have answered many outstanding questions but, naturally, also have raised many new ones. On the basis of current evidence, Fig. 5 summarizes the domain structure, substrate preference, and cell-binding modes of ATX and its closest relatives, ENPP1 and ENNP3. Binding of ATX to activated integrins via its N-terminal SMB2 domain provides one mechanism for localized production of LPA close to its cognate receptors. Binding of ATXα to cell-surface HSPGs, via its polybasic insertion loop, represents an additional isoform-specific mechanism for spatially and temporally restricted LPA production. Precisely how ATX activity is regulated, what the structural determinants of ATX-integrin binding are, and how the LPA product is released are questions that remain to be addressed. And, last but not least, the function of the open tunnel still remains enigmatic. Future structure-function analysis will undoubtedly shed more light on these issues.
      Figure thumbnail gr5
      Fig. 5Cartoon summarizing the domain structure and cell-surface localization of ENPP1, ATX/ENPP2 (α and β isoforms), and ENPP3. PPi, pyrophosphate. For details and discussion see text.

      Acknowledgments

      The authors thank Michael Gelb, Andrew Morris, and Junken Aoki for helpful discussions.

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