ATP–NGF-complex, but not NGF, is the neuroprotective ligand Katja Bettina Ferenz a , Karsten Rose a , Simone König b , Josef Krieglstein a,⇑ a Institut für Pharmazeutische und Medizinische Chemie, Fachbereich Chemie und Pharmazie, Germany b Integrierte Funktionelle Genomik, Interdisziplinäres Zentrum für Klinische Forschung, Medizinische Fakultät, Westfälische Wilhelms-Universität, Münster, Germany article info Article history: Received 27 June 2011 Received in revised form 25 August 2011 Accepted 26 August 2011 Available online 10 September 2011 Keywords: Nerve growth factor ATP Photo affinity labeling 8N 3 ATP Mass spectrometry abstract We have shown previously that nerve growth factor (NGF) requires only low nanomolar ATP concentra- tions in the cell culture medium to protect cortical rat neurons (CRN) from cellular damage induced by staurosporine (STS). We have also demonstrated before that NGF and other growth factors form stable non-covalent complexes with ATP. Here we demonstrated that 8N 1 ATP–NGF, but not NGF, protected CRN against damage. The photo-reactive ATP derivative 8N 3 ATP was incubated with NGF and was trapped in its position by UV irradiation forming a covalent bond. The cross-link with a molar ratio of 1:1 (8N 1 ATP:NGF) was confirmed by mass spectrometry. Circular dichroism experiments revealed that 8N 1 ATP altered the secondary structure of NGF in the same way as ATP did. Covalently bound 8N 1 ATP–NGF was shown to be stable in the presence of the ATP-hydrolyzing enzyme alkaline phospha- tase while the non-covalent ATP–NGF-complex dissociated with the removal of free ATP from the solution. 8N 1 ATP–NGF protected CRN against damage by STS independently of free ATP in the culture medium. These results suggest that the ATP–NGF-complex, but not NGF, is the active ligand. Ó 2011 Elsevier B.V. All rights reserved. 1. Introduction Nerve growth factor (NGF) is one of the best characterized mem- bers of the neurotrophin family (Bradshaw et al., 1993), which plays a pivotal role in development and function of the nervous system. NGF has been extensively shown to promote growth, survival and differentiation of neurons and to protect them from damage after injury (Levi-Montalcini, 1987; Lindsay, 1988; Katz et al., 1990; Diamond et al., 1992; Kaplan and Miller, 2000; Segal, 2003; Culm- see et al., 2002; Klumpp et al., 2006; Hasche et al., 2010). Extracel- lular mechanisms besides receptor activation by NGF have however not been examined as thoroughly in the past. So far, research focused on intracellular signaling cascades downstream receptor activation (Dudek et al., 1997; Kaplan and Miller, 2000; Arevalo and Wu, 2006; Jeon et al., 2010), amino-acid residues contributing to the binding of NGF to its receptors Trk A and p75 (Ibanez et al., 1992; Wiesmann et al., 1999) and adapter proteins for NGF recep- tors (Mukai et al., 2000; Salehi et al., 2000; Barker 2004; Yamashita et al., 2005; Barker 2007). Previously, we demonstrated by radiola- beling (Klumpp et al., 2006; Rose et al., 2008, 2009) and mass spec- trometry (MS) (König et al., 2008) that ATP binds to NGF as well as to other growth factors (GF) such as brain-derived neurotrophic factor and fibroblast growth factor 2 (FGF2). In fact, MS provided evidence that the ATP-GF association is non-covalent in nature and that, nevertheless, the complex is stable during gel electropho- retic separation, desalting by solid phase extraction and desolvation and ionization in MS. It was furthermore shown on primary cul- tures of CRN (PC-CRN) using the model of STS-induced cell damage that ATP needs to be present at concentrations above only 2 nM to enable neuroprotective activity of NGF (Hasche et al., 2010). These concentrations are much too low to activate purinergic P2X or P2Y receptors with dissociation constants for ATP in the micromolar range (Zimmermann, 1994; Boeynaems et al., 2005; Abbracchio et al., 2009). ATP as a neurotransmitter acting on these receptors (Abbracchio et al., 2009) is typically found in the extracellular domain at high nanomolar to micromolar concentration (Zimmermann, 1994; Agteresch et al., 1999; Schwiebert and Zsembery, 2003). Conclusively, a further function of ATP with respect to GF protein activity is indicated. We hypothesized that ATP–NGF-complex formation plays an essential role in this process and used PAL to covalently link 8N 1 ATP to NGF. The activity of this stable assembly was furthermore investigated depleting unbound 0197-0186/$ - see front matter Ó 2011 Elsevier B.V. All rights reserved. doi:10.1016/j.neuint.2011.08.020 Abbreviations: 8N 3 ATP, 8-azidoadenosine 5 0 triphosphate; ANOVA, analysis of variance; AP, alkaline phosphatase; CD, circular dichroism; CRN, cortical rat neurons; DMEM, Dulbecco’ modified Eagle’s medium; ESI, elecrospray ionization; FGF2, fibroblast growth factor 2; GF, growth factor; HBD, heparin binding domain; NB, Neurobasal™ medium; NGF, nerve growth factor; MALDI, matrix associated laser desorption ionization; MS, mass spectrometry; PAGE, polyacrylamide gel electrophoresis; PAL, photo affinity labeling; PBS, phosphate-buffered saline; PC-CRN, primary cultures of cortical rat neurons; rh, recombinant human; SDS, sodium dodecyl sulfate; STS, staurosporine; TFA, trifluoro acetic acid; TOF, time of flight. ⇑ Corresponding author. Address: Institut für Pharmazeutische und Medizinische Chemie, Westfälische Wilhelms-Universität, Hittorfstr. 58-62, D-48149 Münster, Germany. Tel.: +49 6421 85697; fax: +49 6421 8869970. E-mail address: krieglst@uni-muenster.de (J. Krieglstein). Neurochemistry International 59 (2011) 989–995 Contents lists available at SciVerse ScienceDirect Neurochemistry International journal homepage: www.elsevier.com/locate/nci