Biochimica et Biophysica Acta 1854 (2015) 827–833 Contents lists available at ScienceDirect Biochimica et Biophysica Acta journal homepage: www.elsevier.com/locate/bbapap Interaction proteomics of canonical Caspr2 (CNTNAP2) reveals the presence of two Caspr2 isoforms with overlapping interactomes☆ Ning Chen a,b, Frank Koopmans b, Aaron Gordon c, Iryna Paliukhovich a,RemcoV.Klaassena, Roel C. van der Schors a, Elior Peles c, Matthijs Verhage b, August B. Smit a,1,KaWanLia,⁎,1 a Department of Molecular and Cellular Neurobiology, Center for Neurogenomics and Cognitive Research, Neuroscience Campus Amsterdam, VU University, De Boelelaan 1085, Amsterdam 1081 HV, The Netherlands b Department of Functional Genomics, Center for Neurogenomics and Cognitive Research, Neuroscience Campus Amsterdam, VU University, De Boelelaan 1085, Amsterdam 1081 HV, The Netherlands c Department of Molecular Cell Biology, The Weizmann Institute of Science, Rehovot 76100, Israel article info abstract Article history: Autism is a human developmental brain disorder characterized by impaired social interaction and communica- Received 18 September 2014 tion. Contactin-associated protein-like 2 (Caspr2, CNTNAP2) is a known genetic risk factor of autism. However, Received in revised form 31 January 2015 how this protein might contribute to pathology is unclear. In this study, we demonstrate that Caspr2 is abundant- Accepted 11 February 2015 ly present in lipid raft and in the synaptic membrane but is highly depleted in the postsynaptic density. The Available online 21 February 2015 Caspr2 protein level in hippocampus is present at a constant level during synapse formation and myelination Keywords: from P0 to P84. Interaction proteomics revealed the interactors of Caspr2, including CNTN2, KCNAs, members Autism of the ADAM family (ADAM22, ADAM23 and ADAM11), members of LGI family and MAGUKs (DLGs and Caspr2 MPPs). Interestingly, a short form of Caspr2 was detected, which lacks most of the extracellular domains, howev- Interaction proteomics er, is still associated with ADAM22 and to a lesser extent LGI1 and Kv1 channels. The comprehensive Caspr2 Mouse model interactome revealed here might aid in understanding the molecular mechanisms underlying autism. This article Brain is part of a Special Issue titled Neuroproteomics: Applications in Neuroscience and Neurology. © 2015 Elsevier B.V. All rights reserved. 1. Introduction Recent behavioral and physiological studies revealed the prominent role of contactin-associated protein-like 2 (Caspr2, CNTNAP2) in ASD Autism spectrum disorder (ASD) is the most prevalent developmen- [8–10]. In particular, Caspr2 knockout mice exhibited behavioral abnor- tal brain disorder in children. The diagnosis of ASD is based on abnor- malities that mimic some of the core features of ASD [8,11].Atthecellu- malities in social interaction, impairments in language development lar level, knockout mice showed neuronal migration abnormalities, and communication and occurrence of repetitive or stereotyped behav- reduced numbers of interneurons and abnormal neuronal network ac- iors [1]. In addition, many ASD patients have seizures and display intel- tivity [8]. Similar alterations were reproduced independently by the lectual disability. The cause, or causes, of ASD are largely unknown. The knockdown of Caspr2 transcripts in a cortical neuron culture in vitro, high heritability of autism was demonstrated convincingly by which exhibited impaired synapse formation and neural network concordance rates in genetic studies in families and twins [2,3].Consid- assembly leading to a global decrease of synaptic transmission [10]. erable symptom heterogeneity in the autism spectrum exists, which How mutations at the Caspr2 locus and/or reduction of Caspr2 expres- may be indicative of the underlying genetic complexity of the disorder. sion cause ASD remains largely unclear. Currently, over 100 ASD-genes have been reported by genetic analysis Caspr2 is a single pass transmembrane cell adhesion protein. The [4,5]. In particular, mutations in Shank, Caspr2, Neurexin and Cadherin, extracellular region of Caspr2 is composed of several domains common cell adhesion molecules and components of the synapse have been to cell adhesion molecules, including laminin G, EGF repeats and linked to ASD [5–7]. This supports the hypothesis that synaptic dysfunc- discoidin-like domains. Caspr2 interacts with Contactin 2 (CNTN2) tion is an underlying cause, at least in part, of the observed ASD behav- extracellularly, forming a neuron–glia cell adhesion complex [12].The ioral phenotypes. short intracellular region of Caspr2 contains a band 4.1 binding domain and a carboxy-terminal PDZ-binding motif, which might be involved in ☆ This article is part of a Special Issue titled Neuroproteomics: Applications in the clustering of voltage-gated potassium channels 1 (Kv1, KCNA) in Neuroscience and Neurology. myelinated axon membranes [13]. This clustering of Kv1 channels in ⁎ Corresponding author. Tel.:+31 20 5987107; fax: +31 20 5989281. fi E-mail address: [email protected] (K.W. Li). myelinated axon membranes, permits a rapid and ef cient propagation 1 These authors have equal contribution. of action potentials at the juxta-paranodal membrane of the nodes of http://dx.doi.org/10.1016/j.bbapap.2015.02.008 1570-9639/© 2015 Elsevier B.V. All rights reserved. 828 N. Chen et al. / Biochimica et Biophysica Acta 1854 (2015) 827–833 Ranvier [12]. Besides, Caspr2 has been shown to associate with Kv1 75–028) and Kv1.1 (catalog number 75–105) were purchased from channels in the distal region of the axon initial segment of pyramidal Neuromab. neurons, which is crucial for controlling action potential initiation and neural activity [14]. Caspr2 has also been demonstrated in the synaptic 2.3. SDS–PAGE separation of IP samples and in-gel trypsin digestion plasma membrane of brain lysates [15]. To improve understanding of Caspr2 function, we examined the The protein A/G beads with Caspr2 antibody and the bound Caspr2 subcellular localization of Caspr2 by biochemical fractionation and the protein complexes were mixed with 14 μl 2× SDS sample buffer and developmental expression pattern of Caspr2. We then characterized heated to 98 °C for 5 min. To block cysteine residues, 5 μl30%acrylamide the Caspr2 protein complex in two preparations, namely, an extract of was added and incubated at room temperature for 30 min [18].Proteins synapse-enriched fraction of the hippocampus and an extract from were then resolved on a 10% SDS–polyacrylamide gel and stained with whole cortex, in wild-type and Caspr2 knockout mice. In addition to colloidal Coomassie blue G-250 for 30 min. The gel lane of each sample the previously reported Caspr2 interactors, we detected a Caspr2 iso- was cut into five pieces. Each was chopped into smaller pieces using a form 2. In the Caspr2 knockout mice, the short Caspr2 isoform 2, lacking scalpel and transferred to an Eppendorf tube. The gel pieces were most of the extracellular domains, is still present and is capable of destained with 50% acetonitrile in 50 mM ammonium bicarbonate, interacting with a subset of the Caspr2 long isoform interactors. dehydrated in 100% acetonitrile and rehydrated in 50 mM ammonium bicarbonate. The destaining cycle was repeated once. After dehydration in 100% acetonitrile and dried in a speedvac, the gel pieces were 2. Materials and methods incubated with trypsin solution containing 10 μg/mL trypsin (sequence grade; Promega, Madison, USA) in 50 mM ammonium bicarbonate 2.1. Subcellular fractionation of hippocampal lysate overnight at 37 °C. Peptides from the gel pieces were extracted twice with 200 μL 50% acetonitrile in 0.1% Trifluoroacetic acid, dried in a Mature C57Bl6 mice brains were taken at postnatal days P56-P70, SpeedVac and stored at −20 °C for future use. and hippocampi were dissected on ice [16] andstoredat−80 °C for fur- ther use. Frozen hippocampi were homogenized in homogenization 2.4. HPLC-MS/MS analysis buffer (320 mM sucrose, 5 mM HEPES (pH 7.4) and a protease inhibitor mixture(Roche Applied Science)) using a dounce homogenizer on ice, Peptides were re-dissolved in 20 μL 0.1% acetic acid and injected into and centrifuged at 1,000 ×g. The supernatant was further centrifuged an LTQ-Orbitrap mass spectrometer (Thermo Electron, San Jose, CA, at 16,000 ×g for 20 min to obtain the pellet as P2 fraction. The superna- USA) equipped with an HPLC system (Eksigent). Samples were first tant was centrifuged at 85,000 ×g for 2 h to obtain the pellet as micro- trapped on a 5 mm Pepmap 100 C18 (Dionex) column (300 μmID, some fraction. The P2 fraction was centrifuged at 85,000 ×g for 2 h in 5 μm particle size) and then analyzed on a 200 mm Alltima C18 home- a sucrose step gradient to obtain the synaptosome fraction at the made column (100 μmID,3μm particle size). Separation was achieved 0.85 M/1.2 M sucrose interface. Synaptosomes were lysed in hypotonic by using a mobile phase from 5% acetonitrile, 94.9% H O, 0.1% acetic acid solution containing 5 mM HEPES (pH 7.4), and the resulting synaptic 2 (solvent A) and 95% acetonitrile, 4.9% H O and 0.1% acetic acid (solvent membrane fraction was recovered by centrifugation using the sucrose 2 B), and the linear gradient was from 5% to 40% solvent B for 40 min at a gradient as stated above. To obtain postsynaptic density (PSD) and flow rate of 400 nL/min. The LTQ-Orbitrap mass spectrometer was lipid raft, the P2 and microsome fractions were pooled and stirred for 30 min on ice in 50 mM HEPES (pH 7.4) containing 1% Triton X-100. The sample was loaded on top of a sucrose gradient consisting of 0.32 M, 1.0 M, 1.5 M and 2.0 M sucrose and centrifuged at 85,000×g for 2 h. The interface between 1.5 M and 2.0 M was collected as PSD and the interface between the input and 0.32 M as lipid raft.