Contactin-associated protein-like 2 (CNTNAP2) encodes for CASPR2, a multidomain single transmembrane protein belonging to the neurexin superfamily that has been implicated in a broad range of human phenotypes including autism and language impairment. Using a combination of biophysical techniques, including small angle x-ray scattering, single particle electron microscopy, analytical ultracentrifugation, and bio-layer interferometry, we present novel structural and functional data that relate the architecture of the extracellular domain of CASPR2 to a previously unknown ligand, Contactin1 (CNTN1). Structurally, CASPR2 is highly glycosylated and has an overall compact architecture. Functionally, we show that CASPR2 associates with micromolar affinity with CNTN1 but, under the same conditions, it does not interact with any of the other members of the contactin family. Moreover, by using dissociated hippocampal neurons we show that microbeads loaded with CASPR2, but not with a deletion mutant, co-localize with transfected CNTN1, suggesting that CNTN1 is an endogenous ligand for CASPR2. These data provide novel insights into the structure and function of CASPR2, suggesting a complex role of CASPR2 in the nervous system.

From the Child Health Institute of New Jersey and Departments of Neuroscience and Cell Biology and

From the Child Health Institute of New Jersey and Departments of Neuroscience and Cell Biology and; Pediatrics Robert Wood Johnson Medical School Rutgers University New Brunswick New Jersey 08901

the Department of Biochemistry The University of Texas Health Science Center San Antonio Texas 78229

the Department of Biology and Biotechnologies Charles Darwin and Pasteur Institute Cenci Bolognetti Foundation Sapienza University of Rome Rome Italy 00185

the National Center for Microscopy and Imaging Research University of California San Diego La Jolla California 92093 and

the School of Molecular Bioscience University of Sydney New South Wales 2006 Australia

the School of Molecular Bioscience University of Sydney New South Wales 2006 Australia ; the Department of Chemistry University of Utah Salt Lake City Utah 84112

the Structural Biology Programme Central European Institute of Technology Masaryk University 62500 Brno Czech Republic

See more in PubMed

Poliak S., Gollan L., Martinez R., Custer A., Einheber S., Salzer J. L., Trimmer J. S., Shrager P., and Peles E. (1999) Caspr2, a new member of the neurexin superfamily, is localized at the juxtaparanodes of myelinated axons and associates with K+ channels. Neuron 24, 1037–1047 PubMed

Poliak S., Salomon D., Elhanany H., Sabanay H., Kiernan B., Pevny L., Stewart C. L., Xu X., Chiu S. Y., Shrager P., Furley A. J., and Peles E. (2003) Juxtaparanodal clustering of Shaker-like K+ channels in myelinated axons depends on Caspr2 and TAG-1. J. Cell Biol. 162, 1149–1160 PubMed PMC

Traka M., Goutebroze L., Denisenko N., Bessa M., Nifli A., Havaki S., Iwakura Y., Fukamauchi F., Watanabe K., Soliven B., Girault J. A., and Karagogeos D. (2003) Association of TAG-1 with Caspr2 is essential for the molecular organization of juxtaparanodal regions of myelinated fibers. J. Cell Biol. 162, 1161–1172 PubMed PMC

Tzimourakas A., Giasemi S., Mouratidou M., and Karagogeos D. (2007) Structure-function analysis of protein complexes involved in the molecular architecture of juxtaparanodal regions of myelinated fibers. Biotechnol. J. 2, 577–583 PubMed

Strauss K. A., Puffenberger E. G., Huentelman M. J., Gottlieb S., Dobrin S. E., Parod J. M., Stephan D. A., and Morton D. H. (2006) Recessive symptomatic focal epilepsy and mutant contactin-associated protein-like 2. New Engl. J. Med. 354, 1370–1377 PubMed

Jackman C., Horn N. D., Molleston J. P., and Sokol D. K. (2009) Gene associated with seizures, autism, and hepatomegaly in an Amish girl. Pediatr. Neurol. 40, 310–313 PubMed

Falivelli G., De Jaco A., Favaloro F. L., Kim H., Wilson J., Dubi N., Ellisman M. H., Abrahams B. S., Taylor P., and Comoletti D. (2012) Inherited genetic variants in autism-related CNTNAP2 show perturbed trafficking and ATF6 activation. Hum. Mol. Genet. 21, 4761–4773 PubMed PMC

Kastriti M. E., Sargiannidou I., Kleopa K. A., and Karagogeos D. (2015) Differential modulation of the juxtaparanodal complex in multiple sclerosis. Mol. Cell. Neurosci. 67, 93–103 PubMed

Ranscht B. (1988) Sequence of contactin, a 130-kDa glycoprotein concentrated in areas of interneuronal contact, defines a new member of the immunoglobulin supergene family in the nervous system. J. Cell Biol. 107, 1561–1573 PubMed PMC

Faivre-Sarrailh C., Gauthier F., Denisenko-Nehrbass N., Le Bivic A., Rougon G., and Girault J. A. (2000) The glycosylphosphatidyl inositol-anchored adhesion molecule F3/contactin is required for surface transport of paranodin/contactin-associated protein (caspr). J. Cell Biol. 149, 491–502 PubMed PMC

Rios J. C., Melendez-Vasquez C. V., Einheber S., Lustig M., Grumet M., Hemperly J., Peles E., and Salzer J. L. (2000) Contactin-associated protein (Caspr) and contactin form a complex that is targeted to the paranodal junctions during myelination. J. Neurosci. 20, 8354–8364 PubMed PMC

Puzzo D., Bizzoca A., Privitera L., Furnari D., Giunta S., Girolamo F., Pinto M., Gennarini G., and Palmeri A. (2013) F3/Contactin promotes hippocampal neurogenesis, synaptic plasticity, and memory in adult mice. Hippocampus 23, 1367–1382 PubMed

Koch T., Brugger T., Bach A., Gennarini G., and Trotter J. (1997) Expression of the immunoglobulin superfamily cell adhesion molecule F3 by oligodendrocyte-lineage cells. Glia 19, 199–212 PubMed

Parent A. S., Mungenast A. E., Lomniczi A., Sandau U. S., Peles E., Bosch M. A., Rønnekleiv O. K., and Ojeda S. R. (2007) A contactin-receptor-like protein tyrosine phosphatase β complex mediates adhesive communication between astroglial cells and gonadotrophin-releasing hormone neurones. J. Neuroendocrinol. 19, 847–859 PubMed

Çolakoǧlu G., Bergstrom-Tyrberg U., Berglund E. O., and Ranscht B. (2014) Contactin-1 regulates myelination and nodal/paranodal domain organization in the central nervous system. Proc. Natl. Acad. Sci. U.S.A. 111, E394–403 PubMed PMC

Lamprianou S., Chatzopoulou E., Thomas J. L., Bouyain S., and Harroch S. (2011) A complex between contactin-1 and the protein tyrosine phosphatase PTPRZ controls the development of oligodendrocyte precursor cells. Proc. Natl. Acad. Sci. U.S.A. 108, 17498–17503 PubMed PMC

Poliak S., Gollan L., Salomon D., Berglund E. O., Ohara R., Ranscht B., and Peles E. (2001) Localization of Caspr2 in myelinated nerves depends on axon-glia interactions and the generation of barriers along the axon. J. Neurosci. 21, 7568–7575 PubMed PMC

Comoletti D., Grishaev A., Whitten A. E., Tsigelny I., Taylor P., and Trewhella J. (2007) Synaptic arrangement of the neuroligin/β-neurexin complex revealed by x-ray and neutron scattering. Structure 15, 693–705 PubMed PMC

Reeves P. J., Callewaert N., Contreras R., and Khorana H. G. (2002) Structure and function in rhodopsin: high-level expression of rhodopsin with restricted and homogeneous N-glycosylation by a tetracycline-inducible N-acetylglucosaminyltransferase I-negative HEK293S stable mammalian cell line. Proc. Natl. Acad. Sci. U.S.A. 99, 13419–13424 PubMed PMC

Comoletti D., Flynn R. E., Boucard A. A., Demeler B., Schirf V., Shi J., Jennings L. L., Newlin H. R., Südhof T. C., and Taylor P. (2006) Gene selection, alternative splicing, and post-translational processing regulate neuroligin selectivity for β-neurexins. Biochemistry 45, 12816–12827 PubMed

Gasteiger E., Hoogland C., Gattiker A., Duvaud S., Wilkins M. R., Appel R. D., and Bairoch A. (2005) The Proteomics Protocols Handbook (Walker J. M., ed) pp. 571–607, Humana Press, New York

Demeler B. (2010) Methods for the design and analysis of sedimentation velocity and sedimentation equilibrium experiments with proteins. Curr. Protoc. Protein Sci. Chapter 7, Unit 7.13 PubMed PMC

Demeler B., and Gorbet G. (2016) Analytical Ultracentrifugation Data Analysis with UltraScan-III. Analytical Ultracentrifugation: Instrumentation, Software, and Applications, Springer, New York, in press

Brookes E., Cao W., and Demeler B. (2010) A two-dimensional spectrum analysis for sedimentation velocity experiments of mixtures with heterogeneity in molecular weight and shape. Eur. Biophys. J. 39, 405–414 PubMed

Demeler B., and van Holde K. E. (2004) Sedimentation velocity analysis of highly heterogeneous systems. Anal. Biochem. 335, 279–288 PubMed

Brookes E., and Demeler B. (2006) Genetic algorithm optimization for obtaining accurate molecular weight distributions from Sedimentation velocity experiments. Analytical Ultracentrifugation VIII in Progress in Colloid Polymer Science (Wandrey C., and Cölfen H., eds) Vol. 131, pp. 78–82, Springer-Verlag, New York

Brookes E., and Demeler B. (2007) Parsimonious regularization using genetic algorithms applied to the analysis of analytical ultracentrifugation experiments. In GECCO '07 Proceedings of the 9th Annual Conference on Genetic and Evolutionary Computation, pp. 361–368, ACM, New York

Demeler B., and Brookes E. (2008) Monte Carlo analysis of sedimentation experiments. Colloid Polym. Sci. 286, 129–137

Brookes E., and Demeler B. (2008) Parallel computational techniques for the analysis of sedimentation velocity experiments in UltraScan. Colloid Polym. Sci. 286, 138–148

Durchschlag H. (1986) Thermodynamic data for biochemistry and biotechnology (Hinz H. J., ed) pp. 45–128, Springer-Verlag, New York

Jeffries C. M., Whitten A. E., Harris S. P., and Trewhella J. (2008) Small-angle x-ray scattering reveals the N-terminal domain organization of cardiac myosin binding protein C. J. Mol. Biol. 377, 1186–1199 PubMed

Svergun D. I. (1992) Determination of the regularization parameter in indirect-transform methods using perceptual criteria. J. Appl. Crystallogr. 25, 495–503

Krigbaum W. R., and Kügler F. R. (1970) Molecular conformation of egg-white lysozyme and bovine α-lactalbumin in solution. Biochemistry 9, 1216–1223 PubMed

Svergun D. I. (1999) Restoring low resolution structure of biological macromolecules from solution scattering using simulated annealing. Biophys. J. 76, 2879–2886 PubMed PMC

Franke D., Jeffries C. M., and Svergun D. I. (2015) Correlation map, a goodness-of-fit test for one-dimensional x-ray scattering spectra. Nat. Methods 12, 419–422 PubMed

Valentini E., Kikhney A. G., Previtali G., Jeffries C. M., and Svergun D. I. (2015) SASBDB, a repository for biological small-angle scattering data. Nucleic Acids Res. 43, D357–D363 PubMed PMC

Ohi M., Li Y., Cheng Y., and Walz T. (2004) Negative staining and image classification: powerful tools in modern electron microscopy. Biol. Proced. Online 6, 23–34 PubMed PMC

Frank J., Radermacher M., Penczek P., Zhu J., Li Y., Ladjadj M., and Leith A. (1996) SPIDER and WEB: processing and visualization of images in 3D electron microscopy and related fields. J. Struct. Biol. 116, 190–199 PubMed

Demeler B., Brookes E., Wang R., Schirf V., and Kim C. A. (2010) Characterization of reversible associations by sedimentation velocity with UltraScan. Macromol. Biosci. 10, 775–782 PubMed

Guinier A. (1939) La diffraction des rayons X aux tres petits angles; application a l'etude de phenomenes ultramicroscopiques. Ann. Phys. Paris 12, 161–237

Petoukhov M. V., Franke D., Shkumatov A. V., Tria G., Kikhney A. G., Gajda M., Gorba C., Mertens H. D., Konarev P. V., and Svergun D. I. (2012) New developments in the program package for small-angle scattering data analysis. J. Appl. Crystallogr. 45, 342–350 PubMed PMC

Volkov V. V., and Svergun D. I. (2003) Uniqueness of ab initio shape determination in small-angle scattering. J. Appl. Crystallogr. 36, 860–864 PubMed PMC

Comoletti D., Miller M. T., Jeffries C. M., Wilson J., Demeler B., Taylor P., Trewhella J., and Nakagawa T. (2010) The macromolecular architecture of extracellular domain of alphaNRXN1: domain organization, flexibility, and insights into trans-synaptic disposition. Structure 18, 1044–1053 PubMed PMC

Boucard A. A., Chubykin A. A., Comoletti D., Taylor P., and Südhof T. C. (2005) A splice code for trans-synaptic cell adhesion mediated by binding of neuroligin 1 to α- and β-neurexins. Neuron 48, 229–236 PubMed

Abrahams B. S., Tentler D., Perederiy J. V., Oldham M. C., Coppola G., and Geschwind D. H. (2007) Genome-wide analyses of human perisylvian cerebral cortical patterning. Proc. Natl. Acad. Sci. U.S.A. 104, 17849–17854 PubMed PMC

Miller M. T., Mileni M., Comoletti D., Stevens R. C., Harel M., and Taylor P. (2011) The crystal structure of the α-neurexin-1 extracellular region reveals a hinge point for mediating synaptic adhesion and function. Structure 19, 767–778 PubMed PMC

Chen F., Venugopal V., Murray B., and Rudenko G. (2011) The structure of neurexin 1α reveals features promoting a role as synaptic organizer. Structure 19, 779–789 PubMed PMC

van der Merwe P. A., and Barclay A. N. (1994) Transient intercellular adhesion: the importance of weak protein-protein interactions. Trends Biochem. Sci. 19, 354–358 PubMed

Finci L. I., Krüger N., Sun X., Zhang J., Chegkazi M., Wu Y., Schenk G., Mertens H. D., Svergun D. I., Zhang Y., Wang J. H., and Meijers R. (2014) The crystal structure of netrin-1 in complex with DCC reveals the bifunctionality of netrin-1 as a guidance cue. Neuron 83, 839–849 PubMed PMC

Bohne A., Lang E., and von der Lieth C. W. (1999) SWEET: WWW-based rapid 3D construction of oligo- and polysaccharides. Bioinformatics 15, 767–768 PubMed