1 2 3 Monoclonal Antibody DL11C8 Identifies ADAM23 as a Component of 4 Lipid Raft Microdomains 5 Zaine L. M. Borgonovo, a,by Caroline F. Ribeiro, ay Michele D. M. Costa, a,cy Ingrid L. M. Souza, a,b Gustavo R. Rossi, b 6 Monica V. Alcantara, a Max Ingberman, a Luciano G. Braga, a Adriana F. Mercadante, a Lia S. Nakao a,b and 7 Silvio M. Zanata a,b * 8 a Department of Basic Pathology, Universidade Federal do Parana ´, Curitiba, PR, Brazil 9 b Department of Cell Biology, Universidade Federal do Parana ´, Curitiba, PR, Brazil 10 c Department of Structural and Molecular Biology and Genetics, Universidade Estadual de Ponta Grossa, Ponta Grossa, PR, Brazil 11 12 Abstract—A disintegrin and metalloprotease protein 23 (ADAM23) is a transmembrane type I glycoprotein involved with the development and maintenance of the nervous system, including neurite outgrowth, neuronal adhesion and differentiation and regulation of synaptic transmission. In addition, ADAM23 seems to participate in immune response and tumor establishment through interaction with different members of integrin receptors. Here, we describe a novel monoclonal antibody (DL11C8) that specifically recognizes the cysteine-rich domain of both pre-protein (100 kDa) and mature (70 kDa) forms of ADAM23 from different species, including human, rodents and avian orthologs. Using this antibody, we detected both forms of ADAM23 on the cell surface of three neuronal cell lineages (Neuro-2a, SH-SY5Y and CHLA-20), with a higher relative content of ADAM23 100 kDa . Further- more, we demonstrate for the first time that a catalytically inactive member of the ADAM family is present in the membrane signaling platforms, namely lipid rafts. Indeed, the mature ADAM23 70 kDa partitions between raft and non-raft membrane domains, while the pro-protein ADAM23 100 kDa is mainly expressed in non-raft domains. These membranous distributions were observed in both different brain regions homogenates and primary cultured neu- rons lysates from mouse cortex and cerebellum. Taken together, these findings point out ADAM23 as a lipid raft molecular component. Ó 2018 IBRO. Published by Elsevier Ltd. All rights reserved. Keywords: ADAM23, lipid rafts, disintegrins, monoclonal antibody, prion protein, integrin. 13 INTRODUCTION 14 A disintegrin and metalloprotease (ADAM) is a family of 15 type I transmembrane glycoproteins containing a 16 metalloprotease domain followed by disintegrin, 17 cysteine-rich region, EGF-like, transmembrane domain 18 and cytoplasmic tail (Giebeler and Zigrino, 2016; Seals 19 and Courtneidge, 2003). ADAMs are involved in various 20 biological processes, such as sperm–egg interactions, 21 cell differentiation, cell migration, axonal growth, muscular 22 development, and some aspects of immunity. Besides 23 physiological roles, members of this family are involved 24 in pathological conditions, such as cancer, neurological 25 and cardiac diseases, asthma, infection, and inflamma- 26 tion (Edwards et al., 2008; Murphy, 2008; Giebeler and 27 Zigrino, 2016; Seals and Courtneidge, 2003). 28 ADAM23 (MDC3) is an ADAM family member widely 29 expressed in the nervous system, particularly in neurons 30 and Schwann cells of the peripheral nervous system 31 (Dhaunchak et al., 2010; Goldsmith et al., 2004; Kegel 32 et al., 2014; Markus et al., 2011; Owuor et al., 2009; Lin 33 et al., 2008; Sagane et al., 1998; 1999). Similarly to other 34 ADAMs, ADAM23 presents two domains important for its 35 functions: the metalloprotease and disintegrin domains, 36 however the metalloprotease domain is believed to be 37 catalytically inactive since it lacks essential residues 38 responsible for the Zn 2+ coordination to the catalytic motif 39 (Sagane et al., 1998). Therefore, ADAM23 is predicted to 40 act mainly through its disintegrin domain. In fact, this 41 domain interacts in vitro with avb3 and a4 integrin recep- 42 tors, promoting neural (Cal et al., 2000) and immune 43 (Wang et al., 2017) cell adhesion, respectively. In addi- 44 tion, this interaction has been shown to negatively modu- 45 late the avb3 activation in MDA-MB-435 cells and the loss 46 of ADAM23 expression enhances avb3 integrin-mediated 47 cell migration and adhesion (Verbisck et al., 2009). https://doi.org/10.1016/j.neuroscience.2018.05.016 0306-4522/Ó 2018 IBRO. Published by Elsevier Ltd. All rights reserved. * Correspondence to: S. M. Zanata, Department of Basic Pathology, Universidade Federal do Parana´, Curitiba, PR, Brazil. E-mail address: smzanata@ufpr.br (S. M. Zanata). y These authors contributed equally to this work. Abbreviations: ADAM23, a disintegrin and metalloprotease protein 23; CNS, central nervous system; conA, concanavalin A-Sepharose; ELISA, enzyme-linked immunosorbent assay; FCS, fetal calf serum; PrPc, cellular prion protein; TNF, tumor necrosis factor; WGA, wheat germ agglutinin. NEUROSCIENCE RESEARCH ARTICLE Z. L. M. Borgonovo et al. / Neuroscience xxx (2018) xxx–xxx 1 NSC 18453 No. of Pages 13 25 May 2018 Please cite this article in press as: Borgonovo ZLM et al. Monoclonal Antibody DL11C8 Identifies ADAM23 as a Component of Lipid Raft Microdomains. Neuroscience (2018), https://doi.org/10.1016/j. neuroscience.2018.05.016