Cryoelectron tomography of homophilic adhesion mediated by the neural cell adhesion molecule L 1

The neural cell adhesion molecule L1 participates in homophilic interactions that are important for axon guidance and neuronal development. The structural details of homophilic adhesion mediated by L1 and other immunoglobulin superfamily members containing an N-terminal horseshoe arrangement of four immunoglobulin-like domains are unknown. Here we used cryoelectron tomography to study liposomes to which intact or truncated forms of the L1 ectodomain were attached. Tomographic reconstructions revealed an adhesion interface with a regular and repeating pattern consistent with interactions between paired horseshoes contributed by L1 proteins from neighboring liposomes. The characteristics of the pattern, such as the space between horseshoe pairs and/or between adjacent membranes, changed when N-linked carbohydrates were altered by removing sialic acids or converting from complex to high mannose or oligomannose glycans, suggesting a regulatory role for carbohydrates in L1-mediated homophilic adhesion. Using the results from tomograms and crystal structures of L1-related molecules, we present a structural model for L1-mediated homophilic adhesion that depends on protein-protein, protein-carbohydrate, and carbohydrate-carbohydrate interactions. The neural cell adhesion molecule L1 (CD171) is a cell surface protein expressed by predominantly neuronal, as well as some non-neuronal, cells in vertebrate and invertebrate species. L1 and related proteins function in neural development, axonal guidance, myelination, and cell migration by mediating both homophilic and heterophilic adhesion at the cell surface (Hortsch, 1996; Hortsch, 2000; Maness and Schachner, 2007). Heterophilic binding partners of L1 include other cell adhesion molecules, proteoglycans, extracellular matrix-associated molecules, integrins, receptor tyrosine phosphatases, and fibroblast growth factor receptor (Crossin and Krushel, 2000; Hortsch et al., 1998; Moos et al., 1988). Both homophilic and heterophilic interactions mediated by L1 are important in promoting or inhibiting neural outgrowth in vivo (Maness and Schachner, 2007). L1 is a member of the immunoglobulin (Ig) gene superfamily, which includes other neural cell adhesion molecules such as neurofascin, NCAM, and TAG-1/axonin-1, all of which contain tandem Ig-like domains followed in some cases by fibronectin type III (FNIII) domains. L1 is composed of an ~1100 residue ectodomain containing six Ig-like domains followed by five *Corresponding author: E-mail: bjorkman@caltech.edu, Phone: 626-395-8350, Fax: 626-792-3683. Publisher's Disclaimer: This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final citable form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain. Published as: Structure. 2009 March 11; 17(3): 460–471. H H M I Athor M anscript H H M I Athor M anscript H H M I Athor M anscript FNIII repeats, a single-pass transmembrane region, and a 114-residue cytoplasm tail (Figure 1A) (Hortsch, 1996). Based on the crystal structures of two L1 homologues, hemolin (Su et al., 1998) and axonin-1 (Freigang et al., 2000), the four N-terminal Ig-like domains of L1 are predicted to adopt a horseshoe-shaped conformation in which the first and the second domains (D1 and D2) fold back to interact with the fourth and the third domains (D4 and D3), respectively (Figure 1A). The horseshoe conformation may be required for function, as the first four domains of L1 and related proteins are necessary and sufficient for homophilic adhesion (Haspel et al., 2000). The role of the FNIII domains in adhesion remains unclear, although some results suggest that they are involved in cis-interactions that facilitate heterophilic and/or homophilic interactions (Kunz et al., 2002). The tandem arrangement of 11 consecutive domains in the L1 ectodomain suggests a flexible and extended structure. Indeed, electron microscopy (EM) studies of negatively-stained EM samples were consistent with an N-terminal horseshoe structure followed by a flexible extended region representing the remainder of the domains (Schurmann et al., 2001). Rotaryshadowed samples showed a more extended structure that did not include the N-terminal horseshoe, presumably because of interactions between the protein and the mica surface (Schurmann et al., 2001). Like many surface adhesion proteins, human L1 is highly glycosylated, with 21 potential Nlinked glycosylation sites in its ectodomain: four within the N-terminal horseshoe domains, five within the fifth and sixth Ig-like domains, and the remaining 12 sites in the FNIII domains. Carbohydrates have been shown to play a role in L1and N-CAM-mediated neural adhesion (Acheson et al., 1991; Kadmon et al., 1990). In particular, L1 can bind to terminal sialic acids attached to complex carbohydrates, thereby acting as a sialic acid-binding lectin in regulating neurite growth (Crocker, 2002; Kleene et al., 2001). Although X-ray and EM studies have provided some information about the structure of L1, a mechanistic understanding of homophilic adhesion mediated by membrane-bound L1 molecules and the roles of carbohydrates in adhesion remain to be clarified. Here we used cryoelectron tomography (cryoET) to investigate the three-dimensional structure of the homophilic adhesion interface formed between L1 molecules on opposing membranes. Our results suggest a new model for homophilic adhesion mediated by L1 family members in which horseshoes from proteins on opposing membranes meet as trans pairs, which form a regularly spaced lattice due to lateral contacts resulting from protein-carbohydrate and carbohydratecarbohydrate interactions. Taken together with the demonstration that alterations in carbohydrate structure changed the structure of the adhesion interface, this model predicts a primary role for carbohydrates in regulating adhesion mediated by L1 and related proteins.