Antibody fragments directed against different portions of the human neural cell adhesion molecule L1 act as inhibitors or activators of L1 function.

Yan Wang,Ralf Kleene,Ricardo Gouveia,Hong-Chao Pan,Wei-Jiang Zhao,Gabriele Loers,Melitta Schachner,Yan-Qin Shen,Julia Costa

doi:10.1371/journal.pone.0052404

Abstract

The neural cell adhesion molecule L1 plays important roles in neuronal migration and survival, neuritogenesis and synaptogenesis. L1 has also been found in tumors of different origins, with levels of L1 expression correlating positively with the metastatic potential of tumors. To select antibodies targeting the varied functions of L1, we screened the Tomlinson library of recombinant human antibody fragments to identify antibodies binding to recombinant human L1 protein comprising the entire extracellular domain of human L1. We obtained four L1 binding single-chain variable fragment antibodies (scFvs), named I4, I6, I13, and I27 and showed by enzyme-linked immunosorbent assay (ELISA) that scFvs I4 and I6 have high affinity to the immunoglobulin-like (Ig) domains 1–4 of L1, while scFvs I13 and I27 bind strongly to the fibronectin type III homologous (Fn) domains 1–3 of L1. Application of scFvs I4 and I6 to human SK-N-SH neuroblastoma cells reduced proliferation and transmigration of these cells. Treatment of SK-N-SH cells with scFvs I13 and I27 enhanced cell proliferation and migration, neurite outgrowth, and protected against the toxic effects of H2O2 by increasing the ratio of Bcl-2/Bax. In addition, scFvs I4 and I6 inhibited and scFvs I13 and I27 promoted phosphorylation of src and Erk. Our findings indicate that scFvs reacting with the immunoglobulin-like domains 1–4 inhibit L1 functions, whereas scFvs interacting with the fibronectin type III domains 1–3 trigger L1 functions of cultured neuroblastoma cells.

Highlights

The cell adhesion molecule L1, a member of the immunoglobulin superfamily of cell adhesion molecules, plays important roles in cell-cell interactions
We previously identified scFvs against mouse Fn1–2 [24] that do not cross-react with mouse NCAM or CHL1 which are homologous to L1
L1 binds to integrins via the RGD motif in the sixth Ig-like domain to stimulate signal transduction resulting in cell adhesion and migration [26,27,28,29] and can recruit integrins in a non-RGD dependent manner via a dibasic sequence within the third Fn3 domain [30]

Summary

Introduction

The cell adhesion molecule L1 ( called L1CAM or CD171), a member of the immunoglobulin superfamily of cell adhesion molecules, plays important roles in cell-cell interactions. Mutations in the X chromosome-localized L1 gene severely affect nervous system functions in affected males, including mental disabilities, aphasia, shuffling gait, and adducted thumbs (MASA syndrome) [12,13,14]. Besides its functions in the nervous system, L1 plays important roles in tumor progression and metastatis. Because of its pivotal importance in repair of the nervous system and in the metastatic behavior of tumors, we sought to screen for antibodies that, by reacting with different domains of the human L1 molecule, would, on the one hand, trigger its beneficial functions and, on the other hand, inhibit the detrimental functions of the molecule

Methods

Results

Conclusion