Journal zyxwvutsrqpon of Neurochemisrry zyxwvutsrqponm Raven Press, zyxwvutsrqpo Ltd., New York zyxwvutsrqpon 0 1988 International Society for Neurochemistry Biochemical Characterization of Different Molecular Forms of the Neural Cell Adhesion Molecule L1 Karin Sadoul, Rbmy Sadoul, Andreas Faissner, and Melitta Schachner Department of Neitrobiology, University of Heidelberg, Heidelberg, F.R.G. Abstract: The neural cell adhesion molecule LI is a phos- phorylated, integral membrane glycoprotein that is recov- ered from adult mouse brain tissue by immunoaffinity chromatography as a set of polypeptides with apparent mo- lecular masses of 200, 180, 140, and 80 kilodaltons (LI-200, LI-180, LI-140, and LI-80, respectively). It has been shown that LI-140 and the phosphorylated LI-80 is generated from Ll-200 by mild proteolytic treatment of intact cells. In the present study we have investigated the structural relationships between the different molecular forms of L1 and their location with regard to the surface membrane. We could show that LI-200 has two preferred cleavage sites, one that generates the amino terminal, ex- tracellularly exposed L zyxwvutsrq 1 - 140 and the carboxy terminal LI-80 that spans the membrane. Cleavage at the other site leads to the generation of the amino terminally located L zyxwvutsrqp 1 - 180 and the membrane-attached, phosphorylated car- boxy terminal LI-30. This site is cleaved during treatment of live cultured cells with broad-spectrum, protease-free phospholipase C (but not phosphatidylinositol-specific phospholipase C) or exposure to sodium azide or cyanogen bromide. Other conditions that cause damage to cells do not lead to the generation of L 1 - 180 and L 1-30. suggesting a particular cell-intrinsic cleavage mechanism. L 1 - I80 is truly soluble in aqueous solutions, since it can be recovered from culture supernatants and in the supernatant of a crude membrane fraction after incubation for 2 h at 37°C. Al- though trypsin treatment alone does not release LI-140 into the supernatant, combination of phospholipase zyxwvu C and mild tryptic treatment leads to the release of L1-140 and L1-50, the latter being most likely the extracellularly ex- posed domain of LI-80 that is complementary to the membrane-integrated phosphorylated L 1-30. Phase separa- tion experiments with Triton X-114 show that the released forms of L1-180 and L1-140 distribute into the aqueous phase, whereas they distribute into the detergent phase when in association with LI-200 or LI-80. However, when LI-80 is cleaved to yield the soluble L1-50 and membrane- anchored LI-30, L1- 140 is released into the supernatant together with L1-50. A strong affinity of L1-200, L1-140, and L1-80 to each other is also indicated by the fact that they incorporate together into liposomes and separate only under strong detergent conditions. Also, a strong tendency to aggregate is observed for L 1 -containing liposomes, but not for those containing the adhesion molecules neural cell adhesion molecule and myelin-associated glycoprotein. Al- though the physiological roles of the soluble Ll forms, their mode of generation, and the strong affinity for each other remain to be investigated, the availability of soluble forms of LI opens the possibility to use them as probes for the functional properties of L1 in assay systems involving live cells in vitro. Key Words: Antibodies-Membrane disposi- tion-Neural cell adhesion molecule L1-Phospholipase C-Proteolytic cleavage-Triton X- 1 14. Sadoul K. et al. Biochemical characterization of different molecular forms of the neural cell adhesion molecule L1. zyxw J. Neurochern. 50, 5 10-52 1 (1988). The cell adhesion molecule L1 is involved in a Ca*+-independent adhesion mechanism among neural cells (Rathjen and Schachner, 1984). Fab fragments of poly-, but not monoclonal antibodies interfere with aggregation of early postnatal mouse cerebellar and neuroblastoma N2A cells (Faissner et al., 1984~; Rathjen and Schachner, 1984; Rathjen and Rutishauser, 1984). L1 is involved in granule neuron migration (Lindner et al., 1983, 1986) and fasciculation of neurites (Fischer et al., 1986). It does not appear to mediate synapse formation (Mehrke et al., 1984; Orkand et al., 1984) or synaptic or electrical Received May 11, 1987: revised manuscript received July 11. 1987: accepted August 17, 1987. Address correspondence and reprint requests to Dr. M. Schachner at Department of Neurobiology. University of Heidel- berg, Im Neuenheimer Feld 364. 6900 Heidelberg. F.R.G. Abbreviurions used: BSA. bovine serum albumin; CMF-HBSS. Ca2’- and Mg2’-free Hank’s balanced salt solution: HEPES, X-2- hydroxyeth~lpiperazine-N’2-ethanesulfonic acid; kD, kilodalton; MAG, myelin-associated glycoprotein; N-CAM, neural cell adhe- sion molecule; NILE, nerve growth factor inducible large external protein; PBS, phosphate-buffered saline; pCMBS, p-chloromercuri- benzenesulfonic acid; SDS-PAGE. sodium dodecyl sulfate-poly- acrylamide gel electrophoresis.