Abstract
The neural cell adhesion molecule L1 is a phosphorylated, integral membrane glycoprotein that is recovered from adult mouse brain tissue by immunoaffinity chromatography as a set of polypeptides with apparent molecular masses of 200, 180, 140, and 80 kilodaltons (L1–200, L1–180, L1–140, and L1–80, respectively). It has been shown that L1–140 and the phosphorylated L1–80 is generated from L1–200 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 L1–200 has two preferred cleavage sites, one that generates the amino terminal, extracellularly exposed L1–140 and the carboxy terminal L1–80 that spans the membrane. Cleavage at the other site leads to the generation of the amino terminally located L1–180 and the membrane‐attached, phosphorylated carboxy terminal L1–30. 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 L1–180 and L1–30, suggesting a particular cell‐intrinsic cleavage mechanism. L1–180 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. Although trypsin treatment alone does not release L1–140 into the supernatant, combination of phospholipase C and mild tryptic treatment leads to the release of L1–140 and L1–50, the latter being most likely the extracellularly exposed domain of L1–80 that is complementary to the membrane‐integrated phosphorylated L1–30. Phase separation 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 L1–200 or L1–80. However, when L1–80 is cleaved to yield the soluble L1–50 and membrane‐anchored L1–30, 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 L1‐containing liposomes, but not for those containing the adhesion molecules neural cell adhesion molecule and myelin‐associated glycoprotein. Although the physiological roles of the soluble L1 forms, their mode of generation, and the strong affinity for each other remain to be investigated, the availability of soluble forms of L1 opens the possibility to use them as probes for the functional properties of L1 in assay systems involving live cells in vitro.