Biosynthesis and membrane topography of the neural cell adhesion molecule L1.

Abstract

The biosynthesis and membrane topography of the neural cell adhesion molecule L1 have been studied in cerebellar cell cultures by metabolic labeling and immunoprecipitation. Pulse and pulse‐chase experiments with [35S]methionine show that L1 is synthesized in its high mol. wt. form, the 200 kd component. The lower mol. wt. components with 40, 80 and 140 K apparent mol. wts. can be generated by proteolysis in intact cellular membranes. Peptide maps generated by protease treatment of L1 isolated from adult mouse brain show that the 80 and 140 kd components are related to the 200 kd component, but not to each other. The 200, 80 and 40 kd components can be biosynthetically phosphorylated. The 140 kd component is not phosphorylated and not released from the surface membrane during tryspinization. The phosphorylated amino acid is serine. In the presence of tunicamycin the 200 kd component is synthesized as a 150 kd protein. Pulse‐chase experiments in the presence of tunicamycin indicate that the carbohydrate moieties are predominantly N‐glycosidically linked and that the contribution of O‐glycosylation is minimal. The carbohydrate moieties are of the complex type as shown by treatment with endoglycosidase H. Since monensin inhibits processing of the carbohydrate moieties, the 200 kd component appears to be transported to the surface membrane via the Golgi apparatus.

L1 cell adhesion molecule found in human CSF varies as a function of age

“…We interpret these findings as follows: the 200-kDa form is full-length fully glycosylated L1 as described in lysates from whole brain (Faissner et al, 1985;Michelson et al, 2002;Poltorak et al, 1995;Vawter et al, 1998). The protein in this band is immunoreactive with both antibodies to L1CD as well as L1ED, implying the full-length molecule is represented.…”

“…High molecular weight (HMW) bands identified with L1 antibodies are detected in human tissues . These different isoforms may be L1 with immature posttranslational modifications (Faissner et al, 1985;Michelson et al, 2002), or the result of extracellular proteolysis of L1 . These proteolytic fragments may regulate L1 expression and function Michelson et al, 2002;Nayeem et al, 1999).…”

Cell-Surface Glycoprotein of Oligodendrocyte Progenitors Involved in Migration

“…For steady-state radiolabeling of the cell line Oli-neu, cells were incubated overnight with 100 C i /ml L-( 35 S) in vitro labeling mix consisting of 70% methionine and 30% cysteine or 100 C i /ml 35 SO 4 (Amersham-Buchler). Subsequently an immunoprecipitation was performed [basically as described in Faissner et al (1985)], using the monoclonal AN2 antibody and Protein-A Sepharose, which had been preincubated with rabbit anti-rat "bridge" antibody before addition to the cell lysate. For "pulse -chase" labeling, the cells were first incubated for 1 hr in methionine/cysteine-free minimal essential medium (M EM) with 2 mM glutamine.…”

Alterations of hippocampal secreted N-CAM in bipolar disorder and synaptophysin in schizophrenia

“…The affinity purified SEC N-CAM antisera were used to immunoprecipitate brain extracts (membrane and cytosolic) using Sepharose 4B-Protein A and a slight modification of a previously published procedure 42 in which 0.05% Tween-20 was added to Buffer A 27 for washes. The SEC N-CAM immunoprecipitates were also immunoblotted with polyclonal N-CAM antisera Percentage of protein extracted from each brain sample (mg protein mg −1 weight of frozen brain tissue).…”

“…A cytosolic brain extract was immunoprecipitated with polyclonal N-CAM 3732 using the same procedure. 42 …”