Studies on the transmembrane disposition of the neural cell adhesion molecule N‐CAM

Publisher Website
Google Scholar
Abstract
The transmembrane orientation of the polypeptide chains present in preparations of adult and neonatal mouse N‐CAM was studied using, as a model system, liposome‐inserted purified N‐CAM preparations. N‐CAM purified from adult or neonatal mouse brain was 125I‐labeled and reconstituted into artificial lipid vesicles. After trypsin digestion, the peptides that remained associated with the liposomes were isolated by floatation of the vesicles on sucrose gradients. In control experiments the liposomes were lysed before trypsin treatment. Large, overlapping peptides were obtained after this treatment, several of which were protected by the liposome membrane. Sialic‐acid‐bearing peptides were revealed by their sensitivity to neuraminidase. To distinguish between peptides corresponding to intracellular or extracellular domains use was made of the P61 and H28.123 monoclonal antibodies, which recognize determinants located on the cytoplasmic and the extracellular part of the molecules respectively.There was no indication that the N‐CAM chains were inserted in an inside‐out configuration. Peptides protected from trypsin attack by the liposomes and recognized only by P61 had Mr values of 92000, 42000 and 35000. The H28.123 determinant could be mapped to a 32000‐Mr peptide located close to the membrane at the vesicle's exterior. The bulk of the sialic acid seemed to be carried by a rather short sequence distal to the H28.123‐reactive peptide but at some distance from the N terminus. Fragments of very similar Mr were generated from young and adult material. However, a 45000‐Mr peptide from neonatal N‐CAM appeared to migrate in the high‐Mr region of sodium dodecyl sulfate/polyacrylamide gels in its fully sialylated form.It is concluded that (a) identical polypeptide chains are present in young and adult preparation, (b) the 180000‐Mr, 140000‐Mr and 120000‐Mr chains differ by the length of their cytoplasmic extensions and (c) the largest cytoplasmic sequences have a Mr close to 90000. A tentative linear model of the transmembrane topography of the N‐CAM polypeptides is presented.