Interactions between neural cell surfaces seem to be of prime importance during neuroontogenesis, and responsible for the guidance of migrating neuroblasts and growing axons and for the formation of synapses. Little is known about the underlying molecular mechanisms, but most hypotheses imply the existence of cell-surface molecules that mediate the formation of transient or permanent bonds between neural cells. Recently, a membrane glycoprotein called neural cell adhesion molecule (N-CAM) has been characterized in chick and rodent nervous tissue that appears to act as a ligand in adhesion among neural cell bodies or neurites. We have identified a mouse neural surface glycoprotein, named BSP-2 (ref. 7), which by criteriaof electrophoretic migration, developmental changes, amino acid and sugar composition seems to be closely related or identical to N-CAM. Both BSP-2 (refs 8, 9) and N-CAM undergo conversion from an embryonic to an adult form during brain development and it has been suggested that this transition changes the adhesive properties or the binding specificity of the molecule. Using a neuroblastoma line to study functional differences between embryonic and adult BSP-2/N-CAM molecules, we show here that liposomes bearing adult BSP-2 but not those bearing the embryonic form adhere to neuroblastoma cells, demonstrating that the two forms do indeed possess different binding properties.

We have previously identified a cell surface glycoprotein of the mouse nervous system named brain cell surface protein‐2 (BSP‐2). Here we report that this antigen is not a single, discrete entity, but a family of antigenically and structurally related molecules. Three components of 180, 140, and 120 K were characteristic for more mature nervous tissues. Adult cerebral cortex contained the 140‐K and 120‐K antigens, adult spinal cord only the 120‐K, and dorsal root ganglia from young mice mainly the 180‐K component. Very different forms of the antigen that migrated as a diffuse zone from 180‐250‐K in SDS‐polyacrylamide gels were found in immature nervous tissues. A molecule different from the previous ones was found in a neuroblastoma line. Evidence is presented that the structural diversity of BSP‐2 is due to differences in glycosylation. This result indicates that cell type‐ and developmental stage‐specific glycoprotein patterns previously found in the nervous system may in part be due to different glycosylation of identical polypeptides. The finding that a neural cell surface protein may be glycosylated in different ways has important implications for the generation of cell surface specificity.

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 1251-labeled and reconstituted into artificial lipid vesicles. After trypsin digestion, the peptides thar 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-acidbearing 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 M , values of 92000,42000 and 35000. The H28.123 determinant could be mapped to a 32000-M, 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 M , were generated from young and adult material.However, a 45000-M, peptide from neonatal N-CAM appeared to migrate in the high-Mr region of sodium dodecyl sulfatejpolyacrylamide &ells 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.The neural cell adhesion molecules, called N-CAMS, are among the best-characterized cell adhesion proteins [I, 21. Unsolved questions concerning their structure are their mode of interaction with the cell membrane and the exact relationship between the two or three structurally similar polypeptides isolated from chick [3] or rodent [4 -61 brain. Our approach to these problems has been to reconstitute purified mouse N-CAM into artificial lipid vesicles and to analyze the peptides generated by trypsin digestion of these vesicles. A number of previous studies have addressed the question of how intrinsic membrane proteins insert in liposomes (see for instance [7 -9]), and it is generally agreed that the proteins interact with the vesicle bilayer in a way closely resembling their arrangement in the native membrane. However, few studies on the transmembrane topography of proteins have Abbreviations. SDS, sodium dodecyl sulfate; Tris/saline, 50 mM Tris pH 7.4,...

The cell-adhesion molecules N-CAM (neural cell-adhesion molecule) are ligands in the formation of cell-cell bonds and have been shown to play important roles during neuro-ontogenesis. They exist in several molecular forms which differ at the protein and carbohydrate levels. The regulation of the expression of these different forms is an important issue that bears on such questions as to how adhesive interactions between cells are modulated during morphogenesis. In the present study we have used N-CAM cDNA clones to investigate the expression of the cognate mRNAs in the mouse and rat brain and in 2 neural cell lines. The results were compared with the levels of the different N-CAM proteins. We made the following observations. A complex set of 5 size classes of mRNAs--which show developmental, regional, and cell-type-dependent variations in their expression--hybridize to 1 of our cDNA probes. While embryonic brain contains N-CAM gene transcripts 7.4, 6.7, and 4.3 kilobases (kb) in length, 2 additional mRNAs of 5.2 and 2.9 kb appear postnatally. Transformed brain cells of an astrocytic character express predominantly mRNAs of 6.7, 4.3, and 2.9 kb and a neuroblastoma line those of 7.4, 6.7, 4.3, and 2.9 kb. There are important quantitative changes in the amount of N-CAM message expressed during brain development, with a peak around birth, suggesting that N-CAM synthesis is controlled at the transcriptional level. A comparison of N-CAM protein and mRNA levels reveals a striking correlation between the relative concentrations of the Mr 120,000 N-CAM protein (N-CAM120) and the 5.2 kb transcript.(ABSTRACT TRUNCATED AT 250 WORDS)

Interleukin-2 is a major growth factor for activated T lymphocytes, and antibodies reacting with the Tac-chain component of the interleukin-2 receptor can prevent allograft rejection in animals. Because Tac chains are expressed only on a small fraction of activated lymphocytes, monoclonal antibodies against the interleukin-2 receptor may offer a more specific means of immunosuppression than polyclonal antilymphocyte globulin in prophylaxis against graft rejection. Therefore, we compared the immunosuppressive effect of 33B3.1, a rat monoclonal antibody against the human Tac chain, with the effect of a rabbit polyclonal antithymocyte globulin in a randomized study of 100 recipients of first renal transplants. Injections of 33B3.1 (10 mg per day) were tolerated well, whereas major side effects in 15 of 47 patients (32 percent) receiving antithymocyte globulin required discontinuation of treatment before day 14. The incidence of rejection episodes was not statistically different in the two groups at days 14, 30, 60, and 90 after transplantation. Patient and graft survival was also equal in the two groups at one year (96 and 85 percent, respectively, in both groups), and graft function was similar. The total number of infectious episodes within the first three months was lower in the 33B3.1 group than in the antithymocyte group (47 vs. 72). The drop in peripheral-blood lymphocyte concentrations was significantly larger in the patients treated with antithymocyte globulin. The level of circulating Tac-chain-bearing lymphocytes remained below 1 percent during 33B3.1 treatment, as compared with 4 to 5 percent during antithymocyte-globulin treatment (P not significant). We conclude that 33B3.1 is as effective as antithymocyte globulin in the prevention of renal-transplant rejection, and its use results in fewer infections and side effects.

The N‐CAMs are a group of surface glycoproteins involved in adhesive interactions of neurones. Related molecules of the mouse nervous system, identified in our laboratory, have been called BSP‐2 and shown to act as ligands in adhesion of neuroblastoma cells. Results presented in this report show that they are immunochemically identical with N‐CAM. A monoclonal anti‐(N‐CAM) antibody, that recognized a determinant accessible only after permeabilization of intact cells, was used to define the mode of association of the N‐CAMs with the plasma membrane. This antibody bound a 35000‐Mr fragment in lysates of trypsin‐treated neuroblastoma cells. It is concluded that the antibody reacts with a transmembrane or cytoplasmic domain of the molecules. The same antibody recognized the Mr‐180000 and Mr‐140000 proteins but not the Mr‐120000 chain, which copurify from adult mouse brain. The latter polypeptide was detected in the cytosol and could be partially released from brain membranes by osmotic shock. Part or all of the Mr‐120000 protein may thus lack a transmembrane segment. Our conclusion that the N‐CAM forms of higher Mr are transmembrane proteins was further corroborated by our finding that they contain phosphoserine residues, which can be labeled with (32P)phosphate in intact neuroblastoma cells.

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