Individual neural cell types express immunologically distinct N-CAM forms.

The neural cell adhesion molecules, or N-CAMs, are a group of structurally and immunologically related glycoproteins found in vertebrate neural tissues. Adult brain N-CAMs have apparent molecular weights of 180,000, 140,000, and 120,000. In this article we identify, using monoclonal antibody (Mab) 3G6.41, an immunologically distinct adult rat N-CAM form and show that this form is selectively expressed by some clonal neural cell lines. Consecutive immunoprecipitation experiments indicate that rabbit anti-N-CAM can remove from solubilized cerebellar neuron primary cultures all 180,000- and 140,000-mol-wt N-CAM molecules that react with Mab 3G6.41. However Mab 3G6.41 cannot remove all N-CAM molecules that react with rabbit anti-N-CAM. Rabbit anti-N-CAM binds to and immunoprecipitates N-CAM forms from the rat neuronal cell lines B35, B65, and B104, the glial lines B12 and C6, and L6 myoblasts. Mab 3G6.41 does not bind to or immunoprecipitate N-CAM from the B12 and B65 lines but does react with the other four lines by both criteria. Many cells in primary cultures of postnatal rat that express glial fibrillary acidic protein also bind Mab 3G6.41. Thus a unique form of rat N-CAM recognized by Mab 3G6.41 is found on some but not all neuronal, glial, and muscle cells.

Cell-cell interaction is of central importance to the establishment and maintenance of functional connections in the developing nervous system. Specific adhesion between cells has been proposed as the basis for precise neural connectivity (32). The BSP-2 glycoproteins originally identified using a monoclonal antibody against neonatal mouse brain glycoprotein extracts (18) have been implicated in the cell-cell adhesion of neurons (29). BSP-2 is composed of a group of related glycoproteins with apparent molecular weights (Mr'S) of 180,000, 140,000, and 120,000, in the adult mouse cerebellum (17) and is immunologically similar or identical to the previously identified chick neural cell adhesion molecules or N-CAMs I (6,7,12) and the mammalian D2 protein (17). Hence, for uniformity of terminology these mammalian proteins will also hereafter be called N-CAM. It has been suggested that control of the specificity of cell-cell interactions involving these molecules may be due either to temporal ~ Abbreviations used in this paper: DME, Dulbecco's modified Eagle's medium; Mab, monoclonal antibody; N-CAM, neural cell adhesion molecule; Ng-CAM, polypeptide that may mediate the adhesion between neurons and glia. 36 expression of N-CAM or to alterations in the structure of the expressed molecules (9).
In embryonic brain N-CAM is found as a unique polysialylated form of the adult polypeptide (10, 1 l, 26). Liposomes containing sialylated N-CAM show less binding to N-CAM on cell surfaces than do liposomes with nonsialylated N-CAM (19,29). This structural and potential functional difference between embryonic and adult N-CAM forms can be assayed immunologically with a monoclonal antibody (Mab) that binds to the polysialyl chains (6). The adult 180,000-, 140,000-, and 120,000-mol-wt forms have been shown to vary primarily in the size of their cytoplasmic domain (13). However it is not clear whether each of these molecular weight classes represents a single polypeptide or a group of polypeptides of similar Mr. Likewise, the immunologic relationships among the molecular weight forms of adult N-CAM are less clear. Recently differences in the reactivity of Mab's with adult N-CAM from brain and muscle or various brain regions have been reported (6).
Two of us (Drs. Williams and Akeson) have reported that the monoclonal antibody 3G6.41 immunoprecipitated 185,000-and 140,000-Mr polypeptides from iodinated cere-bellar neuronal cultures (34). We report here that the 185,000and 140,000 molecules recognized by Mab 3G6.41 are a subset of those precipitated by rabbit anti-N-CAM from rat primary neuronal cultures. Some individual neural clonal cell lines do not express the form(s) of N-CAM that reacts with Mab 3G6.41 whereas other lines express N-CAM forms that react with both rabbit anti-N-CAM and Mab 3G6.41.
Antibodies and Assay of Antibody Binding to Cells: The monoclonat antibody 3G6.41, isotype -t2b, ~ was produced from the fusion of X63-Ag8.653 myeloma cells (653 myeloma; from Dr. Daryl Doyle, State University of New York at Buffalo) with spleen cells from BALB/c x C57BL6 Ft (Jackson Laboratory, Bar Harbor, ME) mice immunized with adult rat forebrain synaptic plasma membranes as described (34). Polyclonal antiserum against N-CAM polypeptides was generated in rabbits using as an immunogen rat N-CAM that had been affinity purified with a monoclonal anti-N-CAM column (12. 13). This serum blocks cell-cell aggregation. As compared with a rabbit anti-mouse N-CAM serum (7; the gift of Dr. G. M. Edelman, The Rockefeller University) both sera totally remove all iodinated (12) or [SH]methionine-labeled (Goridis, C., unpublished data) molecules that react with the other sera. Some earlier articles used the acronym BSP-2 for the N-CAM molecules, but BSP-2 and mammalian N-CAM are immunologically identical (12). Hence we will use the term N-CAM here.
Cultured cells were removed with EDTA in pH 7.4 phosphate-buffered saline and washed in 0.01 M Tris HCI pH 7.4, 0.15 M NaCI with 0.5% bovine serum albumin. We quantitated binding of Mab 3G6.41 and anti-BSP-2 sera using ~l-labeled protein A in the previously described antibody-excess assay (25). Double-label immunofluoreseence analysis of antibody binding using Mab 3G6.41 and anti-glial fibrillary acidic protein sera (Dako Corp., Santa Barbara, CA) was performed as previously described (1).
Polypeptide Labeling and Immunoprecipitation: Cell-surface radioiodination of clonal cell lines and primary cultures, detergent solubilization of membranes, and immunoprecipitations were done as described (34). Postnatal day 7 cerebellar cultures (prepared as above) were biosynthetically labeled by replacement of the serum-containing medium with DME containing only 1 mg/ml glucose and the supplements described for serum-free culture of neurons (5). After 3 d in culture, 20 uCi of [3H]fucose (ICN Pharmaceuticals Inc., lrvine, CA) were added to the cultures in 2 ml of this serum-free medium. After 43 h of incubation, the cell membranes were solubilized and polypeptides were immunoprecipitated as above.
For immunoblotting proteins from adult rat whole brain membranes were solubilized in 1% Nonidet P-40, immunoprecipitated with monoclonal antibody 3G6.41 as described (34), resolved on SDS gels, and electrophoretically transferred to nitrocellulose strips (33). Immunoblotting of the strips with Mab P61 was done as described (12) except that bound Mab P61 was detected using rabbit anti-rat 1/300 dilution followed by ~2~I protein A 1.6 x 106 cpm/ml (specific activity, 7.7 × 106 cpm/ug protein).

Relationship of Polypeptides Immunoprecipitated by Mab 3G6.41 and Anti-N-CAM
We identified the polypeptides that bind Mab 3G6.41 by lactoperoxidase cell-surface labeling of viable neuronal postnatal rat cerebellum cultures and subsequent solubilization of membrane proteins in nonionic detergent and immunoprecipitation. Mab 3G6.41, but not control ascites containing Mab of the same isotype, precipitated a pair of polypeptides with Mr's of 185,000 and 140,000 ( Fig. 1, lanes A and B). Both polypeptides are glycoproteins since they can also be immunoprecipitated from [3H]fucose biosynthetically labeled cerebellar primary culture (Fig. I, lanes C and D). The Mr's and glycoprotein nature of the polypeptides immunoprecipirated by 3G6.41 suggest these molecules may be similar or identical to the previously described N-CAM (10, 17) class of neuronal cell surface macromolecules.
To compare directly the relationship between these 3G6.41 140,000-and 180,000-mol-wt proteins with the previously described 140,000-and 180,000-mol-wt N-CAM polypeptides, we performed sequential precipitations. In this approach  In a reciprocal experiment rabbit anti-N-CAM or nonimmune rabbit serum was used for three consecutive immunoprecipitations ofiodinated rat cerebral cultures (Fig. 3, panels  1-3). After clearance with S. aureus, a final immunoprecipitation with Mab 3G6.41 gave no bands from anti-N-CAMpretreated supernatants (broken line, panel 5 of Fig. 3). Large amounts of the 3G6.41 reactive bands were left if the first immunoprecipitations were done with nonimmune rabbit serum (solid line, panel 5 of Fig. 3). Therefore all the 3G6.41 reactive molecules from these primary cultures were immunoprecipitated by rabbit anti-N-CAM.
For an additional test of the relationship of the polypeptides precipitated by Mab 3G6.41 and previously defined N-CAMs, we used Mab P61, which has been shown to react exclusively with N-CAMs (12). lmmunoprecipitates of detergent-solubilized unlabeled rat brain proteins were made with Mab 3G6.41 or normal mouse sera, electrophoresed, and transferred to nitrocellulose for immunoblotting. Mab P61 reacts strongly with the 140,000-and 180,000-mol-wt bands precipitated by Mab 3G6.41 (Fig. 4), indicating these molecules express this previously defined N-CAM antigenic site.   muscle cell line because N-CAM or N-CAM-like molecules have been reported to be expressed on chicken muscle (16,22). Six rat cell lines were found to be anti-N-CAM positive: B35, B65, and Bl04 are neuronal cell lines, whereas B12 and C6 are glial in phenotype and L6K is a skeletal myoblast. Four of these lines, B35, Bl04, C6, and L6K, were also found to react with Mab 3G6.41 (Table I) To test if Mab 3G6.41 bound to unique molecular forms on these cell lines, they were surface iodinated and immunoprecipitated with Mab 3G6.41 or rabbit anti-N-CAM in parallel. Anti-N-CAM immunoprecipitates from three cell lines, B35, C6, and L6 that bound both Mab 3G6.41 and anti-N-CAM all showed the presence of the 140,000 and 120,000 M, polypeptides and to a lesser extent of a polypeptide band at ~ 180,000 Mr (Fig. 5, lanes B, D, and F). In the Mab 3G6.41 immunoprecipitates we consistently saw the 140,000-mol-wt component in each of these three cell lines (Fig. 5, lanes A, C, and E). As with the 125I-labeled cerebellar cultures, we did not observe the 120,000 Mr component in any of the Mab 3G6.41 immunoprecipitates. From the C6 glial cell line Mab 3G6.41 immunoprecipitated a band with Mr 180,000-190,000-mol-wt that comigrated with the highest Mr band precipitated by anti-N-CAM. This upper component was not seen in the L6 myoblast or B35 neuronal cell lines.
From the B12 glial cell line, which bound rabbit anti-N-CAM but not Mab 3G6.41, anti-N-CAM immunoprecipitated broad bands of almost identical intensity centered at ~145,000 and 180,000 Mr (Fig. 6, lane B) and a less intense 120,000 M, component. Mab 3G6.41 did not immunoprecipitate these polypeptides from B 12 cells. Rabbit anti-N-CAM also precipitated 140,000-and 120,000-mol-wt components from the B65 neuronal line (Fig. 6, lane F). However, we did not see a 180,000-mol-wt component in this precipitate. Again, as expected from the lack of detectable antibody binding to whole cells, Mab 3G6.41 did not precipitate any of these N-CAM components from the B65 cell line (Fig. 6, lane G). As summarized in Table II, Mab 3G6.41 can immunoprecipitate polypeptides in the 140,000-mol-wt range (all ceils tested) and also the 180,000-mol-wt range (C6 cells and primary neuron cultures). However, the 120,000-mol-wt form of N-CAM does not seem to contain the 3G6.41 antigenic site. The apparent molecular weights of the 140,000-and 180,000-mol-wt region polypeptides precipitated by Mab 3G6.41 and rabbit anti-N-CAM overlap entirely. Thus these experiments do not support the hypothesis that Mab 3G6.41 reacts with a unique size class of N-CAM molecules.

N -C A M Expression by Primary Astrocytes
The reactivity of Mab 3G6.41 with C6 astrocytoma cells as well as our earlier observation that 3G6.41 reacts with cells of non-neuronal morphology in cerebellar primary cultures suggest that rat astrocytes may express N-CAM like surface components. This point has not been entirely clear, as rat cerebellar Bergmann glia (17), mouse glial cell lines (2), and neuroepithelial cells adjacent to the chick embryo visual pathway (3 l) have been reported to stain with anti-N-CAM. In contrast no staining with anti-N-CAM of non-neuronal cells in chick or mouse brain primary cultures was detected    (4). Third passage whole brain astrocyte cultures that contained no neurons had many cells that bound both antibodies (Fig. 7). Most of the anti-glial fibrillary acidic protein-labeled cells also bound Mab 3G6.41. Fluorescence was often particularly intense at regions of cellcell contact (Fig. 7, arrowheads). A cell unlabeled by either antibody is outlined in the figure. Thus rat astrocytes clearly express components immunologically similar to N-CAM.

DISCUSSION
Distinct neural cell adhesion molecules appear to be expressed in the same brain region and in some cases the same cell type. Mouse cerebellar granule cells express the Ll cell adhesion molecules consisting of 200,000-and 140,000-mol-wt glyco-40 THE JOURNAL OF CELL BIOLOGY , VOLUME 101, 1985 proteins (24) and also N-CAM molecules (18). Individual neurons cultured from chick embryo brains have been shown to express both N-CAM and also Ng-CAM, a 135,000-molwt polypeptide that has been suggested to mediate the adhesion between neurons and glia (14, 15). Through the use of specific antibody reagents (23,30), N-CAM and Ll have been shown to be immunologically distinct; however N-CAM and Ng-CAM share at least one antigenic site (15). Further diversity among the 140,000-M~ glycoproteins has been observed using the monoclonal antibody NSP-4 (27), which recognizes a family of molecules in mouse brain distinct from N-CAMs, Ng-CAM, and L I. Sets of nervous system cells that are perhaps functionally or developmentally distinct, may express combinations of these cell adhesion molecules. Diversity in the molecular forms of the N-CAM molecules themselves during neural development is characterized by a shift from highly sialylated N-CAM glycoproteins in embryonic and early postnatal mammals to much less sialylated polypeptides with distinct Mr's of 180,000, 140,000, and 120,000 in adults (17,26). Differences among the lengths of these adult type chains appears to be due to the size of their cytoplasmic extensions. N-CAM of all MTs reconstituted into liposomes and then trypsinized yields different sized fragments which remain liposome bound and hence contain the cytoplasmic domain (12,13). Variations that may occur within each of the components of the adult forms have been less well characterized. One Mab directed against a nonsialic acid portion of N-CAM has been reported to react with adult cerebral but not cerebellar N-CAM (6, 7).
Mab 3G6.41 appears to recognize a subclass of the adult form of N-CAM cell adhesion molecules. This conclusion is based on the following evidence: (a) Mab 3G6.41 and monoclonal anti-N-CAM (18) recognize neuronal cell surface glycoproteins of 140,000 and ~ 180,000 Mr in cerebellar cultures. The immunoprecipitated bands using Mab 3G6.41 were reproducibly less intense on the autoradiographs than the bands immunoprecipitated by anti-N-CAM sera, implying that Mab