Phylogenetic origins of antibody structure. II. Immunoglobulins in the primary immune response of the bullfrog, Rana catesbiana.

The anuran amphibian, Rana catesbiana, has been found to possess at least two kinds of immunoglobulins corresponding to gammaG- and gammaM-classes. These classes have the same chain structures as those of their counterparts in higher animal species. Light chains of both immunoglobulins had molecular weights of 20,000. Heavy chains of the gammaM-class had molecular weights of 72,100; those of the gammaG-class had molecular weights of 53,600. The carbohydrate content of the gammaG-immunoglobulin was 2.1%, and that of the gammaM-protein was 10.8%. The amino acid compositions of the immunoglobulins were generally similar to those of mammalian immunoglobulins. After a single injection of phage antigen (f2), the order of appearance of phage-neutralizing activity in the frog immunoglobulin classes was (a) gammaM-antibodies, and (b) gammaG-antibodies. The results of this and previous studies suggest that the gammaG-immunoglobulins emerged at some point in evolution between the elasmobranchs and the anuran amphibians.

Maturation of the humoral immune response in mice.

In spite of the prenatal appearance of immunoglobulin-bearing lymphocytes and theta-positive lymphocytes in the spleens of Swiss-L mice, these mice are not able to produce detectable levels of humoral antibodies in response to antigen until after 1 wk of age. Adult levels of response are not achieved until 4-8 wk of age. In the presence of bacterial lipopolysaccharides, which can substitute for or enhance T-cell function, the B cells from young Swiss-L mice were found to be indistinguishable in function from adult B cells, both with respect to the numbers of plaque-forming cells (PFC) produced in vitro in response to antigen and with respect to the kinetics of PFC induction. The spleen cells from young Swiss-L mice are significantly less sensitive than adult spleen cells, however, to stimulation by the T cell mitogens, concanavalin A (Con A) and phytohemagglutinin (PHA). Very few Con A-responsive cells could be detected at birth but the numbers increased sharply with age until 3 wk after birth. On the other hand, PHA-responsive cells could not be detected in the spleen until about 3 wk of age. The latter cells were found to respond also to Con A, but at a lower dose (1 microg/ml) than that required for the bulk of the Con A-responsive cells (3 microg/ml). The cells that respond both to PHA and to Con A appear in the spleen at about the time that Swiss-L mice acquire the ability to produce humoral antibodies, and these cells can be depleted from the spleen by the in vivo administration of antithymocyte serum. The development of humoral immune responses in these mice therefore appears to be correlated with the appearance of recirculating T lymphocytes that are responsive both to PHA and to Con A.

Participation of the H-2 antigens of tumor cells in their lysis by syngeneic T cells.

Cytotoxic T lymphocytes were generated in vitro against H-2 compatible or syngeneic tumor cells. In vitro cytotoxic activity was inhibited by specific anti-H2 sera, suggesting that H-2 antigens are involved in cell lysis. Two observations directly demonstrated the participation of the H-2 antigens on the tumor cells in their lysis by H-2-compatible T cells. First, coating of the H-2 antigens on the target tumor cell reduced the number of cells lysed on subsequent exposure to cytotoxic T cells. Second, when cytotoxic T cells were activated against an H-2 compatible tumor and assayed against an H-2-incompatible tumor, anti-H-2 serum that could bind to the target cell, but not to the cytotoxic lymphocyte, inhibited lysis. H-2 antigens were also shown to be present on the cytotoxic lymphocytes. Specific antisera reacting with these H-2 antigens, but not those of the target cell, failed to inhibit lysis when small numbers of effector cells were assayed against H-2-incompatible target cells or when effector cells of F1-hybrid origin and bearing two H-2 haplotypes were assayed against a tumor cell of one of the parental strains. These findings suggest that it is the H-2 antigens on the tumor cell and not those on the cytotoxic lymphocytes that are important in cell-mediated lysis of H-2-compatible tumor cells.

Joint recognition by cytotoxic T cells of inactivated Sendai virus and products of the major histocompatibility complex.

Cytotoxic T cells specific for Sendai virus were generated by culturing murine spleen cells in vitro together with UV-inactivated Sendai virus. In vivo immunization of donor mice with UV-inactivated Sendai virus resulted in an in vitro secondary response of increased magnitude. Cytotoxic activity was demonstrated in a short-term 51Cr-release assay, using syngeneic tumor cells which had been coated with inactivated Sendai virus by incubation at 4 degrees C for 30 min. The lysis of Sendai virus-coated target cells was restricted by the H-2 haplotype of the target cells, suggesting that the H-2 genes of the target cell contributed to the specificity of the lysis. Kinetic experiments showed that susceptibility to lysis by cytotoxic T cells specific for Sendai virus appeared within 30 min after coating target cells with inactivated virus. Furthermore, there was no detectable synthesis of new proteins in cells treated with UV-inactivated Sendai virus. For these reasons, we suggest that neither viral replication nor the synthesis of new proteins are necessary for the production of the antigen recognized by cytotoxic cells specific for Sendai virus. We infer that the virus-specific component on the target cells is probably a preformed virion antigen adsorbed onto or integrated into the cell membrane. These results imply that, if the cytotoxic T cell recognizes a single antigenic determinant specified both by viral and H-2 genes, this determinant is formed by the physical association of H-2 and Sendai virus antigens rather than by their alteration during the processes of synthesis.

Analysis of the stimulation-inhibition paradox exhibited by lymphocytes exposed to concanavalin A.

High doses of Concanavalin A (Con A), which normally inhibit T-lymphocyte stimulation as measured by increases in DNA synthesis, cause these lymphocytes to become committed to mitogenesis while also generating a dominant but reversible negative growth signal. The observed response to the stimulatory signal as measured by the rate of commitment to enter the S phase (i.e., the rate at which the stimulation becomes lectin independent) increases with lectin concentration even in the inhibitory range. The generation of this positive signal is prevented by treating the cells with colchicine. Cells that have become committed but are also simultaneously blocked from entering the S phase by the high doses of Con A can begin synthesizing DNA if the lectin is released by adding a competitive inhibitor of binding. Experiments done in agarose cultures in which lymphocytes are kept from contact with each other suggest that the reversible inhibitory signal is mediated by structures in the individual cells rather than as a result of agglutination. Continuously dividing cells of the lymphoid line P388 are also individually and reversibly inhibited by Con A. These findings are considered in terms of the relation of the inhibitory signal to the microtubular components of cell surface modulating assemblies made up of submembranous arrays of microtubules, microfilaments, and associated proteins.

Isolation by cell-column chromatography of immunoglobulins specific for cell surface carbohydrates.

A new method of affinity chromatography using glutaraldehyde-fixed cells immobilized on Sephadex beads has been used to isolate immunoglobulins (Ig's) specific for cell surface glycoproteins. Ig's that specifically bound and agglutinated the same cells as those originally fixed on the columns were isolated from nonimmune sera of various species. Periodate treatment of the cell-columns and the free cells destroyed their ability to bind the Ig's, and the binding of the Ig's to untreated cells was inhibited by monosaccharides such as D-galactose and sialic acid. The binding of antibodies directed against cell surfaces obtained by immunizing animals with the same mouse tumor cell lines used on the columns (P388 and EL4) was not inhibited by various saccharides. Surface glycoproteins obtained from the mouse tumor cells by immunoprecipitation with the column-isolated Ig's yielded specific electrophoretic patterns that differed from those obtained using Ig's from the sera of rabbits immunized with the tumor cells. The data suggest that the Ig's isolated by cell-column chromatography were directed against carbohydrates, probably those in terminal positions of the polysaccharide portions of the tumor cell surface glycoproteins. Column-isolated Ig's specific for carbohydrates were also useful in studies of cell interactions in nonmammalian systems including Dictyostelium discoideum and Saccharomyces cerevisiae. The cell-column method appears to be adaptable to the isolation of a variety of molecules in addition to antibodies.

Frequency and avidity of specific antigen-binding cells in developing mice.

In order to analyze the development of antibody diversity in which the genes coding for the antigen-specific cells we have compared the binding of diverse antigens by cells in the fetal, neonatal, and adult mouse. Although the numbers of antigen-binding cells present in fetuses and young animals were smaller than in adults, no restriction could be detected in the varity of specificities expressed in the fetuses, either with respect to the kinds of antigens bound, or to the range of avidities of binding. Cells specific for each of the 11 antigens tested could be detected in the fetus only in the last 4 days before birth, at which time they appeared both in the liver and in the spleen. In all cases, these cells disappeared both in the liver and in the spleen. In all cases, these cells disappeared from the liver within a day of birth, but continued to increase in number in the spleen until adulthood...

Phylogenetic origins of antibody structure. 3. Antibodies in the primary immune response of the sea lamprey, Petromyzon marinus.

The sea lamprey, Petromyzon marinus, has been found to produce specific antibodies after immunization with bacteriophage f2. Antibody activity is localized in 6.6S and 14S fractions of lamprey serum. The 6.6S antibodies were purified by a combination of zone electrophoresis, ion exchange chromatography, and gel filtration. Antigenic analysis of the 6.6S antibodies showed them to be free of other serum proteins and antigenically similar or identical to the 14S fraction. Evidence has been obtained which suggests that the 6.6S immunoglobulins consist of light components (molecular weight 25,000) and heavy components (molecular weight 70,000). In the immunoglobulin, these polypeptides appear to be linked via weak interactions but not by interchain disulfide bonds. Molecular weight analyses support the view that the chains can undergo concentration-dependent dissociation in aqueous solutions. Amino acid analyses showed that the compositions of the light and heavy components were similar and that aspartic acid or asparagine was the predominant amino terminal residue. Starch gel electrophoresis indicated that the subunits of lamprey antibodies are diffusely heterogeneous. The heavy chain mobility corresponded to that of micro-chains and resembled that of heavy chains of shark and sting ray immunoglobulins. In the course of the fractionation a 46S natural hemagglutinin composed of lower molecular weight subunits was isolated. This hemagglutinin did not resemble the lamprey immunoglobulin although it had a similar zone electrophoretic mobility in the beta-region. These studies are consistent with the hypothesis that micro-chains were the earliest of the heavy chain classes to emerge and further support the view that the multichain structure of immunoglobulins is a fundamental feature of antibody molecules.

Variation and control of specific antigen-binding cell populations in individual fetal mice.

To determine the extent and nature of individual variation in the development of specific antigen-binding cells, the numbers of cells specific for each of two antigens in the spleens of individual random-bred Swiss-L and inbred CBA/J and BALB/c fetal mice were measured as a function of spleen size. For Swiss-L fetuses, the ratio of antigen-binding cells to nucleateated cells varied more than would arise from sampling fluctuation. For each inbred strain, however, the number of cells specific for a given antigen was a constant proportion of the total number of nucleated cells within sampling error. These proportions varied from antigen to antigen, and from strain to strain. The ratio of the proportions of cells specific for the two antigens, however, differed no more from CBA/J to BALB/c mice than would be expected in repeated samples of cells from the spleen of a single fetus. These results confirm at the level of the individual fetus the uniform pattern of development seen for populations of fetuses. They reveal a surprising precision in the proliferation of specific antigen-binding cell populations and suggest that the development of these cells may be subject to strong genetic controls.

Characterization of splenic lymphoid cells in fetal and newborn mice.

In order to clarify the cellular events that precede the onset of immunological competence in the mouse, we have characterized and quantitated the lymphoid cells of the spleen as a function of age. Our results show that T cells and B cells both appeared in the spleens of Swiss-L mice as early as the 15th-16th day of gestation. Antigen-binding cells specific for each of three different antigens were also first detected during this same 24 h interval. The B cells and three varieties of antigen-binding cells increased in number rapidly and in parallel until about 1 wk after birth. The T cells, which were more numerous than B cells at first, increased in number somewhat more slowly. Coincident with the onset of response to antigen, there was a further increase in B cell numbers and a decrease in the T cell to B cell ratio. The capacity to respond to antigen by cellular proliferation and synthesis of antibody did not arise until about 2 wk after birth although there were no quantitative changes in the total numbers of T cells, B cells, and antigen-binding cells between 1 and 2 wk of age. Some qualitative change, such as the functional maturation of an antigen-reactive cell, may be required during this interval for the onset of this immunological response. Although the numbers of antigen-binding cells present in fetuses and young animals were smaller than in adults, we have as yet been unable to detect any restriction in the variety of specificities that can be expressed in fetuses, either in the kinds of antigens bound or in the range of avidities with which a single antigen is bound.

Synthesis and distribution of H-2 antigens in preimplantation mouse embryos.

Synthesis of H-2 antigens by preimplantation mouse embryos is first detectable at the late blastocyst stage. These antigens were detected using immune precipitation assays of extracts of embryos labeled by incorporation of radioactive amino acids but not by surface iodination. Experiments using isolated inner cell massess and trophoblast vesicles indicate that it is the cells of the inner cell mass that synthesize these antigens. H-2 antigens were not detected in either early blastocysts or at earlier cleavage stages.

The specific antigen-binding cell populations of individual fetal mouse spleens: repertoire composition, size, and genetic control.

In order to analyze the genetic and physiological basis of controls affecting the generation of the repertoire of antigen-binding cells in fetal mice, we have measured the numbers of spleen cells specific for each of four antigens as a function of the total numbers of nucleated and Ig-bearing cells in inbred, hybrid, and random bred fetuses. For each of the two inbred strains BALB/c and CBA/J, the proportion of nucleated cells specific for a given antigen was the same for all individuals of the strain at the 18th day of gestation. The proportion did vary from antigen to antigen, however, and for each antigen the proportion of specific cells observed in CBA/J fetuses was approximately four times that observed in BALB/c fetuses. This difference appeared to be due to a difference between the two strains in the relative size of the repertoire of antigen-binding spleen cells at this stage of development, inasmuch as the frequency of Ig-bearing spleen cells in CBA/J fetuses was likewise approximately four times that observed in BALB/c fetuses. In random bred Swiss-L fetal mice at the 18th day of gestation, the proportion of cells specific for a given antigen varied significantly from one individual to the next. The ratio of proportions of the two antigens observed was constant from individual to individual, however, and this constant ratio differed significantly from the ratio observed for the same two antigens in fetal BALB/c and CBA/J inbred mice. These data suggest that the ontogeny of the repertoire of antigen-binding cells in fetal mice is subject to at least two independent sets of controls, one affecting the relative size of the repertoire in the spleen, and the other affecting the distribution of antigen-binding specificities within that repertoire. Analysis of repertoire size and composition in the spleens of hybrid fetuses confirmed the observation that the two parameters are controlled independently, and suggested further that the control of repertoire size in these fetuses is due to the action of one or a few closely-linked autosomal Mendelian genes. These data are consistent with models for the origin of antibody diversity in which the genes coding for the full repertoire of antibodies are generated somatically from a small number of germ-line genes early in development and in the absence of any strong positive or negative selection with respect to antigenic specificity.

Lymphocyte activation by monovalent fragments of antibodies reactive with cell surface carbohydrates.

Antibodies reactive with cell surface carbohydrates were isolated from normal chicken serum and were found to be mitogenic for mouse splenic lymphocytes as assayed by both blast transformation and [3H]thymidine incorporation. The Fab' fragments of these carbohydrate-binding immunoglobulins were just as mitogenic as the divalent native antibody. Moreover, succinylated Fab' fragments, which probably would not form self-associating aggregates, showed similar mitogenic properties. All of these results indicate that, at least for saccharide-specific ligands, multipoint attachment and receptor cross-linkage on the cell to which the ligand is attached may not be a stringent requirement for activation.

Neuron-glia cell adhesion molecule interacts with neurons and astroglia via different binding mechanisms.

The neuron-glia cell adhesion molecule (Ng-CAM) is present in the central nervous system on postmitotic neurons and in the periphery on neurons and Schwann cells. It has been implicated in binding between neurons and between neurons and glia. To understand the molecular mechanisms of Ng-CAM binding, we analyzed the aggregation of chick Ng-CAM either immobilized on 0.5-micron beads (Covaspheres) or reconstituted into liposomes. The results were correlated with the binding of these particles to different types of cells as well as with cell-cell binding itself. Both Ng-CAM-Covaspheres and Ng-CAM liposomes individually self-aggregated, and antibodies against Ng-CAM strongly inhibited their aggregation; the rate of aggregation increased approximately with the square of the concentration of the beads or the liposomes. Much higher rates of aggregation were observed when the ratio of Ng-CAM to lipid in the liposome was increased. Radioiodinated Ng-CAM on Covaspheres and in liposomes bound both to neurons and to glial cells and in each case antibodies against Ng-CAM inhibited 50-90% of the binding. Control preparations of fibroblasts and meningeal cells did not exhibit significant binding. Adhesion between neurons and glia within and across species (chick and mouse) was explored in cellular assays after defining markers for each cell type, and optimal conditions of shear, temperature, and cell density. As previously noted using chick cells (Grumet, M., S. Hoffman, C.-M. Chuong, and G. M. Edelman. 1984 Proc. Natl. Acad. Sci. USA. 81:7989-7993), anti-Ng-CAM antibodies inhibited neuron-neuron and neuron-glia binding. In cross-species adhesion assays, binding of chick neurons to mouse astroglia and binding of mouse neurons to chick astroglia were both inhibited by anti-Ng-CAM antibodies. To identify whether the cellular ligands for Ng-CAM differed for neuron-neuron and neuron-glia binding, cells were preincubated with specific antibodies, the antibodies were removed by washing, and Ng-CAM-Covasphere binding was measured. Preincubation of neurons with anti-Ng-CAM antibodies inhibited Ng-CAM-Covasphere binding but similar preincubation of astroglial cells did not inhibit binding. In contrast, preincubation of astroglia with anti-astroglial cell antibodies inhibited binding to these cells but preincubation of neurons with these antibodies had no effect. Together with the data on Covaspheres and liposome aggregation, these findings suggested that Ng-CAM-Covaspheres bound to Ng-CAM on neurons but bound to different molecules on astroglia.(ABSTRACT TRUNCATED AT 400 WORDS)

Effects of fasciculation on the outgrowth of neurites from spinal ganglia in culture.

This report describes the influence of neurite fasciculation on two aspects of nerve growth from chick spinal ganglia in vitro: the inhibition of outgrowth by high concentrations of nerve growth factor (NGF) and the preferential growth of neurites toward a capillary tube containing NGF. These studies involved a comparison of cultures of single cells, cell aggregates, and intact ganglia and the use of antibodies against the nerve cell adhesion molecule (CAM) to perturb fasciculation under a variety of conditions. The inhibition of outgrowth, which was observed with ganglia and aggregates but not with single cells, was correlated with a thickening of neurite fascicles. In accord with this observation, anti-CAM, which diminishes fasciculation by inhibiting side-to-side interactions between individual neurites, also partially reversed the inhibition of neurite outgrowth at high NGF concentrations. On the basis of these and other studies, we consider the possibility that neurite bundling causes an increase in the elastic tension of a fascicle without a compensatory increase in its adhesion to substratum. It is proposed that this imbalance could inhibit neurites from growing out from a ganglion and even result in retraction of preexisting outgrowth. In the analysis of NGF-directed growth, it was found that a capillary source of NGF produced a steep but transient NGF gradient that subsided before most neurites had emerged from the ganglion. Nevertheless, the presence of a single NGF capillary caused a dramatic and persistent asymmetry in the outgrowth of neurites from ganglia or cell aggregates. In contrast, processes of individual cells did not appear to orient themselves toward the capillary. The most revealing finding was that anti-CAM antibodies caused a decrease in the asymmetry of neurite outgrowth. These results suggest that side-to-side interactions among neurites can influence the guidance of nerve bundles by sustaining and amplifying an initial directional signal.

Heterotypic binding between neuronal membrane vesicles and glial cells is mediated by a specific cell adhesion molecule.

By means of a multistage quantitative assay, we have identified a new kind of cell adhesion molecule (CAM) on neuronal cells of the chick embryo that is involved in their adhesion to glial cells. The assay used to identify the binding component (which we name neuron-glia CAM or Ng-CAM) was designed to distinguish between homotypic binding (e.g., neuron to neuron) and heterotypic binding (e.g., neuron to glia). This distinction was essential because a single neuron might simultaneously carry different CAMs separately mediating each of these interactions. The adhesion of neuronal cells to glial cells in vitro was previously found to be inhibited by Fab' fragments prepared from antisera against neuronal membranes but not by Fab' fragments against N-CAM, the neural cell adhesion molecule. This suggested that neuron-glia adhesion is mediated by specific cell surface molecules different from previously isolated CAMs . To verify that this was the case, neuronal membrane vesicles were labeled internally with 6-carboxyfluorescein and externally with 125I-labeled antibodies to N-CAM to block their homotypic binding. Labeled vesicles bound to glial cells but not to fibroblasts during a 30-min incubation period. The specific binding of the neuronal vesicles to glial cells was measured by fluorescence microscopy and gamma spectroscopy of the 125I label. Binding increased with increasing concentrations of both glial cells and neuronal vesicles. Fab' fragments prepared from anti-neuronal membrane sera that inhibited binding between neurons and glial cells were also found to inhibit neuronal vesicle binding to glial cells. The inhibitory activity of the Fab' fragments was depleted by preincubation with neuronal cells but not with glial cells. Trypsin treatment of neuronal membrane vesicles released material that neutralized Fab' fragment inhibition; after chromatography, neutralizing activity was enriched 50-fold. This fraction was injected into mice to produce monoclonal antibodies; an antibody was obtained that interacted with neurons, inhibited binding of neuronal membrane vesicles to glial cells, and recognized an Mr = 135,000 band in immunoblots of embryonic chick brain membranes. These results suggest that this molecule is present on the surfaces of neurons and that it directly or indirectly mediates adhesion between neurons and glial cells. Because the monoclonal antibody as well as the original polyspecific antibodies that were active in the assay did not bind to glial cells, we infer that neuron-glial interaction is heterophilic, i.e., it occurs between Ng-CAM on neurons and an as yet unidentified CAM present on glial cells.

Expression sequences and distribution of two primary cell adhesion molecules during embryonic development of Xenopus laevis.

Studies of chicken embryos have demonstrated that cell adhesion molecules are important in embryonic induction and are expressed in defined sequences during embryogenesis and histogenesis. To extend these observations and to provide comparable evidence for heterochronic changes in such sequences during evolution, the local distributions of the neural cell adhesion molecule (N-CAM) and of the liver cell adhesion molecule (L-CAM) were examined in Xenopus laevis embryos by immunohistochemical and biochemical techniques. Because of the technical difficulties presented by the existence of multiple polypeptide forms of CAMs and by autofluorescence of yolk-containing cells, special care was taken in choosing and characterizing antibodies, fluorophores, and embedding procedures. Both N-CAM and L-CAM were found at low levels in pregastrulation embryos. During gastrulation, N-CAM levels increased in the presumptive neural epithelium and decreased in the endoderm, but L-CAM continued to be expressed in all cells including endodermal cells. During neurulation, the level of N-CAM expression in the neural ectoderm increased considerably, while remaining constant in non-neural ectoderm and diminishing in the somites; in the notochord, N-CAM was expressed transiently. Prevalence modulation was also seen at all sites of secondary induction: both CAMs increased in the sensory layer of the ectoderm during condensation of the placodes. During organogenesis, the expression of L-CAM gradually diminished in the nervous system while N-CAM expression remained high. In all other organs examined, the amount of one or the other CAM decreased, so that by stage 50 these two molecules were expressed in non-overlapping territories. Embryonic and adult tissues were compared to search for concordance of CAM expression at later stages. With few exceptions, the tissue distributions of N-CAM and L-CAM were similar in the frog and in the chicken from early times of development. In contrast to previous observations in the chicken and in the mouse, N-CAM expression was found to be high in the adult liver of Xenopus, whereas L-CAM expression was low. In the adult brain, N-CAM was expressed as three components of apparent molecular mass 180, 140, and 120 kD, respectively; in earlier stages of development only the 140-kD component could be detected. In the liver, a single N-CAM band appears at 160 kD, raising the possibility that this band represents an unusual N-CAM polypeptide. L-CAM appeared at all stages as a 124-kD molecule.(ABSTRACT TRUNCATED AT 400 WORDS)

Functional mapping of cytotactin: proteolytic fragments active in cell-substrate adhesion.

Cytotactin is an extracellular matrix glycoprotein with a restricted distribution during development. In electron microscopic images, it appears as a hexabrachion with six arms extending from a central core. Cytotactin binds to other extracellular matrix proteins including a chondroitin sulfate proteoglycan (CTB proteoglycan) and fibronectin. Although cytotactin binds to a variety of cells including fibroblasts and neurons, in some cases it causes cells in culture to round up and it inhibits their migration. To relate these various effects of cytotactin on cell behavior to its binding regions, we have examined its ability to support cell-substrate adhesion and have mapped its cell-binding function onto its structure. In a cell-substrate adhesion assay, fibroblasts bound to cytotactin but remained round. In contrast, they both attached and spread on fibronectin. Neither neurons nor glia bound to cytotactin in this assay. In an assay in which cell-substrate contact was initiated by centrifugation, however, neurons and glia bound well to cytotactin; this binding was blocked by specific anti-cytotactin antibodies. The results suggest that neurons and glia can bind to cytotactin-coated substrates and that these cells, like fibroblasts, possess cell surface ligands for cytotactin. After applying methods of limited proteolysis and fractionation, these assays were used to map the binding functions of cytotactin onto its structure. Fragments produced by limited proteolysis were fractionated into two major pools: one (fraction I) contained disulfide-linked oligomers of a 100-kD fragment and two minor related fragments, and the second (fraction II) contained monomeric 90- and 65-kD fragments. The 90- and 65-kD fragments in fraction II were closely related to each other and were structurally and immunologically distinct from the fragments in fraction I. Only components in fraction I were recognized by mAb M1, which binds to an epitope located in the proximal portion of the arms of the hexabrachion and by a polyclonal antibody prepared against a 75-kD CNBr fragment of intact cytotactin. A mAb (1D8) and a polyclonal antibody prepared against a 35-kD CNBr fragment of cytotactin only recognized components present in fraction II. In cell-binding experiments, fibroblasts, neurons, and glia each adhered to substrates coated with fraction II, but did not adhere to substrates coated with fraction I. Fab fragments of the antibody to the 35-kD CNBr fragment strongly inhibited the binding of cells to cytotactin, supporting the conclusion that fraction II contains a cell-binding region. In addition, Fab fragments of this antibody inhibited the binding of cytotactin to CTB pr

N-CAM at the vertebrate neuromuscular junction.

We have detected the neural cell adhesion molecule, N-CAM, at nerve-muscle contacts in the developing and adult mouse diaphragm. Whereas we found N-CAM staining with fluorescent antibodies consistently to overlap with the pattern of alpha-bungarotoxin staining at nerve-muscle contacts both during development and in the adult, we observed N-CAM staining on the surfaces of developing myofibers and at much lower levels on adult myofibers. Consistent with its function, N-CAM was also detected on axons and axon terminals. Immunoblotting experiments with anti-N-CAM antibodies on detergent extracts of embryonic (E) diaphragm muscle revealed a polydisperse polysialylated N-CAM polypeptide, which in the adult (A) was converted to a discrete form of Mr 140,000; this change, called E-to-A conversion, was previously found to occur in different neural tissues at different rates. The Mr 140,000 component was not recognized by monoclonal antibody anti-N-CAM No. 5, which specifically recognizes antigenic determinants associated with N-linked oligosaccharide determinants on N-CAM from neural tissue. The relative concentration of the Mr 140,000 component prepared from diaphragm muscle increased during fetal development and then decreased sharply to reach adult values. Nevertheless, expression of N-CAM in muscle could be induced after denervation: one week after the sciatic nerve was severed, the relative amount of N-CAM increased dramatically as detected by immunoblots of extracts of whole muscle. Immunofluorescent staining confirmed that there was an increase in N-CAM, both in the cell and at the cell surface; at the same time, however, staining at the motor endplate was diminished. Our findings indicate that, in muscle, in addition to chemical modulation, cell-surface modulation of N-CAM occurs both in amount and distribution during embryogenesis and in response to denervation.

Conversion of embryonic form to adult forms of N-CAM in vitro: results from de novo synthesis of adult forms.

During normal development, the neural cell adhesion molecule N-CAM changes at the cell-surface from a sialic acid-rich embryonic, or E form, to several adult, or A forms that have less sialic acid (E-to-A conversion). To investigate the cellular and molecular mechanisms that underlie these changes, we have established conditions under which E-to-A conversion occurs in cultured explants of central nervous system tissues. Mouse cerebellum, chick spinal cord, and chick retina that express the E form of N-CAM were dissected and cultured on collagen gels. After 3-6 d in culture, increased proportions of A forms were synthesized, as revealed by specific immunoprecipitation and immunoblotting. The rate of E-to-A conversion and the proportions of the different A forms synthesized in vitro were similar to those observed for the tissues in vivo at comparable times. In addition, the explants incorporated radioactive precursors of amino sugars into N-CAM, and the electrophoretic mobilities of the E and A forms of N-CAM were altered by treatment with neuraminidase in a way comparable to that found for N-CAM obtained directly from tissue. These results suggest that the post translational processing in vitro was similar to that in vivo. Logistic studies on cell division and death in the explants suggested that E-to-A conversion resulted mainly from a specific increase in synthesis of A forms in individual cells rather than as a consequence of differential birth or death within distinct cell populations. The data were consistent with the possibility that the increase in synthesis of A forms occurred either in cells that had previously synthesized E forms or in a distinct population of cells that already synthesized A forms. Cells dissociated from embryonic central nervous system tissues and cultured in vitro were also found to undergo E-to-A conversion at the same rate as the explant cultures, which suggests that if intercellular signals were responsible for initiation of the change in synthetic pattern, they had already occurred in vivo before the time of culture. In pulse-chase experiments, the E form of N-CAM that was synthesized during the first day after explantation persisted as E form for several days, at times when newly synthesized N-CAM was predominantly in A forms. These results indicate that in cultured neural tissue, the E form of N-CAM is not processed into A forms but is gradually degraded and replaced by newly synthesized A forms.(ABSTRACT TRUNCATED AT 400 WORDS)