A heparin-binding domain from N-CAM is involved in neural cell- substratum adhesion

Cell-substratum adhesion in the embryonic chicken nervous system has been shown to be mediated in part by a 170,000-mol-wt polypeptide that is a component of adherons. Attachment of retinal cells to the 170,000-mol-wt protein is inhibited by the C1H3 monoclonal antibody and by heparan sulfate (Cole, G. J., D. Schubert, and L. Glaser, 1985, J. Cell Biol., 100:1192-1199). In the present study we have demonstrated that the 170,000-mol-wt C1H3 polypeptide is immunologically identical to the neural cell adhesion molecule N-CAM, and that the 170,000-mol-wt component of N-CAM is preferentially secreted by cells as a component of adherons. We have identified a monoclonal antibody, designated B1A3, that inhibits heparin binding to N-CAM and cell-to-substratum adhesion. A 25,000-mol-wt heparin (heparan sulfate)-binding domain of N-CAM has been identified by limited proteolysis, and this fragment promotes cell attachment when bound to glass surfaces. The fragment also partially inhibits cell binding to adherons when bound to retinal cells, and the B1A3 monoclonal antibody inhibits retinal cell attachment to substrata composed of intact N-CAM or the heparin-binding domain. These data are the first evidence that N-CAM is a multifunctional protein that contains both cell-and heparin (heparan sulfate)-binding domains.     

The properdin-like type I repeats of human thrombospondin contain a cell attachment site

Thrombospondin (TS) is a modular adhesive glycoprotein that contains three domains previously implicated in the attachment of cells to TS. These include the amino-terminal heparin-binding domain, the carboxy terminal cell or platelet-binding domain, and an RGDA sequence of TS. We have characterized a mAb against human TS, designated A4.1, which inhibits the attachment of human melanoma cells (G361) to TS. The epitope for A4.1 lies within the amino terminal half of the central stalklike region of TS which is distinct from the three known cell attachment sites. This region of TS is recovered in a 50-kD peptide after chymotryptic digestion of TS in EDTA. It contains the procollagen-like domain of TS as well as three type I repeats of a 60-residue segment homologous to two malarial proteins and the complement proteins properdin, and factors C6 through C9. The purified chymotryptic fragment is an effective attachment factor for G361 cells. A4.1 blocks adhesion to the 50-kD domain, as do some sulfated glycoconjugates. RGD (and RGE) peptides and mAbs against other domains of TS are not inhibitory. Peptides (19 mers) based on the core homology sequence of the three type I repeats of TS are potent attachment factors for these cells, and this adhesion is also inhibited by sulfated glycoconjugates. A polyclonal antibody raised against one of these peptides inhibits adhesion of G361 cells to the peptides, to the 50-kD fragment and to intact TS. Thus a new cell-adhesion site has been identified in TS whose sequence is very similar to the site identified in region II of the circumsporozoite protein of malaria parasites (Rich, K. A., F. W. George IV, J. L. Law, and W. J. Martin. 1990. Science (Wash. DC) 249:1574-1577. Thus there may be a common receptor which binds TS, malarial proteins, and properdin.     

Topography of N-CAM structural and functional determinants. I. Classification of monoclonal antibody epitopes

12 distinct neural cell adhesion molecule (N-CAM) epitopes, each recognized by a different monoclonal antibody (mAb), have been characterized in terms of the major structural and functional features of the molecule. Seven antibodies, each recognizing the amino-terminal region of the molecule, altered the rate of N-CAM-mediated adhesion. Four of these were inhibitors, two of which also recognized a heparin-binding N-CAM fragment. The other three antibodies specifically enhanced the rate of N-CAM-mediated adhesion. Three epitopes, one polypeptide- and two carbohydrate-dependent, were associated with the sialic acid-rich central portion of the molecule. The remaining two antibodies were found to react with intracellular determinants, and are specific for the largest of the three major N-CAM polypeptide forms. Studies on the ability of one antibody to hinder recognition of native N-CAM by another antibody suggested that the epitopes associated with N-CAM binding functions are in close proximity compared with the other determinants. The classification of these mAb epitopes has allowed the topographical placement of key N-CAM features, as described in the following paper, and provides valuable probes for analysis of both the structure and function of N-CAM.     

Mapping of a cell-binding domain in the cell adhesion molecule gp80 of Dictyostelium discoideum

At the aggregation stage of Dictyostelium discoideum development, a cell surface glycoprotein of Mr 80,000 (gp80) has been found to mediate the EDTA-resistant type of cell-cell adhesion via homophilic interaction (Siu, C.-H., A. Cho, and A. H. C. Choi. 1987. J. Cell Biol. 105:2523-2533). To investigate the structure-function relationships of gp80, we have isolated full length cDNA clones for gp80 and determined the DNA sequence. The deduced structure of gp80 showed three major domains. An amino-terminal globular domain composed of the bulk of the protein is supported by a short stalk region, which is followed by a membrane anchor at the carboxy terminus. Structural analysis suggested that the cell-binding domain of gp80 resides within the globular domain near the amino terminus. To investigate the relationship of the cell-binding activity to this region of the polypeptide, three protein A/gp80 (PA80) gene fusions were constructed using the expression vector pRIT2T. These PA80 fusion proteins were assayed for their ability to bind to aggregation stage cells. Binding of 125I-labeled fusion proteins PA80I (containing the Val123 to Ile514 fragment of gp80) and PA80II (Val123 to Ala258) was dosage dependent and could be inhibited by precoating cells with the cell cohesion-blocking mAb 80L5C4. On the other hand, there was no appreciable binding of PA80III (Ile174 to Ile514) to cells. Reassociation of cells was significantly inhibited in the presence of PA80I or PA80II. In addition, 125I-labeled PA80II exhibited homophilic interaction with immobilized PA80I, PA80II, or gp80. The results of these studies lead to the mapping of a cell-binding domain in the region between Val123 and Leu173 of gp80 and provide direct evidence that the cell-binding activity of gp80 resides in the protein moiety.     

Production and characterization of functional domains of human fibronectin expressed in Escherichia coli.

An efficient expression system was constructed in Escherichia coli that produced a 33-kDa fragment, C-274, of human fibronectin with a strong cell-adhesive activity. The entire sequence of the heparin-binding domain with 271 amino acids, H-271, was also expressed. Deletion analysis of the type III repeats showed that the heparin-binding site was at type III-13. The cell-adhesive activity of a fusion protein, CH-271, containing the cell- and the heparin-binding domains was twice that of C-274 when BHK but not B16-F10 melanoma cells were tested; H-271 alone was inactive. Recombinant proteins containing the CS1 sequence of the IIICS region were more active than C-274 and CH-271 with B16-F10. However, H-296, which contained both H-271 and CS1, was almost inactive with BHK. CH-296, which contained CS1 at the C-terminus of CH-271, was more active with B16-F10 than H-296 and C-CS1, which was produced by the deletion of H-271 from CH-296. Thus, the cell-binding domain was active with both kinds of cells. The heparin-binding domain promoted the adhesion of both kinds of cells only when linked to the cell-binding domain or CS1. CS1 was specific for the adhesion of B16-F10 but was not essential.     

Syndecan-4 deficiency impairs focal adhesion formation only under restricted conditions

Two domains of fibronectin deliver two different but cooperative signals required for focal adhesion formation. The signal from the cell-binding domain is mediated by integrins, whereas the signal from the heparin-binding domain is recognized by heparan sulfate proteoglycans, of which syndecan-4 has been hypothesized to be involved in focal adhesion formation. We generated mice deficient in syndecan-4 to study its role directly. Even in fibroblasts from syndecan-4-deficient mice, focal adhesions were formed, and actin fibers terminated normally at focal adhesions when they were cultured on coverslips coated with fibronectin or with a mixture of its cell-binding and heparin-binding fragments. However, when the cells were cultured on the cell-binding fragment and the heparin-binding fragment was added to the medium, focal adhesion formation was impaired in the syndecan-4 null fibroblasts as compared with that in wild-type cells. Therefore, syndecan-4 is essential for promoting focal adhesion formation only when the signal of the heparin-binding domain of fibronectin is delivered as a soluble form, most probably from the apical surface. When the signal is delivered as a substratum-bound form, other molecule(s) also participate(s) in the signal reception.     

Isolation and characterization of a heparin-binding domain from the amino terminus of platelet thrombospondin

Calcium-replete thrombospondin has been purified from outdated platelets using heparin-Sepharose affinity chromatography, gelatin-Sepharose to remove fibronectin, and gel filtration to eliminate low-molecular-weight heparin-binding proteins. Edman degradation of six different preparations revealed the amino-terminal sequence of thrombospondin (TSP) to be Asn-Arg-Ile-Pro-Glu-Ser-Gly-Gly-Asp-Asn-Ser-Val-Phe-. This sequence was obtained in initial yields as high as 85%, indicating that no blocked chains are present. Cleavage of calcium-replete TSP with thermolysin or plasmin results in the production of relatively stable fragments. Chromatography of these digests on heparin-Sepharose followed by elution with 0.6 M NaCl affords purification of an Mr 25,000 fragment from the thermolysin digest and an Mr 35,000 fragment from the plasmin digest. The binding of these fragments to heparin-Sepharose does not require divalent metal ions. Neither fragment is disulfide-bonded to other fragments present in the digests. The heparin-binding domains from both digests have similar amino acid compositions and their tryptic peptide maps on high performance liquid chromatography are identical with the exception of one peptide unique to each fragment. Automated Edman degradation in a vapor-phase sequenator of the thermolytic heparin-binding domain electroeluted from sodium dodecyl sulfate-gels indicates that the heparin-binding domain resides at the amino terminus of the Mr 180,000 TSP peptide chain.     

Thrombospondin and a 140 kd fragment promote adhesion and neurite outgrowth from embryonic central and peripheral neurons and from PC12 cells

The ability of thrombospondin (TSP), an extracellular matrix glycoprotein, and two proteolytic fragments to support adhesion and neurite outgrowth from embryonic dorsal root ganglia, spinal cord neurons, and PC12 cells was examined. Anti-TSP antibodies or a synthetic peptide (GRGDS) containing an RGD cell-binding region was also added to cells plated on TSP. TSP and its 140 kd fragment were more efficient than laminin controls in supporting adhesion. Neurites formed on laminin, on varying concentrations of TSP, and particularly the 140 kd fragment. The amino-terminal heparin-binding domain supported little adhesion and outgrowth. Both adhesion and process outgrowth on TSP were inhibited by addition of anti-TSP antibodies, but not GRGDS.     

Modulation of haptotactic migration of metastatic melanoma cells by the interaction between heparin and heparin-binding domain of fibronectin

We utilized recombinant fibronectin polypeptides with cell-binding domain and heparin-binding domains (referred to as C-274 and H-271, respectively) and their fusion polypeptide (CH-271) to examine the role of sulfated polysaccharide heparin and/or the functional domains of fibronectin in modulating tumor cell behavior. Both C-274 and CH-271 polypeptides with cell-binding domains promoted the adhesion and migration of B16-BL6 melanoma cells, whereas H-271 did not. Heparin bound to the immobilized polypeptides with heparin-binding domain (H-271, CH-271, and a mixture of C-274 and H-271 or fibronectin) but did not affect the tumor cell adhesion to the substrates. At the same time, heparin or two monoclonal antibodies against the heparin-binding domain were able to inhibit the haptotactic migration to CH-271 or fibronectin, though not to C-274 or a mixture of C-274 and H-271. This suggests that although heparin did not affect tumor cell adhesion to the cell-binding domain near the heparin-binding domain in CH-271 or fibronectin, it did lead to a modulation of cell motility. It seems likely that the regulatory mechanism may depend on interaction between heparin-like molecules on the cell surface and the heparin-binding domain in fibronectin, rather than on simple steric hindrance or on the masking of the cell-binding domain caused by the binding of heparin to heparin-binding domain.     

Adhesion and cytoskeletal organisation of fibroblasts in response to fibronectin fragments.

Fibronectin has been shown previously to promote complete cell adhesion in the absence of other serum components or de novo protein synthesis. Recently a sequence of four amino acids from the cell-binding domain of fibronectin has been termed the 'cell recognition site' of this multidomain molecule since it mediates cell attachment and inhibits cell adhesion to intact fibronectin. We show here, however, that substrata coated with an isolated cell-binding domain of fibronectin are not sufficient for complete cell adhesion; cells attach and spread but, unlike those adhering to intact fibronectin, they do not form stress fibres terminating in focal adhesions. An additional external stimulus is needed for this cytoskeletal reorganisation and may be provided by one of two heparin-binding fragments of fibronectin. The two 'signals' required for complete adhesion need not be provided simultaneously since focal adhesion formation can be promoted by stimulating cells pre-spread on a cell-binding fragment of fibronectin with a soluble heparin-binding fragment. This second stimulation may involve cell membrane heparan sulphate proteoglycans.     

Molecular interactions between fibronectin and integrins during mouse blastocyst outgrowth

To investigate the mechanism of trophoblast adhesion to fibronectin, we cultured blastocysts in serum-free medium on proteolytic fibronectin fragments containing its major functional domains, and localized fibronectin-binding integrins in outgrowing trophoblast cells by immunofluorescent staining. Outgrowth comparable to that obtained with intact fibronectin was observed using a 120 kD chymotryptic fragment containing the central cell-binding domain (FN-120) and the Arg-Gly-Asp (RGD) recognition sequence. A 40 kD COOH-terminal chymotryptic fragment of fibronectin containing both a heparin-binding region and an alternate (non-RGD) cell-binding site was inactive in supporting trophoblast adhesion. Three synthetic peptides derived from the heparin-binding domain, including the CS1 alternate cell-binding site, were also unable to promote trophoblast cell adhesion. A 75 kD recombinant protein, ProNectin F, containing 13 copies of the cell recognition epitope of fibronectin, Val-Thr-Gly-Arg-Gly-Asp-Ser-Pro-Ala-Ser, vigorously supported blastocyst outgrowth. Blastocyst outgrowth was not significantly different when surfaces were precoated with cellular fibronectin, which contains an alternatively spliced type III repeat and is the form actually encountered in vivo. Several putative fibronectin receptors were localized in trophoblast outgrowths by immunofluorescent labeling. Antibodies reactive with integrin subunits α3, α5, αllb, αv, β1 and β3, but not α4, all bound to trophoblast cells. Antibodies raised against either the β1 or β3 integrin subunits significantly inhibited fibronectin-mediated outgrowth. These findings demonstrate the key role of the central cell-binding domain of fibronectin in trophoblast adhesion, and suggest four RGD-binding integrins, α3β1, α5β1, αllbβ3, and αvβ3, that could mediate trophoblast adhesion in vitro and may play an important role during implantation. © 1995 Wiley-Liss, Inc.     

Cloning and sequencing of chicken thrombospondin

Thrombospondin is a multifunction adhesive protein with the ability to bind proteoglycans, cell surface receptors, other proteins, and calcium ions. Several sequence motifs for some of these interactions have been identified in human thrombospondin. To evaluate the potential functional significance of these sequences and to begin a study of the evolution of thrombospondin, we have isolated and sequenced thrombospondin cDNA clones from a chicken embryo library. Comparison of the chicken and human sequences reveals that the NH2-terminal heparin-binding domains are only 34% identical. By contrast, the type 3 repeats and the COOH-terminal domains are 80 and 82% identical, respectively, when comparing human and chicken sequences. Potential cell recognition sequences of RGD and VTCG are conserved, with the chicken sequence containing an additional copy of the VTCG sequence. Whereas substitutions occur in the two potential heparin-binding motifs that have human counterparts, the chicken sequence contains a third potential heparin-binding motif. The results indicate that the evolutionary constraints on the various types of cell-binding motifs may be quite different.     

On the origin of species specificity of fibronectin. Immunological identification of a species-specific domain of human fibronectin

We have investigated whether species-specific epitopes of human fibronectin are localized at a specific domain of fibronectin using rabbit polyclonal antibodies. Tryptic fragments of human fibronectin were tested for reactivity with anti-human fibronectin antibody, which had been previously absorbed with other animal fibronectin to establish species specificity. Human-specific epitopes were found to be present on 75,000, 65,000, and 42,000 dalton fragments. The 42,000-dalton fragment shares almost all the epitopes with the 75,000 and 65,000 dalton fragments. It does not promote BHK cell spreading, whereas the 75,000 and 65,000 dalton fragments do. The amino acid sequence from the amino terminus of the 42,000-dalton fragment is Asp/Gly-Gln/Val-?-Ile-Val-, which is almost identical to the sequence Asp-Gln-Cys-Ile-Val- located in the carboxyl terminal 1/3 of the collagen-binding domain of human fibronectin (Kornblihtt et al. (1985) EMBO J. 4, 1755-1759). These results suggest that human fibronectin bears human-specific epitopes mainly on the amino-terminal half of domain 4 (Hayashi & Yamada (1983) J. Biol. Chem. 258, 3332-3340) located between the collagen and cell binding domains almost at the center of the fibronectin polypeptide. The domain specific for human fibronectin may be a general species-specific domain of animal fibronectins.     

Identification of a heparin binding domain of the neural cell adhesion molecule N-CAM using synthetic peptides

The neural cell adhesion molecule (N-CAM) plays an integral role in cell interactions during neural development, with the binding of heparan sulfate proteoglycan to the amino-terminal region of N-CAM being required for N-CAM function. In the present study we have used synthetic peptides (HBD-1 and HBD-2), derived from the primary amino acid sequence of rat N-CAM, to identify the region of N-CAM that binds heparan sulfate. The 28 amino acid HBD-1 synthetic peptide was shown to bind both [3H]heparin and dissociated retinal cells. Retinal cells also attach to a substratum of HBD-2 peptide, but fail to bind to a control peptide containing a scrambled amino acid sequence of HBD-2. The HBD-2 peptide also inhibits retinal cell adhesion to N-CAM, demonstrating the physiological importance of the amino acid sequence encoded by the HBD peptide. These data therefore permit the localization of a heparin binding domain to a 17 amino acid region of immunoglobulin-like loop 2.     

Human fibronectin contains distinct adhesion- and motility-promoting domains for metastatic melanoma cells

The active migration of tumor cells through extracellular matrices has been proposed to play a role in certain aspects of metastasis. Metastatic tumor cells migrate in vitro in response to substratum-bound adhesive glycoproteins such as fibronectin. The present studies use affinity-purified proteolytic fragments of fibronectin to determine the nature of adhesion- and/or motility-promoting domains within the protein. Two distinct fragments were identified with cell adhesion-promoting activities. By a number of criteria, the adhesive activity promoted by these two fragments was distinct. One fragment, a 75-kD tryptic fragment purified by monoclonal antibody chromatography, promoted the adhesion, spreading, and haptotactic motility of melanoma cells. Experiments using a synthetic cell attachment peptide in solution indicated that at least part of the attachment activity exhibited by the 75-kD fragment is mediated by the sequence arg-gly-asp-ser. It was not possible to demonstrate migration-stimulating activity using a small (11.5 kD) peptic fragment containing this sequence (Pierschbacher, M.D., E. G. Hayman, and E. Ruoslahti, 1981, Cell, 26:259-267) suggesting that another cell-binding activity within the 75 kD fragment distinct from arg-gly-asp-ser might be required for motility. The second fragment that stimulated melanoma adhesion was a 33-kD tryptic/catheptic carboxyl-terminal heparin-binding fragment, which is localized to the A chain of fibronectin. This fragment promotes adhesion and spreading but not the motility of these cells. Melanoma adhesion to this heparin-binding fragment was sensitive to the effects of cycloheximide, which contrasted adhesion to the haptotaxis-promoting fragment. Importantly, these studies illustrate that haptotaxis in response to fibronectin is not due to simple adhesion gradients of this protein. The results are discussed in light of a model for multiple distinct cell surface constituents mediating cell adhesion and motility on fibronectin.     

Localization and chemical synthesis of fibronectin peptides with melanoma adhesion and heparin binding activities

Tumor cell adhesion to the extracellular matrix is an important consideration in tumor metastasis. Recent results show that multiple adhesion-promoting domains for melanoma cells can be purified from proteolytic digests of fibronectin [McCarthy, J. B., Hagen, S. T., & Furcht, L. T. (1986) J. Cell Biol. 102, 179-188]. Monoclonal antibodies were generated against a tryptic/catheptic 33K heparin binding fragment of fibronectin derived from the carboxyl terminal of the A chain. This region contains a tumor cell adhesion-promoting domain(s). The amino-terminal sequence was determined for this fragment, as well as a tryptic 31K fragment which is located to the carboxyl-terminal side of the 33K heparin binding fragment in A chains of fibronectin. The partial sequence data demonstrate that arginyl-glycyl-aspartyl-serine (RGDS) or the related arginyl-glutamyl-aspartyl-valine (REDV) is not present in the 33K heparin binding fragment, confirming earlier results which demonstrated that cells adhere to this fragment by an RGDS-independent mechanism. Two monoclonal antibodies, termed AHB-1 and AHB-2, recognized epitopes common to heparin binding fragments derived from the carboxyl terminus of both the A and B chains of fibronectin. Monoclonal antibody AHB-2 inhibited melanoma adhesion to the 33K heparin binding fragment of fibronectin in a concentration-dependent manner, whereas monoclonal antibody AHB-1 had no effect on adhesion to this fragment. Neither monoclonal antibody inhibited adhesion to intact fibronectin. However, monoclonal AHB-2 potentiated the inhibitory effect of suboptimal levels of exogenous RGDS on cell adhesion to intact fibronectin. AHB-2 recognized an epitope common to both the A- and B-chain carboxyl-terminal heparin binding region of fibronectin.(ABSTRACT TRUNCATED AT 250 WORDS)     

Binding of hepatic lipase to heparin. Identification of specific heparin-binding residues in two distinct positive charge clusters

The interaction of hepatic lipase (HL) with heparan sulfate is critical to the function of this enzyme. The primary amino acid sequence of HL was compared to that of lipoprotein lipase (LPL), a related enzyme that possesses several putative heparin-binding domains. Of the three putative heparin-binding clusters of LPL (J. Biol. Chem. 1994. 269: 4626-4633; J. Lipid Res. 1998. 39: 1310-1315), one was conserved in HL (Cluster 1; residues Lys 297-Arg 300 in rat HL) and two were partially conserved (Cluster 2; residues Asp 307-Phe 320, and Cluster 4; residues Lys 337, and Thr 432-Arg 443). Mutants of HL were generated in which potential heparin-binding residues within Clusters 1 and 4 were changed to Asn. Two chimeras in which the LPL heparin-binding sequences of Clusters 2 and 4 were substituted for the analogous HL sequences were also constructed. These mutants were expressed in Chinese hamster ovary (CHO) cells and assayed for heparin-binding ability using heparin-Sepharose chromatography and a CHO cell-binding assay. The results suggest that residues within the homologous Cluster 1 region (Lys 297, Lys 298, and Arg 300), as well as some residues in the partially conserved Cluster 4 region (Lys 337, Lys 436, and Arg 443), are involved in the heparin binding of hepatic lipase. In the cell-binding assay, heparan sulfate-binding affinity equal to that of LPL was seen for the RHL chimera mutant that possessed the Cluster 4 sequence of LPL. Mutation of Cluster 1 residues of HL resulted in a major reduction in heparin binding ability as seen in both the cell-binding assay and the heparin-Sepharose elution profile. These results suggest that Cluster 1, the N-terminal heparin-binding domain, is of primary significance in RHL. This is different for LPL: mutations in the C-terminal binding domain (Cluster 4) cause a more significant shift in the salt required for elution from heparin-Sepharose than mutations in the N-terminal domain (Cluster 1).     

Adhesion of lymphoid cells to the carboxyl-terminal heparin-binding domains of fibronectin

Previously, we have shown that some lymphoid cell lines adhere to fibronectin (FN)-coated substratum, whereas others do not. In this study, the adhesion of five adherent lymphoid cell lines to different FN domains was examined. These cell lines ranged in their adherence to substratum coated with FN, the cell-binding domain (CBD) fragment, or the heparin-binding domain (HBD) fragments. None of the cell lines adhered to substratum coated with the gelatin-binding domain fragment. Three of the lymphoid cell lines adhered preferentially to HBD over CBD, whereas two other lymphoid cell lines and BHK fibroblasts adhered preferentially to CBD. These results suggest that two distinct adhesive interactions occur between cells and FN and that the pattern of interaction varies among cell types. Using MOPC 315 (which adheres preferentially to HBD) as a cell model to study the cell-HBD interaction, the HBD-promoted adhesion was found to be independent of the RGD sequence and could be inhibited by anti-FN antibodies. Moreover, the MOPC 315-HBD interaction had the following characteristics: (1) adhesion was temperature dependent, (2) presence of divalent cations was necessary, (3) integrity of cellular microfilaments but not microtubules was required, (4) inhibition of protein synthesis abolished adhesion, (5) pretreatment of cells with trypsin inhibited adhesion, and (6) the adhesion was mediated by the carboxyl-terminal HBD.     

Structural and immunological characterization of the amino-terminal domain of mammalian neural cell adhesion molecules

The neural cell adhesion molecules (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 and 140,000 with an additional form at 120,000 in murine brain. In embryonic brain, N-CAMs are represented by a highly sialylated form with an apparent molecular weight greater than 180,000. We have used monoclonal antibodies that cross-react with N-CAMs of various mammalian species to purify N-CAMs from adult murine and bovine brains and from embryonic murine brains. We determined the amino acid sequences of the amino-terminal domains of these molecules: Leu-Gln-Val-Asp-Ile-Val-Pro-Ser-Gln-Gly-Glu-Ile-Ser-Val-Gly-Glu-Ser. This sequence is highly conserved among all three forms of adult murine brain N-CAM as well as embryonic murine brain N-CAM and adult bovine brain N-CAM. Based on this sequence, we synthesized an undecapeptide and used it to raise a site-directed polyclonal antiserum. This antiserum reacted with the intact N-CAM in liquid phase radioimmunoassays, immunoblotting experiments, and immunofluorescent labeling of cells. The antiserum also reacted with N-CAMs in extracts of brain tissues from different species, confirming the highly conserved nature of the amino-terminal domain of mammalian N-CAMs. Immunofluorescence experiments indicated that this domain resides on the outer surfaces of cells that express N-CAMs, in both primary neuronal cell culture and in cell lines.