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Regulation of Cell Adhesion by Polysialic Acid

2001; Elsevier BV; Volume: 276; Issue: 34 Linguagem: Inglês

10.1074/jbc.m104525200

1083-351X

Ichiro Fujimoto, Juan L. Brusés, Urs Rutishauser,

Receptor Mechanisms and Signaling

The polysialylation of neural cell adhesion molecule (NCAM) evolved in vertebrates to carry out biological functions related to changes in cell position and morphology. Many of these effects involve the attenuation of cell interactions that are not mediated through NCAM's own adhesion properties. A proposed mechanism for this global effect on cell interaction is the steric inhibition of membrane-membrane apposition based solely on polysialic acid (PSA) biophysical properties. However, it remains possible that the intrinsic binding or signaling properties of the NCAM polypeptide are also involved. To help resolve this issue, this study uses a quantitative cell detachment assay together with cells engineered to display different adhesion receptors together with a variety of polysialylated NCAM polypeptide isoforms and functional domain deletion mutations. The results obtained indicate that regulation by PSA occurs with adhesion receptors as diverse as an IgCAM, a cadherin and an integrin, and does not require NCAM functional domains other than those minimally required for polysialylation. These findings are most consistent with the cell apposition mechanism for PSA action, as this model predicts that the inhibitory effects of PSA-NCAM on cell adhesion should be independent of the nature of the adhesion system and of any intrinsic binding or signaling properties of the NCAM polypeptide itself. The polysialylation of neural cell adhesion molecule (NCAM) evolved in vertebrates to carry out biological functions related to changes in cell position and morphology. Many of these effects involve the attenuation of cell interactions that are not mediated through NCAM's own adhesion properties. A proposed mechanism for this global effect on cell interaction is the steric inhibition of membrane-membrane apposition based solely on polysialic acid (PSA) biophysical properties. However, it remains possible that the intrinsic binding or signaling properties of the NCAM polypeptide are also involved. To help resolve this issue, this study uses a quantitative cell detachment assay together with cells engineered to display different adhesion receptors together with a variety of polysialylated NCAM polypeptide isoforms and functional domain deletion mutations. The results obtained indicate that regulation by PSA occurs with adhesion receptors as diverse as an IgCAM, a cadherin and an integrin, and does not require NCAM functional domains other than those minimally required for polysialylation. These findings are most consistent with the cell apposition mechanism for PSA action, as this model predicts that the inhibitory effects of PSA-NCAM on cell adhesion should be independent of the nature of the adhesion system and of any intrinsic binding or signaling properties of the NCAM polypeptide itself. neural cell adhesion molecule cell adhesion molecule endoneuraminidase N immunoglobulin (domain) polysialic acid polysialyltransferase monoclonal antibody Chinese hamster ovary polyacrylamide gel electrophoresis cytomegalovirus The polysialylation of NCAM1 is a unique post-translational modification that has appeared during vertebrate development to carry out a variety of biological functions (for review, see Ref. 1Rutishauser U. Landmesser L. Trends Neurosci. 1996; 19: 422-427Abstract Full Text Full Text PDF PubMed Scopus (424) Google Scholar). These functions, which include the facilitation of cell migration, axon pathfinding and synaptogenesis, appear to stem from the ability of PSA-NCAM to reduce cell interactions. Although most abundant during development of both neural and non-neural tissues, PSA-NCAM is also associated with restricted parts of the adult brain that exhibit functional plasticity that may require the rearrangement of neural cells or their interconnections. Remarkably, many of the biological effects of NCAM polysialylation appear to involve cell interactions thought to be mediated by adhesion receptors other than NCAM itself. For example, the regulation of axon fasciculation by PSA expression in the motorneuron plexus of the chick hindlimb can be mimicked by antibodies against the L1 adhesion molecule (2Tang J. Rutishauser U. Landmesser L. Neuron. 1994; 13: 405-414Abstract Full Text PDF PubMed Scopus (237) Google Scholar), and the inhibition of mossy fiber synaptogenesis by PSA expression in the pyramidal cell layer of the hippocampus (3Seki T. Rutishauser U. J. Neurosci. 1998; 18: 3757-3766Crossref PubMed Google Scholar) is likely to involve cadherin-like receptors. These observations are also consistent with a variety of in vitro studies in which PSA removal by a specific endoneuraminidase appears to enhance adhesion involving other CAMs, and can even affect artificially induced cell adherence or agglutination produced by non-physiological ligands such as poly-l-lysine (4Acheson A. Sunshine J.L. Rutishauser U. J. Cell Biol. 1991; 114: 143-153Crossref PubMed Scopus (294) Google Scholar, 5Rutishauser U. Acheson A. Hall A.K. Mann D.M. Sunshine J. Science. 1988; 240: 53-57Crossref PubMed Scopus (670) Google Scholar). Despite the structural simplicity of PSA (it is a long, linear polymer of hydrated α-2,8-linked sialic acid, see Ref. 6Finne J. Trends Biochem. Sci. 1985; 10: 129-132Abstract Full Text PDF Scopus (44) Google Scholar, and is exclusively associated with the Ig5 domain of NCAM (7Cremer H. Lange R. Christoph A. Plomann M. Vopper G. Roes J. Brown R. Baldwin S. Kraemer P. Scheff S. Nature. 1994; 367: 455-459Crossref PubMed Scopus (899) Google Scholar, 8Tomasiewicz H. Ono K. Yee D. Thompson C. Goridis C. Rutishauser U. Magnuson T. Neuron. 1993; 11: 1163-1174Abstract Full Text PDF PubMed Scopus (436) Google Scholar, 9Nelson R.W. Bates P.A. Rutishauser U. J. Biol. Chem. 1995; 270: 17171-17179Abstract Full Text Full Text PDF PubMed Scopus (114) Google Scholar)), the mechanism of action by which PSA-NCAM affects cell interactions remains open to question. In view of PSA's biophysical properties and its ability to affect multiple types of cell interactions, a reasonable case has been made for a simple steric mechanism by which expression of PSA at the cell surface can hinder membrane-membrane and receptor-receptor apposition (10Yang P. Yin X. Rutishauser U. J. Cell Biol. 1992; 116: 1487-1496Crossref PubMed Scopus (158) Google Scholar, 11Yang P. Major D. Rutishauser U. J. Biol. Chem. 1994; 269: 23039-23044Abstract Full Text PDF PubMed Google Scholar). Nevertheless, a variety of NCAM-associated activities have been described that could in principle be affected by this post-translational modification. Fortunately, these potential avenues, which include a proposed NCAM-L1 interaction (12Horstkorte R. Schachner M. Magyar J.P. Vorherr T. Schmitz B. J. Cell Biol. 1993; 121: 1409-1421Crossref PubMed Scopus (191) Google Scholar, 13Kadmon G. Kowitz A. Altevogt P. Schachner M. J. Cell Biol. 1990; 110: 209-218Crossref PubMed Scopus (147) Google Scholar, 14Kadmon G. Kowitz A. Altevogt P. Schachner M. J. Cell Biol. 1990; 110: 193-208Crossref PubMed Scopus (244) Google Scholar), NCAM-proteoglycan binding (15Cole G.J. Akeson R. Neuron. 1989; 2: 1157-1165Abstract Full Text PDF PubMed Scopus (121) Google Scholar, 16Frelinger 3rd, A.L. Rutishauser U. J. Cell Biol. 1986; 103: 1729-1737Crossref PubMed Scopus (64) Google Scholar), NCAM homophilic binding (17Rao Y. Wu X.F. Gariepy J. Rutishauser U. Siu C.H. J. Cell Biol. 1992; 118: 937-949Crossref PubMed Scopus (131) Google Scholar, 18Rao Y. Wu X.F. Yip P. Gariepy J. Siu C.H. J. Biol. Chem. 1993; 268: 20630-20638Abstract Full Text PDF PubMed Google Scholar), association with the cytoskeleton (19Pollerberg G.E. Burridge K. Krebs K.E. Goodman S.R. Schachner M. Cell Tissue Res. 1987; 250: 227-236Crossref PubMed Scopus (252) Google Scholar, 20Pollerberg G.E. Schachner M. Davoust J. Nature. 1986; 324: 462-465Crossref PubMed Scopus (205) Google Scholar), and signaling via CAM association with the fibroblast growth factor receptor (21Saffell J.L. Williams E.J. Mason I.J. Walsh F.S. Doherty P. Neuron. 1997; 18: 231-242Abstract Full Text Full Text PDF PubMed Scopus (288) Google Scholar) or activation of fyn or focal adhesion kinase (22Kolkova K. Novitskaya V. Pedersen N. Berezin V. Bock E. J. Neurosci. 2000; 20: 2238-2246Crossref PubMed Google Scholar, 23Beggs H.E. Baragona S.C. Hemperly J.J. Maness P.F. J. Biol. Chem. 1997; 272: 8310-8319Abstract Full Text Full Text PDF PubMed Scopus (231) Google Scholar), have been assigned to specific regions of the NCAM polypeptide, as summarized in Fig. 1. In this respect, the steric mechanism makes the bold prediction that the effects of PSA on a variety of adhesion receptors should not be affected by deletion of these regions, provided that those deletions do not prevent NCAM polysialylation itself. The goal of this study is to test this prediction in detail. Before addressing the domain deletions within NCAM, it was necessary to establish a rigorous and quantitative assay for PSA function with multiple, defined adhesion systems. Although other adhesion molecules have been implicated in PSA-mediated effects on cells and in tissues, these demonstrations have involved indirect interpretations of results obtained with perturbations by anti-CAM antibodies or by removal of calcium. In the present approach, adhesion is measured between known cell surface receptors and a glass surface coated with the appropriate purified protein binding partner (24Brieher W.M. Yap A.S. Gumbiner B.M. J. Cell Biol. 1996; 135: 487-496Crossref PubMed Scopus (262) Google Scholar, 25Yap A.S. Brieher W.M. Pruschy M. Gumbiner B.M. Curr. Biol. 1997; 7: 308-315Abstract Full Text Full Text PDF PubMed Scopus (301) Google Scholar). In addition, cell detachment as opposed to attachment is used as a measure of adhesive strength, both to obtain better quantitation and because the biological roles of PSA in tissue are more related to the breaking of cell-cell contacts, as in axon defasciculation, cell translocation, and the transient formation of junctions. A second requirement for the study is to obtain polysialylation at the cell surface in the absence of NCAM domains associated with NCAM polypeptide functions. This has been accomplished by transfecting NCAM-negative CHO cells with NCAM or mutated NCAM cDNAs plus a polysialyltransferase (PST) known to be sufficient for the synthesis of PSA-NCAM (26Muhlenhoff M. Eckhardt M. Bethe A. Frosch M. Gerardy-Schahn R. Curr. Biol. 1996; 6: 1188-1191Abstract Full Text Full Text PDF PubMed Scopus (67) Google Scholar, 27Nakayama J. Fukuda M. J. Biol. Chem. 1996; 271: 1829-1832Abstract Full Text Full Text PDF PubMed Scopus (69) Google Scholar). Through the combination of these methods, we are able to provide strong evidence that PSA-NCAM can modulate L1-, cadherin-, and integrin-mediated adhesion, and that this effect is independent of either the cytoplasmic domains or the Ig domains that are the site of NCAM's binding and/or signaling properties. The full-length NCAM 180 and NCAM 140 cDNAs were isolated from an oligo(dT)-primed Zap Express cDNA library (Stratagene) which was generated from St 33 embryonic chick brain poly(A) RNA. A [32P]cDNA probe (508 base pairs) was generated by reverse transcriptase-polymerase chain reaction corresponding to nucleotide positions 2004–2511 of the chick NCAM using the Rediprime random primer method (Amersham Pharmacia Biotech). Hybridizations were performed as suggested by the manufacturer. Positive clones were plaque-purified and then in vivo excised into pBK-CMV-based phagemids by infection with ExAssist helper phage followed by transduction of filamentous phage particles into Escherichia coli XLOLR. The LacZ fragments were removed from these expression vectors. NCAM 120 was isolated from an adult chick brain λgt11 cDNA library (CLONTECH) using a 314-base pair NCAM 120-specific cDNA probe, positions 2104–2417. The longest cDNA inserts were subcloned into pGEM-7zf plasmids and sequenced. The partial NCAM 120 cDNA missing immunoglobulin domains 1–3 was swapped into the NCAM 180 pBK-CMV vector using a PmlI site to make full-length NCAM 120 cDNA. Deletion mutants, D1–3 and D1–4, were generated by removal of nucleotides 66–659 or 66–1219 using ATG sites in the full-length NCAM 140 cDNA. Human ST8SiaIV PST was the kind gift of Dr. Minoru Fukuda (The Burnham Institute, La Jolla, CA) and was subcloned into pcDNA3.1zeo. The cDNA fragments digested out from 5 different NCAMs with a CMV promoter and SV40 poly(A) were subcloned into the PSTpcDNA3.1zeo vector using a BglII site to make a dual CMV promoter vector that can induce expression of both NCAM and the transferase. Human neuroblastoma cell lines, IMR-32 and SK-N-SH, were obtained from the American Type Culture Collection (ATCC, Rockville, MD). The human N-cadherin stably transfected neomycin-resistant CHO cell clone number 35.4 was the kind gift of Dr. Carien Niessen (Memorial Sloan-Kettering Cancer Center, New York). Human L1-IgCAM stably transfected neomycin-resistant CHO cells were generated by transfection of phL1ApcDNA3, which was the kind gift of Dr. Vance Lemmon (Case Western Reserve University, Cleveland, OH). Stable transfections were carried out using the LipofectAMINE 2000 reagent (Life Technologies, Inc.) as described by the manufacturer. In brief, CHO cells were plated in a 24-well culture plate (Falcon), grown to 90% confluency in 0.5 ml of culture medium containing 10% serum. Transfections were carried out at 37 °C by directly adding 0.1 ml of OPTI-MEM medium (Life Technologies, Inc.) containing 2.5 µg of LipofectAMINE 2000 and 8 µg of DNA to each 24-well plate. After 24 h, cells were detached using trypsin, resuspended in serum containing medium, and replated in 100-mm dishes in the presence of 400 µg/ml G418 (Life Technologies, Inc.) for 2 weeks to select for stable transfectants. The expression vectors for pcDNA3.1zeo of chick NCAM 180, 140, 120, D1–3, and D1–4 with human PST were transfected into N-cadherin or L1 stably transfected CHO cells and selected using 200 µg/ml Zeocin (Invitrogen). Independent stable clones were isolated and their expression levels analyzed by SDS-PAGE and Western blotting. Cells and embryonic chick brain tissue (day 9, stage 35) were extracted in 1% Nonidet P-40 lysis buffer (1% Nonidet P-40, 25 mm Hepes, 150 mmNaCl, 1 mm EDTA, pH 7.4) supplemented with a protease inhibitor mixture (Roche Molecular Biochemicals). PSA on NCAM was specifically removed (either on intact cells or in extracts) using purified endoneuraminidase N (endo N, 20 units/ml, 30 min, 25 °C), isolated as described (28Hallenbeck P.C. Vimr E.R., Yu, F. Bassler B. Troy F.A. J. Biol. Chem. 1987; 262: 3553-3561Abstract Full Text PDF PubMed Google Scholar). Samples containing equal quantities of total protein were separated on 7 or 10% SDS-PAGE gels and transferred by electrophoresis onto polyvinylidene difluoride membranes (Millipore). The blots were incubated with monoclonal human N-cadherin antibody (C70320, Transduction Laboratories), polyclonal human L1 antibody (kind gift of Dr. Vance Lemmon, Case Western Reserve University), or monoclonal antibodies against human NCAM (123C3, Santa Cruz Biotechnology Inc.) or chick NCAM (mAbs 5e and 105, which recognize NCAM domains Ig3 and FN2, respectively (16Frelinger 3rd, A.L. Rutishauser U. J. Cell Biol. 1986; 103: 1729-1737Crossref PubMed Scopus (64) Google Scholar)). The membranes were first blocked for 1 h in TBS-T (0.1% Tween 20) containing 5% milk (Bio-Rad), then incubated overnight at 4 °C in the anti-CAM antibodies diluted 1:2000 in blocking buffer, and finally washed 3 times for 5 min in large volumes of TBS-T. The peroxidase-conjugated secondary antibodies were anti-rabbit (Jackson ImmunoResearch) or anti-mouse (Promega), diluted 1:5000 in TBS-T. After incubation with the second antibody for 60 min at room temperature, membranes were washed 4 times for 5 min with TBS-T. Detection was performed using the ECL method (Amersham Pharmacia Biotech). The soluble extracellular segment of Xenopus C-cadherin attached to the Fc region of human IgG was the kind gift of Dr. Carien Niessen (Memorial Sloan-Kettering Cancer Center, New York). Fibronectin and laminin were purchased from Becton Dickinson and Sigma, respectively. The complete extracellular domain of human L1 with a human IgG Fc tail (L1-Fc) was generated in CHO cells (29Fransen E. D'Hooge R. Van Camp G. Verhoye M. Sijbers J. Reyniers E. Soriano P. Kamiguchi H. Willemsen R. Koekkoek S.K. De Zeeuw C.I. De Deyn P.P. Van der Linden A. Lemmon V. Kooy R.F. Willems P.J. Hum. Mol. Genet. 1998; 7: 999-1009Crossref PubMed Scopus (205) Google Scholar) from an expression vector kindly provided by Dr. Vance Lemmon (Case Western Reserve University). Conditioned media from the L1-expressing CHO cell cultures was filtered through a 0.22-µm filter and applied to a HiTrap protein A HP column (Amersham Pharmacia Biotech). The column was washed with 20 column volumes of ImmunoPure IgG binding buffer (Pierce) and the protein eluted by ImmunoPure IgG elution buffer (Pierce). The eluate was immediately adjusted to neutral pH by 1 m Tris buffer (pH 8). Desalting and buffer exchange was performed using a PD-10 column (Amersham Pharmacia Biotech) with 20 mm Hepes (pH 7.4), 50 mm NaCl, 1 mm CaCl2. Laminar flow adhesion assays in glass capillaries were performed as previously described (24Brieher W.M. Yap A.S. Gumbiner B.M. J. Cell Biol. 1996; 135: 487-496Crossref PubMed Scopus (262) Google Scholar, 25Yap A.S. Brieher W.M. Pruschy M. Gumbiner B.M. Curr. Biol. 1997; 7: 308-315Abstract Full Text Full Text PDF PubMed Scopus (301) Google Scholar). Glass capillary tubes (1.1 mm internal diameter; Sutter Instruments) were coated with C-cadherin-Fc (0.1 mg/ml), fibronectin (15 µg/ml), laminin (15 µg/ml), or L1-Fc (0.1 mg/ml) in 100 mm NaCl, 20 mm Hepes, and 1 mm CaCl2 (pH 7.2) overnight at 4 °C in a humidified chamber and then blocked with 5% milk (Bio-Rad) for 2 h at room temperature. The coated capillary was connected to a 60-ml syringe attached to a syringe pump (Harvard Instruments) and mounted on an inverted microscope (Zeiss Inc). For pretreatment with endo N, cells were washed with serum-free Dulbecco's modified Eagle's medium and incubated for 30 min at 25 °C at a final concentration of 20 units/ml. Cells were isolated by incubation with 5 mm EDTA at 37 °C for 10 min, then pipetted and collected by centrifugation and resuspended in Hanks' balanced salt solution without phenol red (Life Technologies, Inc.). The adhesion assay was performed at room temperature as follows: cells were drawn into the coated capillary from a reservoir using the pump. After 1 min, the flow was stopped and the cells were allowed to bind to the surface for 10 min. The number of cells in a selected microscope field was then recorded in a digital photograph. Flow was initiated and the number of cells remaining in the same field was recorded after 30 s. Subsequently, the flow rate was doubled every 30 s, and the number of the cells remaining in the field was recorded at the end of each time period. IMR-32 and SK-N-SH human neuroblastoma cells express similar levels of N-cadherin and NCAM, but the NCAM of IMR-32 cells is much more heavily polysialylated than that of the SK-N-SH cells (Fig. 2,A and B). The level of PSA on the IMR-32 cells, as judged by electrophoretic properties, is similar to that observed for embryonic brain tissue (Fig. 2 A). Therefore a comparison of the adhesion properties of these cells to a cadherin substrate provides an opportunity to assess the effect of endogenous PSA on cadherin homophilic function. These studies were able to use purified C-cadherin as the homophilic partner for N-cadherin, as the adhesion properties of C-cadherin are equivalent to those of N-cadherin. 2C. Niessen and B. Gumbiner, personal communication. As shown in Fig.2, C and D, the strength of the calcium-dependent adhesion of the SK-N-SH cells to C-cadherin is greater than that of the IMR-32 cells. Furthermore, while removal of PSA results in an increase in adhesion for both cell types, the increase is larger for the IMR-32 cells. In fact, after endo N treatment, the adhesion properties of the two cell types are indistinguishable. Together, these findings indicate that the IMR-32 and SK-N-SH cells have similar levels of potential cadherin activity, and that this activity is regulated according to the amount of PSA co-expressed at the cell surface. In addition to N-cadherin, these neuroblastoma cells express αvβ1 integrin and thus exhibit adhesivity for a fibronectin substrate (30Dedhar S. Haqq C. Gray V. J. Biol. Chem. 1989; 264: 4832-4836Abstract Full Text PDF PubMed Google Scholar). As noted in a previous report (31Dedhar S. Gray V. J. Cell Biol. 1990; 110: 2185-2193Crossref PubMed Scopus (64) Google Scholar), this binding is relatively weak for IMR-32 cells as compared with SK-N-SH cells. As shown in Fig. 2 E, the removal of PSA from the IMR-32 cells increases their adhesion to fibronectin, suggesting that the weakness of integrin-mediated binding by these cells is at least in part due to the co-expression of PSA-NCAM. This finding extends the range of endogenous PSA effects to receptors involved in cell-matrix interactions as well as those involving cell-cell adhesion receptors. The above studies indicate that PSA expression is required for the different cadherin adhesive activity of two neuroblastoma cell lines. To establish that PSA is sufficient for regulation of cadherin-mediated adhesion, we exploited the fact that PSA synthesis on NCAM can be conferred by the addition of a single glycosyltransferase (26Muhlenhoff M. Eckhardt M. Bethe A. Frosch M. Gerardy-Schahn R. Curr. Biol. 1996; 6: 1188-1191Abstract Full Text Full Text PDF PubMed Scopus (67) Google Scholar, 27Nakayama J. Fukuda M. J. Biol. Chem. 1996; 271: 1829-1832Abstract Full Text Full Text PDF PubMed Scopus (69) Google Scholar). For this purpose, as well as subsequent studies of NCAM structural mutations, a CHO cell line that has no detectable adhesion systems was used to make transfectants with and without NCAM (180 isoform), polysialyltransferase, and N-cadherin. As shown in Fig. 3A, stably transfected CHO cells expressing NCAM and N-cadherin exhibited significantly less cadherin-mediated adhesion when subsequently transfected with polysialyltransferase cDNA. That the transferase produced PSA expression was confirmed by gel electrophoresis, which revealed a level of polysialylation similar to that found in embryonic brain tissue (Fig. 4A,NCAM 180). That this expression resulted in the decreased adhesion was established by reversing the effect by treatment with endo N (Fig. 3 A).Figure 4The effect of PSA on cadherin-mediated adhesion is independent of NCAMs major alternative splicing variations. A and B, expression of polysialylated NCAM isoforms 180, 140, or 120 and N-cadherin by the appropriately transfected CHO cells was assessed by SDS-PAGE immunoblots using 40 µg of total extracted protein and antibodies against chick NCAM (mAb 5e) or human N-cadherin. Embryonic chick brain tissue is shown as a reference. Endo N was used to remove the PSA from the cells or tissue prior to protein extraction. Note that all three NCAM isoforms are heavily polysialylated (as reflected in the slower and disperse electrophoretic mobility of the NCAM before endo N treatment), and are expressed at similar levels (as judged after treatment with endo N). Cadherin expression levels are also equivalent in each cell line. C-E, adhesion to the cadherin substrate by the above cell lines. For all three polysialylated NCAM isoforms the level of cadherin-mediated adhesion is very similar. After removal of the PSA by endo N, this adhesivity increases by the same amount, regardless of the NCAM isoform being expressed. Data are mean ± S.E. (n = 3).View Large Image Figure ViewerDownload (PPT) As indicated in the Introduction, many of the in vivostudies of PSA pertain to the L1 IgCAM. Although neuroblastoma cells express L1, it is as a splice variant that lacks adhesive activity (32Felding-Habermann B. Silletti S. Mei F. Siu C.H. Yip P.M. Brooks P.C. Cheresh D.A. O'Toole T.E. Ginsberg M.H. Montgomery A.M. J. Cell Biol. 1997; 139: 1567-1581Crossref PubMed Scopus (111) Google Scholar). Therefore, to establish the role of PSA in L1-mediated adhesionin vitro, the CHO cell transfectants described above were also prepared with the adhesive form of L1 rather than N-cadherin. The flow adhesion assay was then performed with an L1-coated substrate. Despite the quantitative and qualitative changes in adhesive properties that accompany a switch from a calcium-dependent cadherin to a calcium-independent IgCAM, the negative regulation of this adhesion as a result of PSA expression (Fig. 3 B) was similar to that obtained for N-cadherin (Fig. 3 A). Again the effectiveness of the transferase was confirmed by electrophoresis (Fig.4 A, NCAM 180), and the role of PSA was established by endo N treatment (Fig. 3 B). These studies were designed to determine if the major NCAM cytoplasmic splicing variants (NCAM 180, 140, and 120, see Fig. 1) affect the ability of PSA to regulate cadherin-mediated adhesion. CHO cells were stably transfected with NCAM 180, 140, or 120 as well as N-cadherin and the polysialyltransferase. Clones of each NCAM isoform having a similar level of NCAM expression, N-cadherin expression, and polysialylation were then selected (Fig. 4 A). Remarkably, the flow detachment profiles for these three cell lines revealed an identical level of cadherin-mediated adhesion in the presence of PSA, and the same increase in this adhesion after the removal of PSA by endo N treatment (Fig. 4, C-E). The level of adhesion obtained after removal of PSA was the same as obtained for cells transfected with N-cadherin alone (compare controls in Fig. 3 A with Fig.4, C-E), suggesting that NCAM itself does not contribute significantly to the adhesiveness of the cells under the conditions of this assay. In order to examine a possible role of NCAM Ig domains (other than those required for polysialylation itself) in PSA regulation of N-cadherin-mediated binding, NCAM deletion mutants lacking Ig domains 1 to 3 (D1–3) or 1 to 4 (D1–4) were generated from NCAM 140. These constructs were then used to stably transfect CHO cells along with N-cadherin and polysialyltransferase. Appropriate levels of NCAM, N-cadherin, and PSA were selected as above (Fig.5A). Although earlier studies suggested that NCAM polysialylation of Ig domain 5 requires the presence of Ig domain 4 (9Nelson R.W. Bates P.A. Rutishauser U. J. Biol. Chem. 1995; 270: 17171-17179Abstract Full Text Full Text PDF PubMed Scopus (114) Google Scholar), we found that the D1–4 mutation had a similar level of PSA on CHO cells to that of the D1–3 mutation or the intact NCAM 140. It should be noted that the NCAM immunoblots in this characterization was carried out with a monoclonal antibody directed against an epitope in a FNIII domain. This epitope is partially masked by PSA (11Yang P. Major D. Rutishauser U. J. Biol. Chem. 1994; 269: 23039-23044Abstract Full Text PDF PubMed Google Scholar), and therefore stains PSA-NCAM less intensely and with greater variability. Nevertheless, the electrophoretic patterns obtained for each NCAM form were indicative of heavily polysialylated NCAM. Despite the concentration of NCAM activities in the first four NCAM Ig domains (Fig. 1), their deletion had no detectable effect on the ability of PSA to regulate N-cadherin-mediated adhesion (Fig. 5,B and C). In fact, comparison of all of the NCAM mutations generated for this study (both cytoplasmic, Fig. 4, and extracellular, Fig. 5) reveals that the flow detachment curves are essentially identical for the five types of PSA-positive cells, as well as for the more adhesive PSA-negative (endo N-treated) cells. The NCAM Ig domain deletion study was extended to L1 IgCAM-mediated homophilic adhesion because interactions have been reported among Ig domains in general and between NCAM and L1 in particular (13Kadmon G. Kowitz A. Altevogt P. Schachner M. J. Cell Biol. 1990; 110: 209-218Crossref PubMed Scopus (147) Google Scholar, 14Kadmon G. Kowitz A. Altevogt P. Schachner M. J. Cell Biol. 1990; 110: 193-208Crossref PubMed Scopus (244) Google Scholar). In this situation, our ability to get effective polysialylation of the Ig1–4 as well as the Ig1–3 deletion was critical, in that Ig4 contains the proposed interaction site (12Horstkorte R. Schachner M. Magyar J.P. Vorherr T. Schmitz B. J. Cell Biol. 1993; 121: 1409-1421Crossref PubMed Scopus (191) Google Scholar). As above, the levels of NCAM and PSA expression were monitored by electrophoresis, as well as that of L1 (Fig.6, A and B). The results were equivalent to those obtained for N-cadherin: the flow assay curves for L1-mediated homophilic adhesion in the presence of PSA-NCAM 140 and its polysialylated D1–3 and D1–4 mutations were identical, and removal of PSA from each NCAM variant produced similar increases in adhesion. The results obtained in this study demonstrate that the inhibitory effects of PSA-NCAM on cell adhesion are independent of the nature of the adhesion system and of all known structural or functional properties of NCAM except those required for PSA expression. That is, the negative regulation occurs equally well with receptor systems as diverse as those mediated by an IgCAM (other than NCAM itself), a cadherin, or an integrin, and does not require NCAM functional domains other than those minimally required for NCAM polysialylation and transport to the cell surface. To obtain definitive evidence, several parameters had to be considered in the choice of an adhesion assay and in the introduction of NCAM molecular variants into cells. The flow shear assay originally devised for cadherins by the Gumbiner laboratory was chosen because of two key features. First, the adhesion occurs between a cell and a surface coated with a specific, purified adhesion ligand, thus allowing for specificity in the mechanism of attachment. Second, this assay quantitates the detachment rather than attachment of cells, which provides a better measure of the strength and stability of the binding and thus is more similar to the effects of PSA on membrane-membrane separation observed in vivo. It should also be noted that the detachment is carried out over a range of shear forces, and thereby yields multiple measurements that together give a superior assessment of effects that alter adhesive strengths. One potential drawback of the substrate detachment assay is that PSA is present on only one of the two interacting surfaces, namely the cell. The effects of PSA on cell adhesion are known to be greater if the carbohydrate is expressed on both membranes during cell-cell contact (33Hoffman S. Edelman G.M. Adv. Exp. Med. Biol. 1984; 181: 147-160Crossref PubMed Scopus (9) Google Scholar), and the possibility exists that we are underestimating its activity in the substrate-cell assay. Another important advance has been the ability to engineer cells simultaneously with respect to adhesion systems, NCAM domain structure, and NCAM polysialylation. These molecular tools would not have been valid unless physiologically relevant levels of NCAM polysialylation could be obtained. Fortunately, the PSA-NCAM produced by transfection of cells with the PST transferase is similar in amount and quality, as judged by its appearance and electrophoretic mobility in SDS-PAGE immunoblots, to that obtained from embryonic brain tissue and PSA-positive neural cell lines. The degree and uniformity of the glycosylation levels obtained suggest that it is relatively easy to get adequate levels of the transferase expressed in these cells, and that other factors, such as decreasing enzyme affinity with polymer length (34Oka S. Bruses J.L. Nelson R.W. Rutishauser U. J. Biol. Chem. 1995; 270: 19357-19463Abstract Full Text Full Text PDF PubMed Scopus (37) Google Scholar), serve to regulate the extent of the polymerization. In the present study, the minimal NCAM structural requirements for polysialylation has been found to be less than that reported previously (9Nelson R.W. Bates P.A. Rutishauser U. J. Biol. Chem. 1995; 270: 17171-17179Abstract Full Text Full Text PDF PubMed Scopus (114) Google Scholar). That is, high levels of glycosylation could be obtained in the absence of Ig domains 1–4 rather than 1–3. The ability to delete Ig domain 4 was useful in that several activities and natural structural variants for NCAM are found in this region of the protein. The difference in the minimal constructs in the two studies has not been determined, but could reflect the cell lines used. In particular, the cell line used previously (F11) has endogenous PSA-NCAM with relatively modest polymer lengths. Moreover, the mutated NCAM in the F11 cells has to compete with endogenous native NCAM as a substrate for the transferase. With establishment of these technical requirements, the interpretation of the results obtained becomes relatively straightforward. It is striking not only that PSA can affect specific adhesion mediated by L1, N-cadherin, and αvβ1 integrin, but that the magnitude of the negative inhibition is very similar in each case (a 40–60% decrease at a shear of 8 ml/min). Because these different adhesion mechanisms occur by completely different molecular interactions, it is unlikely that PSA produces this effect through a specific and direct inactivation of the receptors themselves. On the other hand, a global effect on membrane-membrane apposition would predict exactly this phenomenon, and is consistent with a relatively uniform effect on the different adhesion mechanisms. The first studies of PSA focussed on NCAM itself, and demonstrated a similar inhibition of NCAM homophilic adhesion. In terms of mechanism, however, these findings are difficult to interpret because of the potential for direct effects on NCAM protein conformation, local steric hindrance (11Yang P. Major D. Rutishauser U. J. Biol. Chem. 1994; 269: 23039-23044Abstract Full Text PDF PubMed Google Scholar), and the positive influence of PSA on NCAM-heparin sulfate proteoglycan binding (35Storms S.D. Rutishauser U. J. Biol. Chem. 1998; 273: 27124-27129Abstract Full Text Full Text PDF PubMed Scopus (111) Google Scholar). Moreover, the domains that mediate NCAM homophilic binding also contain other activities and structural features of the protein. Therefore, it is possible that the biology of PSA includes not only a global regulation of cell interactions, but also other effects transmitted through NCAM. But could such effects on NCAM contribute to the observed regulation by PSA of other adhesion systems? The NCAM domain deletion studies reported here address this issue by systematically removing the sites of all known NCAM activities (other than polysialylation) and showing that even with these extensive deletions the PSA-mediated inhibition of cadherin and L1 IgCAM homophilic adhesion is identical to that obtained with intact PSA-NCAM. It should be noted that the two FNIII domains in NCAM were not deleted (at least one of them is required for polysialylation). Therefore, although no activity or notable structural feature has been reported for these domains, either in the intact molecule or as isolated units, we are not able to rule out this possibility. One logical role for these domains might be a homophilic affinity among NCAMs on the same cell. While such an activity could well result in augmentation of NCAM-mediated adhesion, its regulation by PSA would not be expected to affect other adhesion systems. Finally, it is important to remember that this study is limited to effects of PSA on cell adhesion. Therefore the results obtained cannot exclude the possibility that PSA might have other biological activities, such as effects on neurotrophic factor efficacy (36Muller D. Djebbara-Hannas Z. Jourdain P. Vutskits L. Durbec P. Rougon G. Kiss J.Z. Proc. Natl. Acad. Sci. U. S. A. 2000; 97: 4315-4320Crossref PubMed Scopus (194) Google Scholar) or a direct affinity for matrix components (35Storms S.D. Rutishauser U. J. Biol. Chem. 1998; 273: 27124-27129Abstract Full Text Full Text PDF PubMed Scopus (111) Google Scholar), which may or may not involve direct participation by the NCAM polypeptide. In summary, the present studies tackle a difficult logistical problem, trying to provide strong evidence in favor of mechanism for regulation of molecular and cellular function that is not based on binding or enzymatic activity. While some minor caveats remain, the consistency and breadth of the regulation obtained under the stringent criteria and extensive manipulations employed here should provide a basis for interpretation of PSAs role in most biological contexts.