Polysialyltransferase ST8Sia II (STX) polysialylates all of the major isoforms of NCAM and facilitates neurite outgrowth

Isabelle Franceschini1, Kiyohiko Angata, Edgar Ong, Allison Hong, Patrick Doherty3 and Minoru Fukuda2

Glycobiology Program, Cancer Research Center, The Burnham Institute, 10901 North Torrey Pines Road, La Jolla, CA 92037, USA, and 3Molecular Neurobiology Group, Guy’s Hospital, School of Medicine, King’s College, London SE1 9RT, U.K.

Received on September 4, 2000; revised on October 4, 2000; accepted on October 5, 2000.


    Abstract
 Top
 Abstract
 Introduction
 Results
 Discussion
 Materials and methods
 Acknowledgments
 Abbreviations
 References
 
The neural cell adhesion molecule (NCAM) has different isoforms due to different sizes in its polypeptide and plays a significant role in neural development. In neural development, the function of NCAM is modified by polysialylation catalyzed by two polysialyltransferases, ST8Sia II and ST8Sia IV. Previously, it was reported by others that ST8Sia II polysialylates only transmembrane isoforms of the NCAM, such as NCAM-140 and NCAM-180, but not NCAM-120 and NCAM-125 anchored by a glycosylphosphotidylinositol. In the present study, we first discovered that ST8Sia II polysialylates all isoforms of the NCAM examined, and we demonstrated that polysialylation of NCAM expressed on 3T3 cells facilitates neurite outgrowth regardless of isoforms of NCAM, where polysialic acid is attached. We then show that neurite outgrowth is significantly facilitated only when polysialylated NCAM is present in cell membranes. Moreover, the soluble NCAM coated on plates did not have an effect on neurite outgrowth exerted by soluble L1 adhesion molecule coated on plates. These results, taken together, indicate that ST8Sia II plays critical roles in modulating the function of all major isoforms of NCAM. The results also support previous studies showing that a signal cascade initiated by NCAM differs from that initiated by L1 molecule.

Key words: neural cell adhesion molecule/neurite cell outgrowth/polysialic acid/polysialyltransferase/ST8Sia II (STX)


    Introduction
 Top
 Abstract
 Introduction
 Results
 Discussion
 Materials and methods
 Acknowledgments
 Abbreviations
 References
 
In the development of the nervous system, cell–cell interaction mediated by adhesion molecules is one of the most important regulating factors for neuronal projection (Edelman, 1984Go; Schachner and Martini, 1995Go). Among those adhesion molecules, the neural cell adhesion molecule (NCAM) plays a major role through homophilic interaction. As a representative of adhesion molecules in the nervous system, NCAM undergoes important posttranscriptional modification as well as posttranslational modification that regulates its functions (Rutishauser and Landmesser, 1991Go; Walsh and Doherty, 1996Go).

Indeed, NCAM has numerous isoforms due to the difference in splicing of precursor mRNAs. Among them, the major forms are NCAM-180, NCAM-140, NCAM-140(VASE) (Small and Akeson, 1990Go), NCAM-125(MSD), and NCAM-120 (He et al., 1986Go; Hemperly et al., 1986Go; Dickson et al., 1987Go), which differ in polypeptide size. Except for the additional VASE and MSD sequences, these different NCAMs have a common extracellular structure that consists of five immunoglobulin-like domains and two fibronectin type III–like domains (Figure 1). NCAM-180 and NCAM-140 are transmembrane glycoproteins, differing in their cytoplasmic tails (Cunningham et al., 1983Go; Gower et al., 1988Go). NCAM-140(VASE) contains an additional 10 amino acid-sequence (VASE) in the fourth immunoglobulin-like domain of NCAM-140 (Small and Akeson, 1990Go). NCAM-125 and NCAM-120 are anchored to the lipid bilayer through a glycosyl phosphatidylinositol, and NCAM-125 contains an additional so-called muscle-specific domain (MSD) consisting of 37 amino acids between two fibronectin type III–like domains (He et al., 1986Go; Hemperly et al., 1986Go; Dickson et al., 1987Go; Figure 1). NCAM-180, NCAM-140, and NCAM-120 are produced by differential splicing at 3'-region of mRNA precursors of NCAM. The VASE and MSD sequences are also due to additional mRNA segments made through alternative splicing of NCAM mRNAs.



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Fig. 1. Schematic structure of NCAM isoforms. The schematic structure is shown for NCAM-120, NCAM-125(MSD), NCAM-140, NCAM-140(VASE), and NCAM-180. From the amino terminal (N), five immunoglobulin-like domains (Ig 1–5) and two fibronectin-like domains (FN 1 and 2) are shown. MSD and VASE sequences are inserted at indicated positions. NCAM-120 and NCAM-125 are attached to the membrane through a glycosyl phosphatidylinositol. NCAM-140 or NCAM-140(VASE) and NCAM-180 are transmembrane proteins and differ in the length of cytoplasmic domains.

 
As a unique posttranslational modification, NCAM in embryonic tissues contains polysialic acid, which is a linear homopolymer of {alpha}2,8-linked sialic acid (Finne, 1982Go). Although the majority of NCAM in adult tissues lacks this glycan, polysialylated NCAM is still present in the olfactory bulb and hippocampus of adult brain where neural regeneration persists (Seki and Arai, 1991Go; Schachner and Martini, 1995Go). In fact, NCAM-knockout mice exhibit a defect in spatial learning and memory due to an abnormal formation of the hippocampus (Tomasiewicz et al., 1993Go; Cremer et al., 1994Go).

These results are consistent with reports showing that removal of polysialic acid by endoneuraminidase (endo-N), which specifically cleaves polysialic acid, resulted in the prevention of the induction of long-term potentiation displayed in the hippocampus (Muller et al., 1996Go). Other studies demonstrated that mossy fiber interaction and synapse formations are perturbed in NCAM-deficient mice and mice treated with endo-N (Cremer et al., 1997Go; Seki and Rutishauser, 1998Go). It is suggested that spatial learning activates NCAM polysialylation in the corticohippocampal pathway (O'Connell et al., 1997Go). Considered together, these results indicate that polysialylated NCAM plays a critical role during development and neural regeneration, most likely by attenuating the adhesive property of NCAM (Hoffman and Edelman, 1983Go; Sadoul et al., 1983Go).

The cDNAs encoding polysialyltransferases have been cloned, and these enzymes are termed ST8Sia IV (also called PST) (Eckhardt et al., 1995Go; Nakayama et al., 1995Go; Yoshida et al., 1995Go) and ST8Sia II (also called STX) (Livingston and Paulson, 1993Go; Kojima et al., 1995Go; Scheidegger et al., 1995Go). ST8Sia IV and ST8Sia II, which belong to the sialyltransferase gene family, are highly homologous to each other and have 59% identity at the amino acid level (Eckhardt et al., 1995Go; Nakayama et al., 1995Go).

It has been shown that both ST8Sia IV and ST8Sia II can polysialylate NCAM-140 and polysialylated NCAM-140 facilitates neurite outgrowth better than nonpolysialylated NCAM-140 (Nakayama et al., 1995Go; Angata et al., 1997Go). Research has also demonstrated that NCAM-140 polysialylated by ST8Sia IV serves as a slightly better substrate for neurite outgrowth than NCAM-140 polysialylated by ST8Sia II (Angata et al., 1997Go). This finding is consistent with the fact that ST8Sia IV adds more polysialic acid to NCAM than does ST8Sia II (Kojima et al., 1996Go; Angata et al., 1998Go). On the other hand, Kojima et al. (1997)Go reported that ST8Sia IV can polysialylate all isoforms of NCAM, whereas ST8Sia II can polysialylate only NCAM-180, NCAM-140(VASE), and NCAM-140, but not NCAM-125 or NCAM-120. If this were true, ST8Sia II and ST8Sia IV would differ in acceptor recognition, depending on whether NCAM is synthesized as a transmembrane protein or a glycosyl phosphatidylinositol–anchored protein.

In the present study, we describe first that ST8Sia II polysialylate NCAM-180, NCAM-140(VASE), NCAM-140, NCAM-125(MSD), and NCAM-120 expressed on 3T3 cells. We then demonstrate that polysialylation of all of these isoforms facilitated neurite outgrowth almost equally and had no significant difference in enhancing neurite outgrowth among different isoforms of NCAM. We also observed that neurite outgrowth was marginally enhanced when a soluble chimeric form of polysialylated NCAM coated on plates was used as a substratum compared with control NCAM substratum. The soluble NCAM coated on plates, on the other hand, did not enhance or decrease the neurite outgrouth exerted by L1 molecules, supporting the idea that a signal cascade initiated by NCAM differs from that initiated by L1 molecules.


    Results
 Top
 Abstract
 Introduction
 Results
 Discussion
 Materials and methods
 Acknowledgments
 Abbreviations
 References
 
Establishment of 3T3 cells expressing different isoforms of NCAM and ST8Sia II or ST8Sia IV
3T3 cells expressing different isoforms of NCAM had been established in previous studies using neomycin as a selection marker (Doherty et al., 1990Go, 1992). In the present study, pcDNA3.1/Hygro-ST8Sia II or pcDNA3.1/Hygro-ST8Sia IV was introduced to these cells, and those cells stably expressing both NCAM and polysialic acid were established.

Figure 2 shows the results of immunofluorescent staining of NCAM or polysialic acid in representative experiments. These cells were judged to express significant amounts of polysialic acid and NCAM after transfecting pcDNA3.1/Hygro-ST8Sia II.



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Fig. 2. Immunofluorescence staining of 3T3-NCAM cells transfected with pcDNA3.1/Hygro-ST8Sia II. 3T3-NCAM-125, 3T3-NCAM-140, and 3T3-NCAM-180 were stably transfected with pcDNA3.1/Hygro-ST8Sia II and stained with Eric-1 anti-NCAM antibody (NCAM) or 12F8 anti–polysialic acid antibody (PSA), followed by respective secondary antibody. The parent 3T3-NCAM-140 is shown as a control. The cells were stained before they reached confluency. Bar = 10 µm.

 
NCAM is polysialylated in transfected 3T3 cells
To determine if NCAM is polysialylated in 3T3-NCAM cells transfected with pcDNA3.1/Hygro-ST8Sia II, glycoproteins from these cells were separated by SDS–polyacrylamide gel electrophoresis and subjected to Western blot analysis using anti-NCAM antibody.

As shown in Figure 3, NCAM molecules from parental 3T3-NCAM cells displayed a relatively sharp band (lanes 1 for each isoform). In contrast, NCAM from 3T3-NCAM cells transfected with ST8Sia II displayed a broad, high molecular weight band (lanes 2 in Figure 3, left half). These heterogeneous bands were sharpened after endo-N treatment (lanes 3 in Figure 3), indicating that all of the isoforms expressed contain polysialic acid after transfection of ST8Sia II cDNA. It is noteworthy that NCAM-120 and NCAM-125 isoforms were polysialylated as much as NCAM-140, NCAM-140(VASE), or NCAM-180 isoforms were.



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Fig. 3. Western blot analysis of NCAM expressed in 3T3-NCAM cells stably transfected with ST8Sia II or ST8Sia IV cDNA. The glycoproteins were isolated from different 3T3-NCAM·ST8Sia II and 3T3-NCAM·ST8Sia IV cells before or after endo-N treatment and separated by SDS–polyacrylamide gel electrophoresis. After blotting onto PVDF membrane, the membrane was incubated with Eric-1 anti-NCAM antibody and visualized using an ECL kit. Lanes 1, 2, and 3 represent NCAM in the parent 3T3-NCAM cells, 3T3-NCAM-ST8Sia II (left panel) or 3T3-NCAM·ST8Sia IV (right panel) cells, and those treated with endo-N, respectively. The migration positions of molecular mass markers are shown in the middle.

 
When NCAM from 3T3-NCAM cells transfected with ST8Sia IV cDNA were examined, almost identical results were obtained (Figure 3, right half). These combined results indicate that both ST8Sia II and ST8Sia IV can polysialylate all isoforms of NCAM.

Effect of MSD and VASE domains on polysialylation
To determine if ST8Sia II and ST8Sia IV may differ in polysialylation of different extracellular domains, plasmid harboring NCAM·IgG chimeric protein with MSD or VASE was constructed. Those plasmids encoding NCAM(MSD)·IgG, NCAM(VASE)·IgG or NCAM·IgG were transiently transfected into HeLa-ST8Sia II or HeLa-ST8Sia IV cells, and soluble chimeric NCAMs were isolated from the culture medium.

The results shown in Figure 4A demonstrate that ST8Sia II or ST8Sia IV added polysialic acid almost equally to NCAM isoforms containing different extracellular domains (lanes 2 and 3 in different NCAM). Moreover, no obvious differences were observed between ST8Sia IV and ST8Sia II for polysialylation of these different NCAM·IgG chimeras (compare lanes 2 and 3).



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Fig. 4. Western blot analysis of extracellular domains of NCAM and L1. A: NCAM(VASE, MSD)·IgG, NCAM(MSD)·IgG, NCAM(VASE)·IgG, and NCAM·IgG were expressed in HeLa-ST8Sia IV (lanes 2) or HeLa-ST8Sia II (lanes 3) cells. Released NCAM·IgG chimeric proteins were isolated by protein A affinity column and subjected to SDS–polyacrylamide gel electrophoresis and Western blot analysis. The chimeric proteins were detected by peroxidase-conjugated goat anti-human Fc antibodies. B: L1·IgG chimera was analyzed using anti-human Fc antibodies.

 
Neurite outgrowth on 3T3 cells expressing different isoforms of NCAM containing polysialic acid
Although all NCAM isoforms can be polysialylated by ST8Sia II, it would be still possible that the effect of polysialic acid on neurite outgrowth may differ depending on different isoforms of NCAM. To determine whether or not that is the case, neurite outgrowth assay was carried out using 3T3 cells as a substratum. Figure 5 illustrates an example of neurons on 3T3-NCAM-125 or 3T3-NCAM-125·ST8Sia II. This figure shows that neurons on 3T3-NCAM-125·ST8Sia II are longer than those on 3T3-NCAM-125.



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Fig. 5. Neurite outgrowth on confluent 3T3 cells expressing NCAM 125 and NCAM-ST8Sia II. 3T3 cells stably espressing NCAM 125 (left) and NCAM 125 plus ST8Sia II (right) were used as substratum for neurite outgrowth. Cerebellar neurons from 5-day-old mice were seeded and cultured on these substrata for 24 h. Neurons were visualized by immunofluorescent staining for GAP-43 protein. Bar = 100 µm.

 
As shown in Figure 6, polysialylation of all isoforms of NCAM facilitated neurite outgrowth. Second, the increase in neurite extension appears to differ somewhat, depending on the isoforms. For example, the difference in neurite outgrowth between polysialylated and nonpolysialylated forms is more statistically significant in NCAM-140(VASE) and NCAM-125(MSD) than the difference observed in NCAM-120, NCAM-140, and NCAM-180 (Figure 6). This difference can be observed better when the number of neurons that are longer than a given length is plotted, as seen in Figure 7. Third, neurite outgrowth on parent 3T3-NCAM expressing different isoforms of NCAM differ to some extent, but the difference in the effect of different isoforms is not as obvious as the difference due to ST8Sia II expression. Neurite outgrowth on polysialylated NCAM was always enhanced compared to nonpolysialylated NCAM, regardless of the isoforms of NCAM (Figure 7).



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Fig. 6. Comparison of neurite outgrowth lengths on 3T3-NCAM·ST8Sia II cells. 3T3 cells expressing different isoforms of NCAM, before or after transfection of ST8Sia II cDNA, were used as substrata for neurite outgrowth. The lengths of 100 neurites that are longer than 35 µm are compared. Experiments were carried out on two different occasions in triplicate, and the results obtained in one experiment are shown. Standard errors are shown, and the statistically significant difference is denoted by * (for p < 0.05) and ** (for p < 0.01). P-values for the others are accessed by t test or anova, 0.138 for 120/120·ST8Sia II, 0.094 for 140/140·ST8Sia II and 0.228 for 180/180·ST8Sia II.

 


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Fig. 7. Distribution of neurite length on different substrata. Neurite lengths on 3T3-NCAM-120 (A), 3T3-NCAM-125 (B), 3T3-NCAM-140 (VASE) (C), and 3T3-NCAM-180 (D) were compared to neurite length on the corresponding 3T3 cells containing polysialic acid synthesized by ST8Sia II. The vertical line indicates the number of neurites in %, which are identical to or longer than a given size shown in the horizontal line.

 
Because polysialylated forms were established by transfecting the parent 3T3-NCAM cells with ST8Sia II, the results ensured that the differences in neurite outgrowth was due to the attachment of polysialic acid. However, the effect of polysialylation may be still influenced by the expression levels of NCAM (see Discussion) and polysialic acid, although immunofluorescent staining of these different cell lines showed similar amounts of NCAM (see Figure 2).

Polysialylated NCAM on intact cells is most efficient in facilitation of neurite outgrowth
We then tested whether or not NCAM substratum alone is sufficient to facilitate neurite outgrowth and if polysialylated NCAM is better than NCAM as a substratum. For these experiments, we took advantage of the fact that polysialylated NCAM can be obtained by transfecting cDNA encoding NCAM·IgG chimera into HeLa-ST8Sia II or HeLa-ST8Sia IV cells expressing polysialic acid. As shown in Figure 4A, NCAM·IgG chimeric protein synthesized in HeLa-ST8Sia II or HeLa-ST8Sia IV cells displayed a large, broad band indicative of polysialic acid. As a positive control, L1·IgG chimeric protein was also produced (Figure 4B).

Figure 8A demonstrated that the soluble chimeric L1 protein coated on plates has a significant effect on neurite outgrowth compared to a control substratum coated only with polylysine and anti-Fc antibody. These results are consistent with previous reports that L1 facilitates neurite outgrowth (Doherty et al., 1995Go). Moreover, the addition of NCAM·IgG to L1·IgG did not have any additional enhancing effect on neurite outgrowth (Figure 8A). On the other hand, NCAM (0.2 µg) coated on plates had a marginal enhancing effect, as shown in Figure 8B, compared with a significant effect by L1 with the same concentration (0.2 µg) in Figure 8A. This enhanced effect by NCAM increased slightly when polysialylated NCAM was coated on plates (Figure 8B). However, this increase in neurite outgrowth by polysialylation was not as evident as the increase on polysialylated NCAM in the cell (compare Figures 6 and 8B).



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Fig. 8. Neurite outgrowth on L1·IgG and NCAM·IgG chimera proteins coated on plates. Neurite outgrowth assays were carried out using the following substrata. (A) Neurite outgrowth on L1·IgG (L1), L1·IgG plus NCAM·IgG (NCAM), and L1·IgG plus polysialylated NCAM·IgG (PSA-NCAM). NCAM·IgG molecules (200 ng/ml) were immobilized after L1·IgG (40 ng/ml) was immobilized. Polylysine (PLL) or polylysine then anti-human IgG (anti-Fc) were coated in the control experiments. In one experiment, L1·IgG (200 ng/ml) was immobilized for comparison to the experiments shown in B. (B) Neurite outgrowth on NCAM·IgG or polysialylated NCAM•IgG coated on plates. NCAM·IgG was isolated from HeLa, HeLa-ST8Sia II, or HeLa-ST8Sia IV cells and coated on plates at the concentration of 200 ng/ml. Statistical difference is p < 0.06 between L1·IgG and anti-Fc (A) and 0.18 or 0.19 between NCAM and NCAM-ST8Sia IV or NCAM-STSia II (B).

 

    Discussion
 Top
 Abstract
 Introduction
 Results
 Discussion
 Materials and methods
 Acknowledgments
 Abbreviations
 References
 
The present study demonstrated that both ST8Sia II and ST8Sia IV can polysialylate all of the major isoforms of NCAM, such as NCAM-180, NCAM-145(VASE), NCAM-140, NCAM-125(MSD), and NCAM-120. During these studies, we did not encounter any problem in making 3T3 cell lines containing NCAM-120 (or -125) positive for polysialic acid after transfecting with pcDNA3.1/Hygro-ST8Sia II. Moreover, our research demonstrated that the addition of an MSD or VASE sequence does not influence polysialylation. Similarly, it has been reported that the presence of VASE did not affect polysialylation using chicken brain extracts as an enzyme source (Oka et al., 1995Go) and NCAM-125(MSD) is polysialylated in various cells (Moore et al., 1987Go; Fredette et al., 1993Go). These combined results indicate that ST8Sia II and ST8Sia IV have no preference in utilizing the different isoforms of NCAM as acceptors. In contrast, Kojima et al. (1997)Go previously reported that ST8Sia II can polysialylate only NCAM-180 and NCAM-140 but not NCAM-120, while ST8Sia IV can polysialylate all of these NCAM isoforms. This result may have been obtained by the lower expression levels of ST8Sia II compared to ST8Sia IV in their work.

As shown previously, ST8Sia II and ST8Sia IV differ in several aspects of polysialylation. First, ST8Sia IV is more efficient in NCAM polysialylation and forms a larger polysialylated NCAM than does ST8Sia II (Angata et al., 1997Go). This is particularly evident when NCAM·IgG chimeric protein was used as an acceptor for in vitro reaction (Kojima et al., 1996Go; Angata et al., 1998Go). Second, ST8Sia IV more preferentially adds polysialic acid to the sixth N-glycosylation site of NCAM than does ST8Sia II (see Figure 1 also) (Nelson et al., 1995Go; Angata et al., 1998Go). Because of this difference in the site specificity, the amount of polysialic acid is apparently increased when both ST8Sia II and ST8Sia IV are present compared to when only ST8Sia II or ST8Sia IV is present (Angata et al., 1998Go). ST8Sia II and ST8Sia IV are differentially expressed during development, and ST8Sia IV appears to play a more dominant role in adult brain (Phillips et al., 1997Go; Kurosawa et al., 1997Go; Hildebrandt et al., 1998Go; Ong et al., 1998bGo; Eckhardt et al., 2000Go). Recent studies demonstrated that inactivation of ST8Sia IV in mouse led to impaired long-term potentiation and long-term depression in Schaffer collateral-CA1 synapses of the adult hippocampus (Eckhardt et al., 2000Go). Loss of polysialic acid, particularly in embryonic brain, was not apparent, because ST8Sia II compensates the inactivation of ST8Sia IV. Taken together, these previous results and those obtained in the present study indicate that both ST8Sia II and ST8Sia IV can act on all major isoforms of NCAM, but they may differentially play a role in different tissues and different developmental stages.

The present study also demonstrated that polysialylation of all NCAM isoforms tested facilitates neurite outgrowth. Previously, we have shown that NCAM-140 can be polysialylated by both ST8Sia II and ST8Sia IV and that polysialylated NCAM-140 facilitates neurite outgrowth better than nonpolysialylated NCAM (Nakayama et al., 1995Go; Angata et al., 1997Go). The present work extended the previous findings so that all NCAM isoforms tested increased the neurite outgrowth after polysialylation. The results clearly indicate that polysialylation provides a unique, universal tool for modulation of the roles of NCAM, regardless of whether or not the polypeptide portion of NCAM molecules is the same.

The present study addressed the effect of NCAM polysialylation on neurite outgrowth but did not address the effect of different isoforms of NCAM on neurite outgrowth. To address the latter points, it is necessary to determine precisely the amount of different isoforms of NCAM expressed. This is not an easy task because we found that the reactivity of anti-NCAM antibody is invariably influenced by the glycosylation of NCAM, in particular, polysialylation. We thus focused our efforts to determine the effect of NCAM polysialylation on neurite outgrowth.

We found that NCAM·IgG and L1·IgG coated on plates did not enhance neurite outgrowth compared to the control, where only L1·IgG was coated. In relation to this finding, it was reported that NCAM-180 facilitates neurite outgrowth less than does NCAM-140 (Doherty et al., 1992Go). This difference was attributed to the fact that lateral mobility of NCAM-180 is lower than that of NCAM-140 (Pollerberg et al., 1986Go), probably because the longer cytoplasmic segment of NCAM-180 is closely associated with cytoskeletal proteins, such as brain spectrin (Pollerberg et al., 1987Go). Because NCAM·IgG coated on plates does not have any lateral mobility, these combined results indicate that the lateral mobility of NCAM on substratum cells may be essential for axonal extension facilitated through NCAM–NCAM interaction. This finding is consistent with the fact that NCAM-180 is more enriched in adult brain than embryonic brain, while the opposite is true for NCAM-140 (Sunshine et al., 1987Go).

Present results are consistent with a previous report that L1·IgG chimeric protein coated on plates facilitates neurite outgrowth (Bourne et al., 1991Go). Moreover, the results also indicate that L1 and NCAM do not influence each other because the addition of NCAM to L1 on plates did not inhibit or enhance the neurite outgrowth exerted by L1 (Figure 8A). These results suggest that L1 and NCAM on substratum acts in a different manner. It has been proposed that L1 and NCAM stimulate neurite outgrowth by activating the FGF signal transduction cascade (Saffell et al., 1997Go). However, the Src-family tyrosine kinase p59 fyn appears to play a role in the NCAM but not L1 response (Beggs et al., 1994Go, 1997).

These combined results suggest that NCAM coated on plates cannot provide the same signal cascade that a "membrane-bound" NCAM provides. The results also suggest that NCAM·IgG does not provide additional cascade signals than what L1 already provides under the conditions employed in the present studies. These results suggest an exciting possibility for future studies to dissect signaling cascade events leading to neurite outgrowth by comparison of neurons on NCAM-expressing substratum versus neurons on NCAM·IgG immobilized on plates.


    Materials and methods
 Top
 Abstract
 Introduction
 Results
 Discussion
 Materials and methods
 Acknowledgments
 Abbreviations
 References
 
Construction of soluble NCAM of different isoforms
pIG-NCAM(VASE, MSD) encoding a soluble NCAM-125 fused with a hinge and constant regions of human IgG, NCAM(VASE, MSD)·IgG, was kindly provided by Dr. David Simmons at Oxford University (Simmons, 1993Go). This vector, originally designated as pIG-NCAM, was found to contain both VASE and MSD sequences. Because such an NCAM(VASE, MSD) is unusual, we decided to delete the MSD and VASE sequences as follows.

First, an EcoRI-KpnI fragment (550 bp) harboring an MSD sequence of pIG-NCAM(VASE, MSD) was replaced with an EcoRI-KpnI fragment (442 bp) of pCDM8-NCAM-140 (Eckhardt et al., 1995Go), resulting in pIG-NCAM(VASE). Because this pIG-NCAM(VASE) still contains a VASE sequence, a BglII fragment (1063 bp) of pIG-NCAM(VASE) was replaced with a BglII fragment (1023 bp) from NCAM-140, resulting in pIG-NCAM. The resulting pIG-NCAM encodes the extracellular domain common to NCAM-180, -140, and -120, which is fused with the hinge and constant regions of human IgG. To construct a vector containing only the MSD domain in NCAM, the 1063 bp DNA sequence containing the VASE sequence was excised with BglII from pIG-NCAM(VASE, MSD) and was replaced with the corresponding BglII fragment of 1023 bp from the NCAM-140 sequence (nucleotides 333–1361), resulting in pIG-NCAM(MSD).

Construction of pcDNAI-L1·IgG
A soluble L1·IgG chimeric protein was constructed as follows. First, a human L1 cDNA insert from pcDNA1.1/amp-L1 (Dahlin-Huppe et al., 1997Go) was excised by HindIII and XbaI digestion and cloned into the same sites of pcDNAI vector (Invitrogen), resulting in pcDNAI-L1. From pcDNAI-L1, the 3'-end portion of L1 cDNA was excised at the SmaI site within the cDNA and XbaI in the vector cloning site. In parallel, the sequence encompassing nucleotides 2056–3341 of L1 cDNA was amplified by PCR using pcDNAI-L1 as a template (nucleotides 1–3 encode the initiation of methionine). For this PCR, the 3'-primer is 5'-CGCGGATCCGGAGGCAGCCTCACGCTGGC-3' (BamHI site is underlined), whereas the 5'-primer corresponds to nucleotides 2056–2075. After TA cloning of this PCR product into pBluescript II (Stratagene) and confirming the sequence, the cDNA fragment was excised by SmaI-BamHI digestion.

This SmaI-BamHI fragment of L1 cDNA and the BamHI-XbaI fragment of human IgG sequence from pcDNAI-ICAM·IgG (Tsuboi and Fukuda, 1997Go) were then ligated into SmaI/XbaI-digested pcDNAI-L1, which contained the 5'-portion of L1 cDNA. This vector is designated as pcDNAI-L1·IgG.

Expression of ST8Sia IV and ST8Sia II in 3T3 cells
pcDNAI-ST8Sia IV harboring cDNA encoding a full-length human ST8Sia IV was cloned by expression cloning as described before (Nakayama et al., 1995Go). pcDNAI-ST8Sia II harboring cDNA encoding a full-length human ST8Sia II was cloned by reverse transcriptase PCR as described previously (Angata et al., 1997Go). cDNA inserts of pcDNAI-ST8Sia IV and pcDNAI-ST8Sia II were excised by HindIII-XbaI and HindIII-XhoI, respectively, and cloned into corresponding sites of pcDNA3.1/Hygro. These plasmids are called pcDNA3.1/Hygro-ST8Sia IV and pcDNA3.1/Hygro-ST8Sia II, respectively.

Mouse 3T3 cells expressing NCAM-180, NCAM-140, NCAM-140(VASE), NCAM-125(MSD), or NCAM-120 were established previously by transfecting pHßApr-1-neo mammalian expression vector harboring cDNA encoding the different isoforms of human NCAM (Doherty et al., 1990Go, 1992). To these different 3T3 cell lines, pcDNA3.1/Hygro-ST8Sia IV or pcDNA3.1/Hygro-ST8Sia II was transfected, and the transfected cells were selected by hygromycin. Clonal cells expressing polysialic acid were chosen after staining with 12F8 anti–polysialic acid antibody (Chung et al., 1991Go).

Before we used these cells as substrata in neurite outgrowth assays, the cells were examined by anti-NCAM antibody (Eric-1) (Bourne et al., 1991Go) or anti–polysialic acid (12F8) followed by phycoerythrin-conjugated anti-mouse IgG (for anti-NCAM) or fluorescein isothiocyanate–conjugated anti-rat IgM (for 12F8)

Western blot analysis
3T3 cells expressing ST8Sia II or ST8Sia IV and various isoforms of NCAM were subjected to Western analysis using the ECL Western blot detection system (Amersham Pharmacia Biotech) as described (Ong et al., 1998aGo). Briefly, the cell pellet was recovered by centrifugation, and a portion of the pellet was digested with endo-N. The cells were then dissolved in 1% Nonidet P-40 containing a protease inhibitor mixture (Roche Molecular Biochemicals) in phosphate-buffered saline, and sialoglycoproteins were concentrated using Microcon 30 spin filters (EY Laboratories). The proteins (50 µg estimated by ABC kit from Pierce) were separated by SDS–polyacrylamide gel (5%) electrophoresis, and blotted onto PVDF membrane as described previously (Ong et al., 1998aGo).

For detection of polysialylated NCAM, Eric-1 antibody was used because we found that this antibody is less influenced by polysialic acid attached to NCAM than the other antibodies tested. The addition of Eric-1 antibody was followed by peroxidase-conjugated goat IgG specific to mouse IgG, and a positive reaction was visualized using an ECL kit.

Expression of NCAM·IgG chimeric protein
HeLa cells stably transfected with pcDNAI-ST8Sia IV or pcDNAI-ST8Sia II were established as described (Nakayama et al., 1995Go; Angata et al., 1997Go). Each construct of pIG-NCAM was transiently transfected to these cells using LipofectAMINE (Life Technologies) as described previously (Angata et al., 1998Go). Twenty-four hours after the transfection, the medium was replaced with Opti-MEM (Life Technologies) and cultured for an additional 24 h. NCAM·IgG chimeric protein in the spent medium was adsorbed to protein A-agarose (Pierce) and eluted with a gentle Ag/Ab elution buffer (Pierce) as described elsewhere (Angata et al., 1998Go). The eluted material was desalted by repeated concentration and washing with 20 mM Tris–HCl, pH 7.4, containing 0.05% Tween 20 using Microcon 30 (Amicon). The concentration of each NCAM·IgG was adjusted after measuring the amount by Western blot analysis using peroxidase-conjugated goat IgG specific to the Fc portion of human IgG (ICN) and an ECL kit. Human IgG protein (Sigma) was used as a standard as described previously (Angata et al., 1998Go).

Neurite outgrowth assay
Monolayers of 3T3 cells were established by seeding cells at 100,000 cells per chamber onto wells of eight-chamber tissue culture slides (Lab-Tek) that had been coated with poly D-lysine (Sigma) and maintained for 24 h before the addition of neurons (Doherty et al., 1990Go, 1991; Zhang et al., 1992Go). Cerebella from 5-day-old mice were removed and dissected free of meninges, minced with a scalpel blade, and subsequently digested with trypsin (3000 U/ml) for 15 min at 37°C (Groves et al., 1993Go). SBTI-DNase was then added and the cell suspension centrifuged for 5 min at 200 x g. The cell pellet was resuspended in HBSS Ca2+, Mg2+–free and dissociated by trituration through 21G and 25G needles. The dissociated cells were centrifuged through a 3-ml cushion of DMEM supplemented with 10% fetal calf serum (DMEM-FCS) containing 4% BSA. The pellet was resuspended in DMEM-FCS and the cells seeded out on PLL-coated flasks and incubated at 37°C for 30 min. The flask was then gently shaked, and the medium, containing a population enriched for granule neurons, was removed. After a 5-min centrifugation at 200 x g, the pellet was resuspended in Sato medium (adapted from Bottenstein and Sato, 1979Go). Cultures were established by seeding 3000 cerebellar neurons in 500 µl of SATO medium into confluent monolayer 3T3 cultures. After 24 h, the co-cultures were fixed with paraformaldehyde and neurons were stained with anti-GAP-43 antibody (Doherty et al., 1995Go). The length of the longest neurite for each GAP-43 positive neuron was determined by computer-assisted digital image analysis as described previously (Nakayama et al., 1995Go; Angata et al., 1997Go).

Neurite outgrowth on NCAM·IgG coated on plates
Individual wells of eight-chamber tissue culture slides were coated with polylysine followed by goat anti-human IgG (Fc-specific) (10 µg/ml) in DMEM for 60 min at 37°C (Doherty et al., 1995Go). After wells were blocked with DMEM containing 2% fetal calf serum, 200 µl of the NCAM·IgG chimera diluted in DMEM containing 2% fetal calf serum was added as described previously (Doherty et al., 1991Go). Alternatively, L1·IgG chimera was coated on the wells. For the L1·IgG chimera, a concentration of 40 ng/ml was used because this provides 50% of the maximum effect of L1·IgG chimera on neurite outgrowth under the conditions used. In parallel, NCAM·IgG chimera was coated on the plates after L1·IgG chimera was coated and used as a substratum.


    Acknowledgments
 Top
 Abstract
 Introduction
 Results
 Discussion
 Materials and methods
 Acknowledgments
 Abbreviations
 References
 
We thank Drs. Rita Gerardy-Schahn, David Simmons, and William Stallcup for the kind gifts of pCDM8-NCAM-140, pIG-NCAM(VASE, MSD), and pcDNA1.1/Amp-L1. We also thank Joseph P. Henig and Risa Tabata for organizing the manuscript. This work was supported by grant CA33895 awarded by the National Cancer Institute and fellowship (to I.F.) from the International Agency for Research on Cancer, Lyon, France.


    Abbreviations
 Top
 Abstract
 Introduction
 Results
 Discussion
 Materials and methods
 Acknowledgments
 Abbreviations
 References
 
NCAM, neural cell adhesion molecule; VASE, variable alternatively spliced exon; MSD, muscle-specific domain; endo-N, endoneuraminidase; PCR, polymerase chain reaction.


    Footnotes
 
1 Present address: Departement de Virologie, Institute Pasteur, 75724 Paris, Cedex, France Back

2 To whom correspondence should be addressed Back


    References
 Top
 Abstract
 Introduction
 Results
 Discussion
 Materials and methods
 Acknowledgments
 Abbreviations
 References
 
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