©1995 by The American Society for Biochemistry and Molecular Biology, Inc.
Protein Determinants for Specific Polysialylation of the Neural Cell Adhesion Molecule (*)

(Received for publication, April 4, 1995)

Richard W. Nelson (1)(§), Paul A. Bates (2), Urs Rutishauser (1)(¶)

From the  (1)Departments of Molecular Biology and Microbiology and Genetics, Case Western Reserve University, Cleveland, Ohio 44106 and the (2)Biomolecular Modeling Laboratory, Imperial Cancer Research Fund, London WC2A 3PX, United Kingdom

ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
FOOTNOTES
ACKNOWLEDGEMENTS
REFERENCES

ABSTRACT

Expression of polysialic acid (PSA) involves its specific attachment to the neural cell adhesion molecule (NCAM). Here we identify the amino acid residues within NCAM that are polysialylated and structural domains of the NCAM polypeptide that are required for addition of PSA in cells. Chicken NCAM cDNAs containing amino acid mutations, domain deletions, and domain substitutions were expressed in the F11 rat/mouse hybrid cell line, which can produce polysialylated NCAM. Polysialylation of the chicken NCAM was evaluated by immunopurification and electrophoresis. Mutation of all three potential N-glycosylation sites within the fifth immunoglobulin domain (Ig5) abrogated polysialylation. Analysis of paired mutations revealed that Asn-459 is heavily polysialylated, Asn-430 has a lower level of substitution, and Asn-404 receives little or no PSA. Analysis of domain deletions established that the intracellular domain, Ig domains 1-3, and the COOH-terminal fibronectin-type III (FNIII) repeat are not required for polysialylation, but that deletion of either the adjacent Ig4 or FNIII-type domain prevented addition of PSA. Accordingly, a minimal polypeptide for polysialylation was found to contain Ig domains 4 and 5, the adjacent FNIII repeat, plus a membrane attachment. These results suggest that although all PSA is located within Ig5, regions outside Ig5 also play a role in PSA addition to NCAM. Furthermore, molecular modeling indicates spatial proximity of Asn-430 and Asn-459 and a tight-locking arrangement between Ig4, Ig5, and FNIII#1 that would be consistent with their formation of a spatially discrete enzyme recognition site for polysialylation.


INTRODUCTION

Neural cell adhesion molecule (NCAM)()is a broadly expressed cell surface glycoprotein containing tandem repeats of both immunoglobulin-like (Ig) and fibronectin-type III (FNIII) domains(1) . Although NCAM can serve as a homophilic receptor in promotion of cell-cell interactions, it is also expressed in an alternative form containing long chains of -2,8-linked polysialic acid (PSA) (2, 3) that converts it into an anti-adhesive agent(4, 5) . The pattern of expression of PSA on NCAM is highly regulated during embryonic development, and the resulting attenuation of cell interactions is a critical event in a variety of cell rearrangements and axonal behaviors(5, 6, 7, 8, 9, 10) . Its persistence or reappearance in adult tissues is also associated with events requiring remodeling or repair of tissue structures(11, 12, 13) . Because of the biological significance of this post-translational modification, it is important to identify the biosynthetic pathways that control PSA synthesis (11, 14, 15, 16, 17) and the relationship of this regulation to other cellular processes. An essential part of that effort is to identify the structural features of NCAM that allow for its specific polysialylation.

Many complex carbohydrates attached to cell surface proteins do not display a strict specificity for a particular protein. For example, the HNK-1 determinant, which can be attached to NCAM, is also present on a wide range of cell surface receptors and matrix components(18, 19) . By contrast, the expression of the long polymers (n = 8 to possibly over 100) of PSA is largely if not entirely restricted to the NCAM polypeptide in vertebrate embryos, being linked to the protein through what is thought to be a typical N-linked carbohydrate core(2, 20, 21, 22) . Moreover, of the seven potential N-glycosylation sites in chicken NCAM, expression of PSA is believed to be associated with one or more of the three potential N-linked glycosylation sites within the fifth immunoglobulin domain(1, 23) . Thus, in addition to the biological significance of PSA, the process of NCAM polysialylation represents a valuable system in which to study protein determinants associated with specific glycosylation.

In this study, we have sought to define the precise sites of polysialic acid addition and identify the structural characteristics of the largest form polypeptide form of NCAM (NCAM-180) that are involved in recognition by PSA biosynthetic enzyme(s). The approach has been to transfect rodent cells capable of producing PSA on NCAM with chicken NCAM cDNA constructs bearing a variety of relevant site-directed and domain mutations. Specific anti-chicken NCAM antibodies are then used to isolate and identify the chicken protein produced, followed by SDS-gel electrophoresis to evaluate the degree of polysialylation. The results obtained are combined with molecular modeling of NCAM domains to summarize the spatial features of a putative recognition site for PSA addition.


EXPERIMENTAL PROCEDURES

Materials

The pCMV plasmid was purchased from Invitrogen (San Diego, CA). The pEC1401 plasmid, which contains the entire coding region of chicken NCAM-180 cDNA, was kindly provided by Dr. Ben Murray (University of California, Irvine). A cDNA encoding human L1 was kindly provided by Dr. Vance Lemmon (Case Western Reserve University). VENT polymerase was purchased from New England Biolabs (Beverly, MA). Custom-synthesized oligonucleotides were purchased from DNA International (Lake Oswego, OR). Escherichia coli strain BMH 71-18 mutS was purchased from Clontech (Palo Alto, CA). Lipofectin reagent and Geneticin were purchased from Life Technologies, Inc. Fluorescein isothiocyanate-conjugated secondary antibodies were purchased from Cappel (West Chester, PA).

Construction of Deletion and Substitution Mutations in Chicken NCAM cDNA

NaeI/NcoI digestion of pEC1401 generated a 3.4-kb fragment that contains the entire NCAM-180 coding sequence. A eukaryotic expression vector for NCAM-180 (pCMVchNCAM-180) was generated by cloning the 3.4-kb NaeI/NcoI fragment from pEC1401 into the XbaI site of the polylinker region of the eukaryotic expression vector pCMV by blunt end ligation after treatment of vector and insert with Klenow fragment. pCMV utilizes the human cytomegalovirus promoter to drive expression in a wide variety of cell types, and contains a neo gene cassette that allows for G418 selection of stable transfectants.

Polymerase chain reaction was utilized to generate deletions and substitutions of regions of the chicken NCAM cDNA. Deletions and substitution sites were placed in interdomain regions based on structural predictions and predicted domain boundaries(24, 25) . In all PCR reactions, VENT polymerase was used with pCMVchNCAM-180 as template. In 100-µl PCR reactions, 0.1 µg of template DNA, 0.1 nmol each primer, 0.8 µM dNTPs and 2 units of VENT polymerase were carried through 15 denaturation/annealing/extension cycles (95 °C, 1 min; 50 °C, 1 min; 75 °C, 1 min/kb amplified product). The sequences and relative binding positions of oligonucleotides used for all described deletions and substitutions are shown in Fig. 1. With all PCR-derived constructs, sequencing of amplified DNA was carried out using the dideoxy method (26) .


Figure 1: Oligonucleotides used for mutant chicken NCAM construction. A, names and binding positions of oligonucleotide primers to the chicken NCAM or human L1 cDNAs used in PCR reactions are designated by bent lines, with f or r suffix indicating ``forward'' (left to right) or ``reverse'' (right to left) orientation of primed DNA synthesis. Sequences encoding Ig domains (white boxes), FNIII repeats (black boxes), and intracellular domains (diagonal hatching) are indicated. The thin segment upstream of intracellular domains represent transmembrane domain-encoding regions. Very thin lines at the 5` and 3` ends of the chicken NCAM schematic represent pCMV vector sequences. B, sequences of all oligonucleotides utilized for PCR and site-directed mutagenesis. Introduced restriction sites are underlined, and mismatches for site-directed mutations are indicated by lowercase letters. Sequences which are complementary to chicken NCAM or human L1 sequences are in bold.



A pCMV plasmid directing synthesis of a mutant NCAM cDNA that lacks sequence encoding the intracellular domain was constructed by replacing the 2-kb ApaI fragment from pCMVchNCAM-180 with a 1-kb fragment generated by ApaI digestion of a PCR product that was amplified using primers 5f and 5r. Primer 5r introduces an ApaI site at the 3` end of amplified product and introduces a stop codon that terminates protein synthesis at amino acid 737, 8 amino acids carboxyl-terminal to the transmembrane domain of chicken NCAM. Correct orientation of insertion was confirmed with restriction enzyme analysis.

To construct nested deletions of regions encoding Ig domains 1-4, PCR product generated by primers 1f and 1r was cut with HindIII and XbaI and ligated into HindIII/XbaI cut pCMV plasmid. pCMV plasmid containing the 1r/1f insert was cut with XbaI and then ligated to XbaI-treated PCR products generated with primer pairs 2f/4r, 3f/4r, 4f/4r, or 5f/4r. These ligations resulted in plasmids encoding NCAM lacking Ig domain 1 (pCMVIg1), Ig domains 1-2 (pCMVIg1-2), Ig domains 1-3 (pCMVIg1-3), or Ig domains 1-4 (pCMVIg1-4), respectively. These constructs also lack the intracellular domain. The method of deletion described introduced an XbaI site at each deletion site, which resulted in the insertion of two amino acids, serine and arginine, in the encoded protein.

To construct deletions within the region of the chicken NCAM cDNA encoding the FNIII repeats, PCR product generated by primer pairs 1f/2r and 1f/3r were cut with HindIII and XbaI and ligated into the HindIII/XbaI site in the polylinker of the pCMV plasmid. pCMV plasmid containing the 1f/2r insert was cut with XbaI and then ligated to XbaI-treated PCR product generated with primer pair 6f/6r. This ligation results in a plasmid encoding NCAM lacking FNIII repeats 1-2 (FNIII#1-2). Correct orientation was confirmed by restriction enzyme analysis. To delete FNIII repeat #2 alone, XbaI-treated PCR product generated using primer pair 6f/6r was ligated into the XbaI site of pCMV containing the 1f/3r insert. Correct orientation was determined by restriction enzyme analysis. This ligation resulted in a plasmid encoding NCAM lacking FNIII repeat #2 (pCMVFNIII#2). The procedure described for constructing FNIII repeat deletions introduced an XbaI site at the site of each deletion, resulting in the insertion of two amino acids, serine and arginine, at the site of deletion in expressed protein.

cDNAs were constructed in which Ig domains or fibronectin repeats of chicken NCAM were replaced with analogous domains from the Ig superfamily adhesion molecule, human L1(27) . A fragment of the human L1 cDNA encoding L1 Ig domains 1-4 was PCR amplified with primers 7f and 7r, which introduced HindIII sites at both the 5` and 3` end of the PCR product. This product was cut with HindIII and inserted into the vector portion of HindIII-digested pCMVchNCAM,Intra (resulting in the plasmid, pCMVIg1-4Swap, Intra). Correct orientation of insert was confirmed by restriction digestion. A PCR fragment containing the region encoding Ig domains 1-5 was amplified using primers 1f and 2r. This fragment was treated with HindIII and XbaI and inserted into the polylinker region of pCMV. A PCR fragment encoding the intracellular, transmembrane, and two carboxyl-terminal FNIII repeats was amplified using primers 8f and 8r, which introduced XbaI sites at both the 5` and 3` end of the PCR product. This product was cut with XbaI and inserted into the pCMV 1f/2r construct (resulting in the plasmid, pCMV-FNIII/intra swap). The use of the primer 8f for this construct resulted in an XbaI site at the site of the transition from NCAM to L1 sequence, resulting in the insertion of two amino acids, serine and arginine, at the transition site.

An expression vector encoding chicken NCAM lacking Ig1-3 and FNIII#2 (pCMVIg1-3, FNIII#2) was generated by cloning the 900-base pair HindIII fragment from pCMVIg1-3 into the equivalent position of pCMVFNIII#2 (see above). Correct orientation of insertion was confirmed by restriction enzyme analysis.

Construction of Site-directed Asn Mutations

Targeted mutations of asparaginyl residues within the fifth immunoglobulin domain were generated in double-stranded DNA as described(28) , with slight modifications. A mutant cDNA for chicken NCAM lacking the intracellular domain in the pCMV vector (described above) was used as template for all mutagenesis reactions. Phosphorylated mutagenic primers (see Fig. 1B) were synthesized to transform codons for Asn within Ig5 into codons for Gln. A phosphorylated ``selection'' primer was designed to transform the NotI site of the pCMV polylinker into a SacII site, or in subsequent rounds of mutagenesis, the SacII site back into a NotI site (see Fig. 1). In 20 µl of solution containing Tris-HCl (20 mM, pH 7.5), MgCl (10 mM), and NaCl (50 mM), 100 ng of template was heat denatured (99 °C, 5 min) and quickly cooled to 4 °C in the presence of 100 ng of mutagenic and 100 ng of selection primer. DNA synthesis was directed from the mutagenic and selection primers by the addition of 3 µl of a solution containing Tris-HCl (100 mM, pH 7.5), dNTPs (5 mM each), ATP (10 mM), and dithiothreitol (20 mM), 3 units (1 µl) of T4 DNA polymerase, and 6 units (1 µl) of T4 DNA ligase. Extension and ligation was carried out at 37 °C for 2 h, and reaction was terminated by incubation of the reaction mix at 70 °C for 10 min. NotI (in the initial mutagenesis reaction) or KspI digests (in subsequent mutagenesis reactions) of resultant DNA was carried out after adjusting NaCl to concentrations optimal for each enzyme. Linearized DNA resulting from restriction enzyme digestion was digested with Exonuclease III for 2 h, 37 °C. The remaining circular DNA (enriched in molecules which have mismatches due to the incorporation of DNA derived from the mutagenic primers) was then used to chemically transform competent E. coli strain BMH 71-18 mutS, which is defective in mismatch repair. Transformed mutS bacteria were grown in LB + 100 µg/ml ampicillin overnight, followed by DNA miniprep. Restriction digestion and Exonuclease III treatment was repeated, and resultant DNA was transformed into chemically competent DH5 E. coli. Transformed DH5 were plated onto LB + 100 µg/ml ampicillin, and plasmids from isolated clones were analyzed for the presence of a mutated selection primer site. Plasmids with mutations at the selection primer site (determined by restriction enzyme analysis) were analyzed by DNA sequencing for mutations at targeted Asn codons within the NCAM sequence. Sequencing was carried out in the region of the site-directed mutations by double strand sequencing using the dideoxy chain termination method(26) . Double and triple Ig5 Asn mutations were derived with repeated rounds of site-directed mutagenesis.

F-11 Cell Culture and Transfection

The F11 rat dorsal root ganglion/mouse neuroblastoma (29) cell line was kindly provided by Dr. Lloyd Culp (Case Western Reserve University). Cells were grown in Dulbecco's modified Eagle's medium, 10% fetal bovine serum at 37 °C under 5% CO in 10-cm tissue culture plates. At approximately 70% confluence, cells were transfected with 10 µg of plasmid DNAs using Lipofectin. 24 h after introduction of lipidDNA complexes, medium was removed and cells were grown for 2 days in normal culture medium. Cells were then passaged 1:1, and stably transfected cells were selected using 350 µg/ml (active) geneticin. Within 7-10 days, selection was complete, and the stably transfected cell population was expanded for use in subsequent analysis.

Antibodies

Monoclonal antibodies directed against localized chicken-specific epitopes (30, 31) were utilized for indirect immunofluorescent staining of cells and for the immunopurification and concentration of chicken NCAM from cellular extracts. Monoclonal antibody 5e, which binds to the Ig region of NCAM was used for constructs lacking FN repeats and/or the intracellular domain. Monoclonal antibody 105, which binds to the FNIII repeat region of NCAM, was utilized when expressed chicken polypeptides lacked one or more Ig domain. Monoclonal antibody 4d, which binds specifically to the intracellular domain of NCAM-180, was used to purify NCAM molecules lacking both Ig domains and FNIII repeats. Chicken NCAM was detected on Western blots using polyclonal rabbit antibodies against affinity-purified chicken NCAM.

Immunofluorescence Staining of Chicken NCAM

F11 cells were assessed for cell surface expression of various constructs using indirect immunofluorescence staining of live cells. Cells growing on tissue culture plastic were washed with Dulbecco's modified Eagle's medium containing HEPES (15 mM) and 10% normal goat serum, then incubated for 30 min at room temperature with 5 µg/ml monoclonal antibody 5e or monoclonal antibody 105. Cells were washed three times with Dulbecco's modified Eagle's medium, 10% normal goat serum, then incubated for 30 min at room temperature with 100 µg/ml fluorescein isothiocyanate-conjugated goat anti-mouse IgG. Cells were washed as above, and fluorescence staining was visualized and photographed using a Nikon Optiphot fluorescence microscope.

Imunoadsorbtion and PSA Removal

Chicken NCAM expressed by F11 cells was solubilized from cell membranes using Nonidet P-40 extraction buffer (150 mM NaCl, 50 mM HEPES, 1 mM EDTA, 100 KIU/ml aprotinin, 2 mM phenylmethylsulfonyl fluoride, 1% Nonidet P-40, pH 7.4), and purified by immunoaffinity adsorption to Sepharose-4B beads (Pharmacia) derivitized with monoclonal antibodies specific to chicken NCAM. Immunoadsorbtion was carried out overnight with rotation at 4 °C. After washing with PBS + aprotinin, PSA was removed from adsorbed proteins by treatment with a purified phage endoneuraminidase (Endo N) (32) that specifically cleaves -2,8-polysialic acid. 0.5 µl of purified Endo N was added to 250 µl of bead slurry (in PBS + aprotinin, pH 7.4), then incubated 4-6 h at 4 °C.

Electrophoresis and Immunoblotting

Equivalent amounts of Endo N-treated and -untreated proteins isolated by immunoadsorbtion were separated by 6%-7.5% SDS-PAGE(33) . Affinity resin slurry with bound chicken NCAM was divided into two equal volume aliquots. The beads were then spun down, and overlying PBS/aprotinin was removed. Immunoadsorbed protein was released from beads by denaturation with Laemmli sample buffer and loaded directly into gel sample wells. After electrophoresis, proteins were electrotransferred to nitrocellulose and detected by immunoblotting(34, 35) . Nonspecific binding sites were blocked with Blotto (36) for 30 min at 37 °C. Blots were then incubated with 2 µg/ml anti-chicken NCAM polyclonal antibody in PBS, 0.1% Tween-20 (PBS-T) for 1 h at room temperature. After washing with PBS-T, blots were incubated with 0.2 µg/ml horseradish peroxidase-conjugated goat anti-rabbit IgG (Cappel) for 1 h at room temperature. Filters were washed thoroughly and detection of antigen carried out using enhanced chemiluminescence (ECL Kit; Amersham Corp.). After addition of peroxidase substrate, luminescent blots were exposed to autoradiographic film (Hyperfilm-ECL, Amersham) for between 30 s and 10 min.

Modeling of the Extracellular Domains of NCAM

Sequence alignments based on NCAM Ig domains having an ``I'' domain topology (37) were carried out using procedures previously described for carcinoembryonic antigen(38) . The first five Ig-type folds of NCAM were compared to and modeled after four x-ray crystal structure templates that are based on the variable (V) domain topology. The two FNIII repeats of chicken NCAM were structurally modeled using the third FNIII repeat of tenascin (39) as the single template for modeling.

The seven extracellular domains of NCAM (five Ig domains and two FNIII repeats) were packed together by manual manipulations. The packing was designed to avoid steric clashes and to allow for formation of potentially energetically favorable interactions, such as salt bridges, that have been reported between other Ig folds(40, 41) . The predicted length of linker domains was indicated by sequence alignment (above), the energetic stability of each domain, and the predicted docking of each domain.

A PSA polymer consisting of eight N-acetylneuraminic acid monomers was modeled into a G1+ (helical) conformation, which is one of the more populated conformations of PSA(42) . The core N-linked glycosylation structure was modeled after a similar conformation to carbohydrate found within the crystal structure of an immunoglobulin Fc fragment(40) .


RESULTS

Assay for Polysialylation of Mutated NCAM Constructs

This assay allowed a qualitative assessment of the polysialylation state of specifically mutated chicken NCAM molecules in a rodent cell line (Fig. 2). After chicken NCAM constructs were expressed in the rodent cells, species-specific monoclonal antibodies were used to immunopurify the expressed chicken protein. A portion of the purified NCAM was then treated with the PSA-specific Endo N, which only attacks -2,8-linked polymers of 8 residues or more, leaving 4-5 residues attached to the core carbohydrate(20, 32, 43) . Equivalent amounts of the Endo N-treated and -untreated NCAM were subjected to gel electrophoresis in SDS and detected by immunoblotting with polyclonal anti-NCAM. Previous studies have shown that electrophoretic mobility in SDS provides a qualitative measure of the degree of polysialylation (44) . Such an Endo N-sensitive decrease in mobility could in principle reflect either an increase in the polymer length of existing chains or an increase in the number of chains/molecule.


Figure 2: Assay for determination of polysialyation of chicken NCAM expressed in F11 cells. Cells from stably transfected cell populations expressing various chicken NCAM constructs were subjected to detergent lysis, and chicken NCAM was immunopurified and concentrated. Endo N-treated and -untreated samples were subjected to SDS-PAGE and Western blotting. In this analysis, polysialylation of chicken NCAM-180 is indicated by the loss of a polydisperse region of immunostaining (-Endo N lane, bracket) and a corresponding increase in the amount of material in higher mobility bands that represent desialylated material (+Endo N lane, arrow).



In this assay, it is necessary that the recipient cell express the glycosylation components for polysialylation of NCAM. In addition, it is essential that rodent polysialylation enzymes be capable of recognizing and modifying chicken NCAM. Many commonly used recipient cell lines (such as 3T3 and L-cells as well as PC12 and N2A neuroblastomas) are not capable of adding significant amounts of PSA to NCAM, and are therefore not useful in our approach. However, the F11 hybrid cell line (mouse neuroblastoma/rat dorsal root ganglion neuron) (5, 29) does possess a functional apparatus for polysialylation, as the NCAM expressed in these cells exhibits the lower electrophoretic mobility associated with PSA, and these cells express PSA-specific antigens on their surface(5) . They also have desirable characteristics in culture (good survival, rapid division, and loose attachment to tissue culture plastic) for transfection and expansion of stable transfectants. However, it should be noted that the amount of PSA on F11 cells, as with other PSA-expressing cell lines(5, 15) , is less than the maximal levels observed in vivo, as for example in embryonic brain(44) . As is shown in Fig. 2, chicken NCAM-180 expressed in F11 cells is polysialylated, as indicated by a shift in its electrophoretic mobility from a smear to a more condensed band after Endo N treatment. Moreover, the degree of polysialylation obtained was similar to that observed with endogenous F11 cell NCAM (not shown). Thus all essential components required for polysialylation are shared between chicken and rodent NCAM.

Another important control in the use of transfected constructs involving large deletions is to determine whether the protein is transported to the cell surface. Therefore each of the wide variety of mutations generated for this study were tested for surface expression by immunocytochemistry using the appropriate anti-chicken NCAM antibody. In all cases surface expression was obtained, as shown for representative mutations in Fig. 3.


Figure 3: Confirmation of cell surface expression of mutant polypeptides. Stably transfected F11 cell populations were stained live using monoclonal antibodies 105 or 5e, which bind specifically to chicken NCAM. mAb 5e binds to the Ig region of chicken NCAM and was used to stain chicken NCAM constructs with FNIII domain alterations. mAb105 binds in the FNIII region of chicken NCAM, and was used to stain constructs with Ig domain alterations. Staining with fluorescein isothiocyanate-conjugated secondary Ab revealed cell surface expression of all deletion mutation and substitution polypeptides, a representative sample of which is shown here. No staining was observed with non-transfected cells. As transfected cells were not clonally derived, varying levels of expression within the population were observed.



PSA Addition Does Not Require the NCAM Intracellular Domain

Chicken NCAM protein that lacks all but the amino-terminal 8 amino acids of the intracellular domain was found to be polysialylated in F11 cells, as indicated by the reduction with Endo N treatment of a broad smear in SDS-PAGE to a more focused band of approximately 125 kDa (Fig. 4). Polysialylation of NCAM lacking the intracellular domain (Intra) demonstrated that the intracellular domain does not play an essential role in the polysialylation mechanism. This result was not unexpected, as NCAM linked to the membrane via a PI linkage can be polysialylated(45, 46) . In subsequent studies of extracellular domains, the Intra mutation was often combined with these mutations, in that changes in polysialylation were more easily detected when attached to a smaller polypeptide.


Figure 4: Identification of polysialylated Asn residues. Mutant chicken NCAM polypeptides were prepared and analyzed as described under ``Experimental Procedures.'' Aliquots of each mutant polypeptide were treated with Endo N to remove -2,8-polsialic acid. Equivalent amounts of treated (+E-N) and untreated material were run on 7% SDS-PAGE and were detected using polyclonal antibody specific to chicken NCAM. Polysialylation is indicated by an Endo N-induced shift in electrophoretic mobility. Intra lacks the intracellular domain, but retains an intact extracellular domain. Constructs containing site-directed mutations at Asn positions in Ig5 (denoted mAsn) contain Asn to Gln mutations at indicated residues.



Restriction of PSA to the Ig5 Domain and Localization of Specific Polysialylated Asparaginyl Residues

Potentially polysialylated Asn residues within NCAM Ig5 are located at positions 404, 430, and 459 in the chicken NCAM primary sequence. When all 3 Asn residues within Ig5 were mutated to Gln within the Intra mutation, the resulting construct (mAsn-(404,430,459)) was no longer capable of being polysialylated in the F11 cells (Fig. 4). This result confirms previous reports that PSA is associated with Ig5 (1, 23) and also establishes that these residues are the sole sites of detectable polysialylation.

Paired mutations of these 3 Asn residues, again to Gln and carried out on Intra NCAM cDNA, were also evaluated for polysialylation (Fig. 4). Protein retaining only Asn residue 459 (mAsn-404,430) was found to have a degree of polysialylation similar to that of the Intra construct containing no Asn mutations. A lesser but significant degree of polysialylation was detected on the mutant protein containing only Asn-430 (mAsn-404,459). In contrast, polysialylation of NCAM retaining only Asn residue 404 (mAsn-430,459) was barely detectable.

Effects of Ig and FNIII Domain Deletions and Substitutions on Polysialylation

In this study, a set of chicken NCAM expression vectors were constructed in which alterations (both large scale deletions and substitutions) were introduced in the Ig domain and FNIII repeat regions. In preparing these constructs, breakpoints in the cDNA sequences of deletional and substitutional mutations were designed to involve interdomain sequences, so as to mimimize the possibility of altered polysialylation due to conformational distortion of Ig5.

A series of cDNAs containing deletions and substututions in the Ig domain region were constructed using the Intra chicken NCAM cDNA (Fig. 5A). When expressed in F11 cells and analyzed for the addition of polysialic acid (Fig. 5A), constructs lacking Ig domain 1 (Ig1) and Ig domains 1-3 (Ig1-3) were found to be polysialyated to a degree similar to that observed with the Intra molecule. In contrast, NCAM lacking Ig domains 1-4 (Ig1-4) was found not to have detectable polysialic acid, suggesting an involvement of Ig4 in NCAM polysialylation. In order to address the nature of the requirement for Ig4, we assessed the polysialylation of a chimeric construct in which Ig domains 1-4 of NCAM were replaced by Ig domains 1-4 of a structurally related adhesion molecule, human L1(27) . This chimeric protein was found to contain low but detectable amounts of PSA in F11 cells, suggesting that L1 Ig1-4 can to some extent substitute for NCAM Ig4.


Figure 5: Analysis of polysialylation of polypeptides with domain deletions and substitutions. Electrophoretic assessment of polysialyation state (see ``Experimental Procedures'') was carried out on chicken NCAM proteins with alterations in the Ig domain region (A), FNIII repeat region (B), or with a combined Ig1-3 + FNIII#2 deletion (C). Schematic representations of mutations are shown to the left. Polysialylation was detected by a characteristic shift in electrophoretic mobility after Endo N treatment (+E-N lanes).



Molecules containing alterations in the fibronectin repeat region, which for technical reasons were constructed with an intact intracellular domain, were also produced in F11 cells and analyzed for the presence of polysialic acid (Fig. 5B). NCAM lacking both fibronectin repeats (FNIII#1-2) did not contain detectable PSA, whereas NCAM lacking only the more carboxyl-terminal FNIII repeat (FNIII#2) was fully polysialylated. Thus it would appear that FNIII#1 is involved in the addition of PSA. Unlike the situation with the Ig4 domain, replacement of the FNIII#1 domain with an FNIII domain from human L1 (FN, intra swap) did not restore any PSA to the polypeptide. Thus the dependence of polysialylation on FNIII#1 appeared to be more direct than for Ig4.

As a complement to the above results, a construct containing only Ig domains 4 and 5, plus the FNIII#1 domain and a transmembrane segment, was tested. As would be predicted, this small polypeptide contained substantial amounts of PSA (Fig. 5C). Thus these three domains (along with a possible requirement for membrane attachment) are not only necessary, but also sufficient for polysialylation.

Structural Modeling of the Extracellular Domains of Chicken NCAM and Placement of Potential N-Linked Sites within Ig5

The predicted structural environment of the domains that we have implicated in NCAM polysialylation was explored using structural modeling of the entire extracellular domain of chicken NCAM. The amino acid sequence of the five Ig folds of NCAM were compared with V-type domains of TLK (telokin)(52) , CD4(2)(47) , CD4(1)(47) , and REI (Bence-Jones dimer VL kappa)(48) . CD4(1) and REI are prototypical V domains while CD4(2) and TLK (telokin) can be considered truncated V domains. CD4(2) is lacking a D strand and is termed a ``C2'' domain(49) , while telokin is lacking the C` and C`` strands and is termed an ``I'' domain(37) . The Ig folds of NCAM appear to be more closely related to an I domain topology as is exemplified by the structure of telokin (Fig. 6A). However, all of the V domain templates were used in model building as this facilitated data base searching for fragment replacements.


Figure 6: Structural modeling of NCAM extracellular domains. Relevant structural details are revealed by sequence alignments and three-dimensional structural analysis. A, multiple sequence alignments of the five NCAM Ig domains with the x-ray crystal templates CD4(1), CD4(2), TLK, and REI, as well as of the two FNIII repeats of NCAM with the single FNIII repeat template, TEN. Sequence identities that helped to align the sequences are shown in bold; asterisks denote residues greater than 95% buried. A conserved salt bridge between the bottom of strand D and the E to F loop for each of the NCAM folds is shown. B, predicted spatial proximity of NCAM extracellular domains and predicted relative orientations of N-linked glycosylations of Ig5. Arrows mark extended spacer regions found between the first three Ig folds and FNIII repeats. PSA chains are depicted on Asn residues 430 and 459 (10 residue polymers are shown; dots represent the full extent of the chain). A core glycosylation with no PSA is shown on Asn residue 404 (oriented into the page). The diagram was produced with the aid of the display program, MOLSCRIPT(50) .



With steric and energetic considerations taken into account, a structural model of the relationships between extracellular domains of NCAM was manually conformed with the aid of computer graphics. While such methodology cannot be considered to be of extremely high accuracy (domain boundaries can show different degrees of flexibility depending on such variables as protein-protein interactions, etc.), the length of linker regions between domains is likely to be a significant determinant in the ability of adjacent domains to interact cooperatively in binding events. As is shown in Fig. 6B, the length of the spacer regions apparent between the first three Ig folds and between the two FNIII repeats are significantly longer than those found between Ig4, Ig5, and FNIII#1.

The structural model of Ig5 allows prediction of the distribution and accessibility of potential N-glycosylation sites (Fig. 6B). In our model based on I domain structure, core glycosylation of the three sites project as equally spaced ``spokes on a wheel,'' with the linear arrangement of protein domains being the axle.


DISCUSSION

The addition of PSA to NCAM involves two levels of specificity, the selection of NCAM in the vertebrate embryo as a unique target for this unusual glycosylation, and the association of the PSA polymer with particular N-glycosylation sites within the NCAM polypeptide. In the present study, we have identified the precise sites of NCAM polysialylation and have defined a subregion of the NCAM polypeptide that contains all required recognition elements necessary for this glycosylation in cells. This analysis utilized a novel assay in which the glycosylation apparatus of a PSA-expressing cell line is used to process a variety of NCAM constructs containing precise mutations, deletions, and substitutions. The findings extend the current understanding of the topology of PSA addition and provide a basis for study of the specific recognition of NCAM as a target for polysialylation.

Previous studies have identified the Ig5 domain as a site for polysialylation and noted that this domain contains 3 potential residues for N-glycosylation(1, 23) . The approach used, however, could not determine which residues were actually substituted and whether these were the only sites for addition of PSA. The present studies establish that, in the F11 cell line, detectable PSA is associated with Asn residues 430 and 459 of Ig5. The possibility remains that the apparent low level or absence of polysialylation at Asn residue 404 was caused by a disruption of normal protein folding from the combined mutations (to Gln) at Asn-430 and Asn-459. However, the combination of either of these mutations with a mutation at Asn-404 did not prevent NCAM polysialylation.

In this study, we have also implicated regions outside Ig5 in the targeting of NCAM for polysialylation. Deletional analyses presented here demonstrate that all structural requirements for recognition and polysialylation of NCAM are localized to Ig5 and its two adjacent domains, Ig4 and FNIII#1. It is possible that all specific recognition of NCAM resides within Ig5 and that the flanking domains only help Ig5 to establish or stabilize its conformation. The independent and compact folding of most Ig domains, and the care taken in targeting deletion sites to polypeptide regions between domains, would argue against this being the major mechanism involved. Moreover, substitution of NCAM FNIII#1 with an FNIII domain of the non-polysialylated adhesion molecule L1 did not allow detectable addition of PSA, and a similar substitution of NCAM Ig4 with an L1 Ig domain produced only a low level polysialylation. That the Ig1-4 substitution did allow some glycosylation, however, suggests that some stabilization of Ig5 by an adjacent Ig domain might promote optimal polysialylation. Alternatively, some specific recognition determinants could be shared by the two Ig domains. While this possibility is difficult to rule out, it seems unlikely that L1, in not being polysialylated, would carry such a specific recognition structure. Moreover, the low degree of sequence homology between these two domains (<25%) suggests that their similarity probably does not extend much beyond the basic structure of an Ig domain (see Fig. 6A).

The recent identification of a vertebrate -2,8-polysialyltransferase as a type 2 membrane protein (51) suggests that its catalytic site may be localized at a certain distance from the membrane. In such a case, the FNIII#1-2 deletion might prevent polysialylation simply by decreasing the distance of Ig5 from the membrane. However, the absence of polysialylation of the FNIII swap protein argues that the dependence of polysialylation on FNIII#1 is not a simple steric effect. Nevertheless, it remains possible that appropriate spacing from the membrane may be a factor in the recognition process. Interestingly, both the polysialyltransferase enzyme and the extracellular region of NCAM extending from the transmembrane region through Ig4 contain approximately 400 amino acids. Thus, assuming a linear domain structure for the enzyme similar to that of NCAM, it is feasible this enzyme could interact with each of the domains constituting the NCAM recognition and polysialylation sites described in this study. However, if spacing is a necessary requirement for polysialylation, it appears there is at least some flexibility in the exact length of the spacing, as the FNIII#2 protein was polysialylated.

Having refined the location of PSA addition and established structural requirements for this glycosylation, one can begin to speculate on the processes by which this specificity is achieved. The possibility that the 2nd and 3rd Asn residues are sterically favored for polysialylation can be considered in terms of the positioning of these residues within the Ig5 domain structure. Structural predictions for NCAM Ig5 based on the conformation of Ig I-type domains suggests that the core glycosylations on the 3 Asn residues in Ig5 are arranged like spokes on a wheel, with the linear arrangement of protein domains as an axle (Fig. 6B). In this arrangement the large, flexible carbohydrate cores extending from Asn-459 and Asn-430 could easily be accessible to (and perhaps favored for) recognition by the same glycosylation apparatus.

This structural analysis also reveals an interesting spatial relationship between domains that are involved in NCAM polysialylation. By our analysis, Ig5 and the two proximal domains (Ig4 and FNIII#1) form what appears to be a relatively compact unit (Fig. 6B). The proximity of these domains is due to the absence of the extended spacer regions that are found between the first three Ig folds and between the two FNIII repeats in the NCAM molecule. The close arrangement of these three domains makes it more feasible that they may contribute to a spatially discrete recognition site for polysialylation.

Given these structural observations, it is inviting to propose that the mechanism by which PSA is restricted to 2 Asn residues within Ig5 of NCAM is coupled with or the same as the mechanism by which NCAM itself is recognized for polysialylation. A schematic representation that links these two levels of regulation of polysialic acid is shown in Fig. 7. Simply stated, Ig5 and its two proximal domains cooperate to form a structure that is recognized by polysialyltransferase enzyme(s). Within this multidomain structure, the tertiary structure of Ig5 spatially sequesters two N-linked cores and preferentially presents them to the catalytic site of polysialyltransferase enzyme(s), probably including the recently identified -2,8-polysialyltransferase enzyme(51) . Now that a putative polysialyltransferase for NCAM has been identified, the logical next steps will be to test for a direct enzyme-substrate affinity and then to use a more refined mutational analysis to define the precise nature of the enzyme-NCAM association.


Figure 7: Schematic model of two levels of polysialic acid regulation. Hypothetical model in which structural determinants of Ig5, as well as Ig4 and FNIII#1, contribute to recognition of NCAM by a polysialylation enzyme apparatus. Steric constraints of this recognition complex could restrict enzymatic activity of the polysialylation enzyme to core glycosylation sites on Asn residues 430 and 459 (triangles).




FOOTNOTES

*
This work was supported in part by National Institutes of Health Grants HD18369 and EY06107. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore by hereby marked ``advertisement'' in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

§
Predoctoral fellow on NIH Training Grant HD07104.

To whom correspondence should be addressed: Dept. of Genetics, School of Medicine, Case Western Reserve University, 10900 Euclid Ave., Cleveland, OH 44106-4955. Tel.: 216-368-2428; Fax: 216-368-3182; uxr{at}po.cwru.edu

The abbreviations used are: NCAM, neural cell adhesion molecule; NCAM-180, the polypeptide isoform of NCAM having the largest intracellular domain; PSA, polysialic acid; Endo N, (endoneuraminidase-N), an -2,8-polysialic acid-specific neuraminidase; PAGE, polyacrylamide gel electrophoresis; kb, kilobase(s); PCR, polymerase chain reaction; FN, fibronectin.


ACKNOWLEDGEMENTS

We thank Denice Major for excellent technical assistance.


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