(Received for publication, April 4, 1995)
From the
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.
Neural cell adhesion molecule (NCAM)
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.
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.
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 (pCMV
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 (
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,
An expression vector encoding
chicken NCAM lacking Ig1-3 and FNIII#2 (pCMV
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) .
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).
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.
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
Paired mutations of
these 3 Asn residues, again to Gln and carried out on
A series of cDNAs containing
deletions and substututions in the Ig domain region were constructed
using the
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
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.
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) .
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.
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
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
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).
We thank Denice Major for excellent technical
assistance.
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
FOOTNOTES
ACKNOWLEDGEMENTS
REFERENCES
(
)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.
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.
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.
Ig1), Ig domains 1-2
(pCMV
Ig1-2), Ig domains 1-3 (pCMV
Ig1-3), or
Ig domains 1-4 (pCMV
Ig1-4), 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.
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 (pCMV
FNIII#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.
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.
Ig1-3,
FNIII#2) was generated by cloning the 900-base pair HindIII
fragment from pCMV
Ig1-3 into the equivalent position of
pCMV
FNIII#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 lipid
DNA 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.
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.
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.
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.
-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.
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.
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.
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.
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.
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.
-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.
-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.
-2,8-polysialic acid-specific neuraminidase; PAGE, polyacrylamide
gel electrophoresis; kb, kilobase(s); PCR, polymerase chain reaction;
FN, fibronectin.
©1995 by The American Society for Biochemistry and Molecular Biology, Inc.