(Received for publication, June 6, 1996, and in revised form, December 26, 1996)
From the Protein Laboratory, To study the function of the first immunoglobulin
(Ig)-like domain of the neural cell adhesion molecule (NCAM), it was
produced as a recombinant fusion protein in a bacterial expression
system and as a recombinant protein in a eukaryotic expression system of the yeast Pichia pastoris. For comparison, other NCAM
domains were also produced as fusion proteins. By means of surface
plasmon resonance analysis, it was shown that the first Ig-like NCAM
domain binds the second Ig-like NCAM domain with a dissociation
constant 5.5 ± 1.6 × 10 The neural cell adhesion molecule
(NCAM)1 is a cell surface glycoprotein
belonging to the immunoglobulin (Ig) superfamily (1, 2). It consists of
five Ig-like domains and two fibronectin type III repeats (3, 4). The
structure of the first Ig-like domain (IgI) belongs to the
intermediate set of Ig-like domains (5). NCAM is expressed as three
major isoforms: two transmembrane isoforms NCAM-A (180 kDa) and NCAM-B
(140 kDa), and a glycosylphoshatidylinositol-anchored NCAM-C (120-kDa)
isoform.
NCAM is known to mediate cell-cell interactions by a
Ca2+-independent homophilic binding mechanism (6-8). The
mechanism of this binding is not clear. It has been reported that a
sequence of 10 amino acids in the IgIII domain of chick NCAM is
involved in the homophilic binding (9, 10). In another study, it was demonstrated that all five Ig-like domains of chick NCAM were involved
in this binding (11). However, analysis of the structure of the IgI
domain, determined by NMR spectroscopy, and the predicted structure of
the IgII domain of mouse NCAM suggests that an acidic cluster of
residues at the NH2-terminal of the IgI domain (aspartic acid 29, 32, 34, 58, 59, 84; glutamic acid 85) and a basic cluster of
residues of the IgII domain (lysine 135, 137, 185; arginine 139, 171)
may be involved in a symmetrical double-reciprocal binding (5).
NCAM also mediates cell-substratum interactions. Different types of
collagen have been shown to bind NCAM (12), but a collagen binding site
in NCAM has not been identified. NCAM also binds heparin/heparan
sulfate (13-15) and chondroitin sulfate proteoglycans (15, 16). The
heparin binding domain was initially localized to a 25-kDa
NH2-terminal fragment of NCAM (17) and, later, mapped to a
17-amino acid long region of the IgII domain (18).
The IgI domain of NCAM has been produced as a recombinant polypeptide
in a bacterial expression system (19). When used as a substratum, this
construct promoted adhesion of neuronal cell bodies, modified the
migration pattern of cells from cerebellar microexplants, and
increased metabolism of inositol phosphates and intracellular
concentrations of Ca2+ and pH, thus indicating a functional
importance of this domain (19).
The limited data concerning binding properties of the IgI NCAM domain
have prompted us to investigate further the role of this domain in
NCAM-mediated binding interactions. We therefore produced the IgI NCAM
domain as a recombinant fusion protein in a bacterial expression system
and as a recombinant protein in a eukaryotic expression system of the
yeast Pichia pastoris. For comparison, other NCAM domains
were also produced as fusion proteins. Both the IgI and IgII domains
inhibited aggregation of cerebellar neurons, whereas other Ig-like
domains of NCAM did not, indicating that the IgI and IgII domains were
functionally active. Using surface plasmon resonance analysis, we show
that the IgI domain binds the immobilized IgII domain and, vice
versa, the IgII domain binds the immobilized IgI domain. We also
show that IgI and IgII domains bind heparin and, via heparin, collagen
type I but not collagen type I directly.
Production of Fusion Proteins of NCAM Fragments in a Bacterial
Expression System
Six fusion proteins were
produced using mouse NCAM cDNA encoding the NCAM-C 120-kDa isoform
(20) kindly provided by Dr. C. Goridis (Laboratoire de Genetique et
Physiologie du Development, CNRS-Universite Aix-Marseille-II, Luminy)
or the NCAM-B 140-kDa isoform (21) kindly provided by Dr. W. Wille
(Institut für Genetik der Universität zu Köln). The
fusion proteins consisted of an NCAM fragment fused between
Escherichia coli maltose-binding protein (MBP) and an
NH2-terminal fragment (65 amino acids) of E. coli E.
coli cells propagating the recombinant and control pMAL-c plasmids
were grown in Terrific Broth (Sigma) containing 50 µg/ml ampicillin
at 37 °C. Cells were induced for 2 h with 1 mM
isopropyl- An amylose resin column (New England Biolabs) was equilibrated with
column buffer 1 (10 mM sodium phosphate, pH 7.4, 0.5 M NaCl, 2 mM EDTA). The supernatant was diluted
to 2-3 mg/ml total protein with column buffer 1 and loaded onto the
column. The column was first washed with column buffer 1 until
A280 was less than 0.03 and then with 3 column
volumes of column buffer 2 (10 mM sodium phosphate, pH 7.4, 100 mM NaCl). Finally, the fusion protein was eluted with
column buffer 2 containing 10 mM maltose. The eluted
protein was concentrated to 5-10 mg/ml and stored frozen at
Production of the IgI NCAM Domain in the P. pastoris Yeast
Expression System
The IgI domain of
NCAM was also produced as a recombinant protein (without MBP as a
fusion partner) in P. pastoris. The cDNA fragment was
synthesized by polymerase chain reaction. The amplified cDNA was
subcloned into an XboI/BamHI site of the
pHIL-S1 plasmid (Invitrogen Corporation, San Diego). An E. coli strain Top 10 F Cells
were grown in 50 ml of medium A (13.4 g of yeast nitrogen base without
amino acids (Difco, Detroit), 2 ml of 0.02% biotin, 20 g of
glycerol, 40 mg of tryptophan, 50 mg of glutamic acid, 50 mg of
methionine, 50 mg of lysine, 50 mg of leucine, 50 mg of
isoleucine/liter of 100 mM potassium phosphate, pH 6.0) at 30 °C until saturation (A600 Production of Antibodies
Polyclonal rabbit antibodies were raised against IgI-MBP.
Rabbits were immunized as described (23).
Immunoblotting
Proteins, separated on 7.5% (w/v) polyacrylamide gels (24),
were electroblotted onto a polyvinylidene difluoride membrane (Millipore, Bedford, MA). Nonspecific binding was blocked by incubation with 2% Tween 20 in washing buffer (50 mM Tris-HCl, pH
10.2, 450 mM NaCl, 0.2 mM phenylmethylsulfonyl
fluoride). After washing, the membrane was incubated overnight on a
shaking platform with either a rabbit antiserum against IgI-MBP diluted
1:200 or with polyclonal rabbit antibodies against rat NCAM (25)
diluted 1:2,000. Alkaline phosphatase-conjugated swine anti-rabbit Igs
(Dakopatts, Denmark) were used as secondary antibodies in a dilution of
1:2,000. After washing, the membrane was incubated in a staining buffer consisting of 0.1 M ethanolamine HCl, pH 9.6, 0.1 mM nitroblue tetrazolium, 0.15 mM
5-bromo-4-chloro-3-indolyl phosphate, 2 mM MgCl2, 1% methanol, 0.5% acetone.
Assay for Aggregation of Cerebellar Neurons
Mixed primary microwell cultures of dissociated mouse cerebellum
were prepared as described previously (26). Briefly, a suspension of
dissociated cells from cerebella of 6-day-old mice was plated onto an
uncoated 60-well microtiter plate (Nunc, Denmark) at a density of
45,000 cells/well in a volume of 10 µl. Cells were grown for 24 h in a culture medium consisting of Dulbecco's modified Eagle's
medium supplemented with 19 mM KCl, 25 mM
glucose, 0.2 mM glutamine, 0.1 international unit/liter
insulin, 0.15 mM p-aminobenzoate, 100 international units/ml penicillin, and 10% (v/v) fetal calf serum.
Various protein constructs were added to the culture medium before
plating at the indicated concentrations. The number of aggregates,
defined as clusters of more than 50 cells, was determined after 24 h of incubation.
Surface Plasmon Resonance Analysis
Real time biomolecular interaction analysis was performed using
a BIAlite instrument (Pharmacia Biosensor AB, Sweden). All of the
experiments were performed at 25 °C using Hepes-buffered saline (10 mM Hepes, pH 7.4, 150 mM NaCl, 3.4 mM EDTA, 0.005% v/v Surfactant P20 (Pharmacia Biosensor))
as a running buffer. The flow rate was, unless otherwise specified, 5 µl/min.
Collagen type I, purified
from rat tails (27), was immobilized on a sensor chip CM5 (Pharmacia
Biosensor) using the following procedure. The chip was activated by 10 µl of 0.05 M N-hydroxysuccinimide, 0.2 M
N-ethyl-N Heparin was bound to collagen type I immobilized on a CM5
sensor chip using 50 µl of 10 mg/ml heparin from porcine intestinal mucosa (Sigma) dissolved in MilliQ water. Subsequently, 65 µl of a
fusion protein at 100 µg/ml concentration was applied. In a
competition experiment, IgI-MBP or IgII-MBP at 100 µg/ml
concentration was preincubated with 100 µg/ml heparin for 15 min. The
chip was regenerated by four 5-µl pulses of 50 mM
NaOH.
Heparin was biotinylated by mixing 1 mg of heparin/ml
with 0.75 mM biotinamidocaproate
N-hydroxysuccinimide ester (Sigma), 0.1 M sodium
borate buffer, pH 8.8, for 1 h at room temperature. Biotinylated
heparin was separated from the free biotinylation reagent by gel
filtration using a Sephadex G-25 M column (Pharmacia). Heparin was
immobilized injecting 25 µl of 10 µg/ml biotinylated heparin into
an SA5 sensor chip (Pharmacia Biosensor) with preimmobilized streptavidin. Subsequently, 60 µl of IgI P. pastoris at a
flow rate of 30 µl/min or 70 µl of a fusion protein at a flow rate of 5 µl/min was applied using the indicated concentrations. In a
competition experiment, IgI-MBP or IgII-MBP at a 100 µg/ml
concentration was preincubated with either 500 µg/ml heparin or
polysialic acid (PSA) (Sigma) for 15 min, and IgI P. pastoris at a concentration of 100 µg/ml was preincubated with
200 µg/ml heparin for 5 min. Finally the chip was regenerated by
three 5-µl pulses of 50 mM NaOH.
IgI-MBP and IgII-MBP were biotinylated by mixing 1 mg of
fusion protein/ml with 0.05 mM biotinamidocaproate
N-hydroxysuccinimide ester in Hepes-buffered saline for
1 h at 4 °C. The biotinylated fusion protein was separated from
the free biotinylation reagent by gel filtration using a Sephadex G-25
M column. The biotinylated IgI-MBP or IgII-MBP was immobilized
injecting 100 µl of 10 µg/ml biotinylated fusion protein into an
SA5 sensor chip at a flow rate of 2 µl/min. Subsequently, different
fusion proteins of NCAM fragments in Hepes-buffered saline containing
2.2 mM maltose were injected into a sensor chip with either
immobilized IgI-MBP (70 µl injection) or IgII-MBP (50 µl injection)
at the indicated concentrations. Finally, the chip was regenerated by
three 5-µl pulses of 5 mM NaOH.
Data were analyzed by nonlinear curve fitting using the BIAevaluation
Software Set (Pharmacia Biosensor). The dissociation rate constant
(kd) was calculated by fitting
dissociation data to Equation 1, using
kd as a floating parameter
5 M.
Furthermore, it was found that the first Ig-like domain binds heparin.
It was also demonstrated that the second Ig-like NCAM domain binds
heparin and that both domains bind collagen type I via heparin but not
collagen type I directly.
-galactosidase. The cDNA for the IgI domain was
excised from NCAM-C cDNA with the PstI and
HincII restriction enzymes, and the cDNA fragments for
NCAM domains IgII, IgIII, IgIV (including exon VASE), IgV, and the
intracellular part of NCAM-B (Ex16,17,19) were synthesized by the
polymerase chain reaction. The cDNA fragments were subcloned into a
pMAL-c expression vector (New England Biolabs). The template for
polymerase chain reaction amplification of the cDNA for Ex16,17,19
was the NCAM-B cDNA. For the other constructs the template was the
NCAM-C cDNA. An XL1-Blue strain of E. coli (Stratagene)
was used for the transformation with the recombinant and control pMAL-c
plasmids. The recombinant clones were identified by small scale
expression of the fusion proteins. Production of a fusion protein
containing NCAM fibronectin type III domains I and II has been
described previously (22). The fusion proteins were designated
Ign-MBP (Ig-like domain number n produced in
E. coli as a fusion protein with MBP as a fusion partner);
Ex16,17,19-MBP (polypeptide encoded by NCAM exons 16, 17, and 19 produced in E. coli as a fusion protein with MBP as a fusion
partner); and F3I,II-MBP (fibronectin type III domains I and II
produced in E. coli as a fusion protein with MBP as a fusion
partner). For IgI-MBP, IgII-MBP, and IgIII-MBP, the recombinant
pMAL-c plasmids were sequenced. IgII-MBP E. coli did not
contain the
-galactosidase fragment because insertion of the IgII
cDNA into the pMAL-c plasmid resulted in a shift of the reading
frame terminating the
-galactosidase after seven amino acids.
-D-thiogalactopyranoside and thereafter
harvested by centrifugation and resuspended in 10 mM sodium
phosphate, pH 7.4, 30 mM NaCl, 10 mM EDTA.
Cells were subsequently frozen at
20 °C and thawed at room
temperature. The sample was sonicated intermittently for 2 min on ice
and centrifuged at 27,000 × g for 30 min at 4 °C.
The supernatant was saved for purification of the fusion protein.
20 °C. The yield was 30-100 mg of purified protein/liter of bacterial culture.
(Invitrogen) was used for transformation,
and the recombinant clones were identified by restriction analysis of
the plasmid DNA. The recombinant plasmid was linearized with
NsiI and used for transfection of a P. pastoris
strain His 4 GS-115 (Invitrogen). Transfection and selection were
performed according to a Pichia expression kit manual
supplied by the manufacturer. The recombinant protein was designated as
IgI P. pastoris (Ig-like domain I produced in P. pastoris). The authenticity of IgI P. pastoris was
secured by amino acid sequencing and mass spectroscopy confirming the expected molecular mass of 11 kDa.
5.0). 10 ml
of the saturated culture was transferred into 300 ml of medium A and
incubated for 24 h at 30 °C. Cells were then centrifuged at
2,000 × g for 10 min at 20 °C and resuspended in 50 ml of medium B (the same as medium A except that 20 g of 100%
methanol was used instead of 20 g of glycerol). After incubation
for 24 h at 30 °C, cells were pelleted by centrifugation at
3,000 × g for 20 min at 20 °C, and the supernatant was filtered through a 0.22-µm filter and concentrated to 2-3 mg of
total protein/ml. The IgI P. pastoris was purified by gel filtration using a Sephadex G-50 column (Pharmacia Biotech Inc.) and
stored frozen at
20 °C. The yield was 40-50 mg of purified protein/liter of cell culture.
-(dimethylaminopropyl)carbodiimide.
Collagen type I was immobilized using 15 µl of 40 µg/ml collagen
type I in 10 mM sodium acetate buffer, pH 5.5. Finally the
chip was blocked by 15 µl of 1 M ethanolamine HCl, pH
8.5.
where R is response, t is time,
R0 is the response at the start of dissociation,
and t0 is the start time for the dissociation. The association rate constant (ka)
was calculated by fitting data to Equation 2, using
ka and Req (steady-state
response level) as floating parameters
(Eq. 1)
where C is concentration of the binding protein and
t0 is the start time for the association. The
initial binding rate (r0) was calculated by
fitting data to Equation 3, using r0 and
ks as floating parameters
(Eq. 2)
where ks = kaC + kd.
(Eq. 3)
Estimation of the solution affinity of IgII-MBP to IgI P. pastoris was performed as described (35). Briefly, the initial binding rate (r0) of soluble IgII-MBP to immobilized IgI-MBP was determined at 1.79, 3.57, 5.36, 7.14, and 8.93 µM concentrations of IgII-MBP. r0 was plotted versus the concentration of IgII-MBP, fitted to a straight line, and the intercept (I) and slope (k) were estimated. Next, IgII-MBP at a 8.93 µM concentration was mixed with IgI P. pastoris at 300, 150, 75, 37.5, 18.8, 9.4, and 4.7 µM concentrations and incubated for 4 h at room temperature to reach equilibrium. r0 of the binding of unbound IgII-MBP to immobilized IgI-MBP was determined for each concentration of IgI P. pastoris. r0 was plotted versus total concentration (C) of IgI P. pastoris, and the data were fitted to Equation 4, using KD (dissociation constant in solution) and rm (initial binding rate of IgII-MBP in the absence of IgI P. pastoris) as floating parameters.
![]() |
(Eq. 4) |
![]() |
To study the function of the IgI NCAM domain, it was synthesized
in a bacterial expression system of E. coli as a recombinant fusion protein (IgI-MBP) and as a recombinant domain in a eukaryotic expression system of the yeast P. pastoris (IgI P. pastoris). The fusion protein consisted of the IgI domain fused
between E. coli MBP and an NH2-terminal fragment
(65 amino acids) of E. coli -galactosidase. This
expression system was chosen because it allows production of a large
amount of soluble recombinant protein that can be purified by affinity
chromatography using the natural affinity of MBP for maltose. The
P. pastoris expression system was selected because P. pastoris is capable of protein folding and processing similar to
higher eukaryotes (28), and the protein secreted into the medium can be
purified easily. For comparison, we also produced other NCAM domains as
fusion proteins: IgII-MBP, IgIII-MBP, IgIV-MBP, IgV-MBP, F3I,II-MBP,
and Ex16,17,19-MBP. The sequences of the various recombinant NCAM
constructs are given in Fig. 1.
The recombinant NCAM constructs were purified and analyzed by
SDS-polyacrylamide gel electrophoresis under reducing conditions as
shown in Fig. 2, a and b. The
purified proteins appeared to be more than 95% pure. The expected
molecular masses for the constructed proteins were: MBP, ~52 kDa;
IgII-MBP, ~56 kDa; IgI-MBP, IgIII-MBP, IgIV-MBP, and IgV-MBP,
~61-63 kDa; Ex16,17,19-MBP, ~64 kDa, and IgI P. pastoris, ~11 kDa. Only very small differences were observed between the calculated and the actual molecular masses probably because
of aberrant electrophoretic migration. Characterization of F3I,II-MBP
has been described previously (22).
Polyclonal rabbit antibodies were raised against IgI-MBP. The
antibodies of the first bleeding recognized NCAM from rat brain under
nonreducing conditions but not NCAM treated with -mercaptoethanol (Fig. 2c). The antibodies of the following bleedings
recognized both nonreduced and reduced NCAM. This means that the
antibodies of the first bleeding reacted with epitopes sensitive to
reduction of the disulfide bond, indicating that they probably are
conformational epitopes and that IgI-MBP employed for immunization was
folded correctly.
Since NCAM mediates cell-cell interactions by a Ca2+-independent homophilic binding mechanism (6-8) and also cell-substratum interactions (12, 14, 16), we investigated a possible role of the IgI NCAM domain in these binding interactions.
The IgI and IgII NCAM Domains Inhibit Aggregation of Mouse Cerebellar NeuronsTo test whether IgI-MBP and IgI P. pastoris were functionally active, the effects of the various NCAM
domains on neuronal cell aggregation were tested. Since immobilized IgI
and IgII domains have been demonstrated to be most efficient (compared
with other Ig-like NCAM domains) for adhesion of neuronal cell bodies
(19), soluble IgI-MBP or IgI P. pastoris and IgII-MBP were
expected to interfere with neuronal aggregation. Dissociated cells from cerebella of 6-day-old mice were plated on a microtiter plate and grown
in the presence of various protein constructs; the number of cell
aggregates was measured after 24 h of incubation. In the absence
of any addition to the culture medium, cells initially spread evenly
over the surface of the well, but after 24 h, large aggregates
were formed. From Fig. 3a it appears that
addition of IgI-MBP, IgII-MBP, and IgI P. pastoris inhibited
the formation of aggregates and therefore led to a decrease in the size
of aggregates and, as a result, to an increase in the number of smaller
aggregates. IgIII-MBP and IgV-MBP did not have any effect when compared
with MBP or phosphate-buffered saline, and IgIV-MBP had only a slight effect. To test whether MBP affects biological activity of the IgI
domain in the IgI-MBP construct, a dose response of MBP, IgI-MBP, and
IgI P. pastoris was studied. As appears from Fig.
3b, IgI-MBP and IgI P. pastoris had essentially
the same dose-response curves, MBP alone had no effect at any of the
tested doses, indicating that MBP does not affect biological activity
of the IgI domain in the IgI-MBP construct. Thus, IgI-MBP, IgI P. pastoris, and IgII-MBP NCAM domains inhibited aggregation of mouse
cerebellar neurons, indicating that these protein constructs were
functionally active and therefore suited for the subsequent binding
assays.
The IgI Domain of NCAM Binds to Heparin
To test the
possibility that the IgI NCAM domain is involved in NCAM-mediated
heterophilic interactions, we used surface plasmon resonance analysis
for studying the possible binding of this domain to some putative NCAM
ligands. No binding was found to laminin, collagen type I, and
fibronectin (not shown), whereas an affinity was found for heparin
(Fig. 4a). To estimate the dissociation and
association rate constants, we used the highest practically reasonable
flow rate of 30 µl/min to overcome possible mass transport limitation. The apparent constant of dissociation for IgI P. pastoris and the apparent association and dissociation rate
constants, in the context of the model used (see "Experimental
Procedures"), were 9.0 ± 2.0 × 107
M, 1.1 ± 0.3 × 104
M
1 s
1, and 8.4 ± 0.4 × 10
3 s
1, respectively. The goodness of
fit between the fitted curves and the experimental data was assessed by
means of the
2 value defined as
![]() |
(Eq. 5) |
To compare the heparin binding of the IgI domain to the well documented heparin binding of the IgII domain (18), we used IgI-MBP and IgII-MBP and five control proteins: MBP alone, Ex16,17,19-MBP, IgIII-MBP, IgIV-MBP, and IgV-MBP. The IgII-MBP exhibited a stronger binding to heparin than IgI-MBP; but the control proteins, MBP and Ex16,17,19-MBP, both exhibited a very low, probably unspecific, binding (Fig. 4b). The binding of IgIII-MBP, IgIV-MBP, and IgV-MBP was at the same level as that of MBP and Ex16,17,19-MBP (not shown). In a competition experiment, IgI-MBP and IgII-MBP were preincubated with either soluble heparin or a control negatively charged carbohydrate, PSA, for 15 min. Heparin completely inhibited subsequent binding of the fusion proteins to immobilized heparin (Fig. 4b), whereas PSA had no effect (not shown). We were, however, unable to show binding of soluble heparin to either immobilized IgII-MBP or IgI-MBP. This may be because the surface of a CM5 sensor chip is negatively charged at pH > 3. Since heparin molecules also are negatively charged, electrostatic repulsion between the surface of the sensor chip and heparin molecules may have precluded binding of heparin to IgI-MBP or IgII-MBP. It is also possible that the heparin binding was not detected because of the low molecular mass of heparin since the observed signal in surface plasmon resonance analysis is proportional to the molecular mass of the soluble ligand, and/or the employed immobilization procedure may have inactivated the heparin binding sites of IgII-MBP and IgI-MBP.
We also tested whether IgI-MBP and IgII-MBP were able to bind a collagen-heparin complex. This was done by immobilizing collagen type I on the sensor chip and subsequently applying heparin, which is a well known ligand of collagen (29). Although neither IgI-MBP nor IgII-MBP was able to bind to collagen type I directly, we found binding of IgI-MBP and IgII-MBP to the collagen-heparin complex (Fig. 4c). Interestingly, the maximal binding of IgII-MBP to heparin alone (Fig. 4b) was about ~5 times higher than the maximal binding of IgI-MBP, whereas the maximal binding of IgII-MBP to the collagen-heparin complex (Fig. 4c) was only 1.3 times higher than the maximal binding of IgI-MBP, indicating that IgI-MBP bound the collagen-heparin complex almost as well as IgII-MBP. This indicates that the IgI NCAM domain possibly also binds collagen type I although with a very low affinity.
In a competition experiment, IgI-MBP and IgII-MBP were preincubated with soluble heparin for 15 min. This completely inhibited subsequent binding of the fusion proteins to the collagen-heparin complex (Fig. 4c), indicating that it was heparin and not collagen type I that bound to IgI-MBP and IgII-MBP. The control proteins, MBP and Ex16,17,19-MBP, exhibited a very low, presumably unspecific binding to the sensor chip carrying the immobilized collagen-heparin complex (Fig. 4c); and the binding of IgIII-MBP, IgIV-MBP, IgV-MBP, and F3I,II-MBP was the same as that of the control proteins (not shown).
It was not possible to extract reliable kinetic data from the binding
of IgI-MBP and IgII-MBP to either heparin alone or the collagen-heparin
complex because of the interference of the MBP portion of IgI-MBP and
IgII-MBP. MBP exhibited a low unspecific binding to heparin and the
collagen-heparin complex compared with the binding of IgI-MBP and
IgII-MBP (Fig. 4, b and c); however, the small
amount of MBP that bound to heparin, did not dissociate. The
dissociation of MBP and IgI-MBP appeared to be identical (Fig. 4b), even though IgI P. pastoris dissociated from
heparin very quickly with a dissociation rate constant of approximately
102 s
1. We therefore believe that the
dissociation rate of IgI-MBP was determined by the dissociation rate of
the IgI domain and by the rate of very slow desorption of MBP.
Therefore, it was not possible to calculate kinetic data reliably from
the binding of fusion proteins containing MBP as a fusion partner. It
should be mentioned that the maximal binding of IgI-MBP to heparin
alone was approximately 900 resonance units, whereas the maximal
binding of IgI-MBP to the collagen-heparin complex was only 120 resonance units. This was because in the former experiment, much more
heparin was immobilized per area unit of the sensor chip than in the
latter.
Thus, the IgI domain of NCAM bound heparin, although apparently with a lower affinity than the IgII NCAM domain. However, when heparin was bound to collagen type I, the IgI domain bound heparin almost as well as the IgII domain, indicating that the IgI domain possibly also recognized collagen type I although with a very low affinity.
The IgI NCAM Domain Is Directly Involved in NCAM-mediated Homophilic BindingWe finally tested the possibility that the IgI
NCAM domain may be involved in NCAM-mediated homophilic binding.
IgI-MBP was immobilized on the sensor chip, and the binding of fusion
proteins of various NCAM domains was tested at a concentration of 1 mg/ml. From Fig. 5a, it appears that IgII-MBP
bound strongly to immobilized IgI-MBP, whereas IgI-MBP, IgV-MBP, and
MBP exhibited low unspecific binding. The binding of IgIII-MBP,
IgIV-MBP, and F3I,II-MBP was approximately the same as that of IgI-MBP,
IgV-MBP, and MBP (not shown). Since soluble IgII-MBP binds to
immobilized IgI-MBP, the reverse should also be expected, namely that
soluble IgI-MBP binds to immobilized IgII-MBP. Therefore, we also
immobilized IgII-MBP on the sensor chip and tested it for binding of
various NCAM fusion proteins at a concentration of 1 mg/ml (Fig.
5b). IgI-MBP bound to immobilized IgII-MBP more strongly
than IgII-MBP, IgV-MBP, and MBP, the maximal binding being at least two
times higher than that of the other proteins. The binding of IgIII-MBP,
IgIV-MBP, and F3I,II-MBP was approximately the same as that of the
IgII-MBP, IgV-MBP, and MBP (not shown). It is easy to see that binding
of soluble IgII-MBP to immobilized IgI-MBP was higher than binding of
soluble IgI-MBP to immobilized IgII-MBP (Fig. 5, a and
b). Since approximately the same amount of protein was
immobilized in both cases, this difference in binding may be the result
of partial inactivation of IgII-MBP and, possibly to a lesser extent, of IgI-MBP during immobilization. IgII-MBP might be more predisposed to
partial inactivation during immobilization compared with IgI-MBP, because the fusion proteins were immobilized via amino groups of lysine
residues, and the potential binding site of IgII-MBP is a cluster of
basic amino acid residues consisting of lysine 135, 137, 185, and
arginine 139 and 171 (5).
To determine the affinity of the IgI-IgII interaction, binding of
soluble IgII-MBP to immobilized IgI-MBP was measured using various
concentrations of IgII-MBP (Fig. 6a). The
apparent dissociation constant and the apparent association and
dissociation rate constants, in the context of the model used (see
"Experimental Procedures"), were 1.3 ± 0.3 × 105 M, 1.4 ± 0.3 × 102 M
1 s
1, and
1.2 ± 0.1 × 10
3 s
1,
respectively. The estimation of kinetic parameters from this experiment
may not be reliable because of the interference of MBP. The rate of
dissociation of IgII-MBP is determined by the rate of dissociation of
the IgII domain and by the rate of unspecific desorption of MBP (see
explanation above). This leads to underestimation of the dissociation
rate constant (apparent slower dissociation) and, as a result, to
overestimation of the affinity. Thus, the true value of the
dissociation constant is higher than 1.3 ± 0.3 × 10
5 M.
To estimate the affinity of the IgI-IgII interaction more reliably, we
therefore measured binding of IgII-MBP to IgI P. pastoris in
solution, thus avoiding the problem of unspecific interaction of MBP
with the sensor chip. IgII-MBP at an 8.93 µM (0.5 mg/ml) concentration was mixed with various concentrations of IgI P. pastoris, incubated for 4 h at room temperature to reach
equilibrium, and the binding of unbound IgII-MBP to immobilized IgI-MBP
was measured at each concentration of IgI P. pastoris (Fig.
6b). To determine the concentration of unbound IgII-MBP in
the mixture with IgI P. pastoris, a calibration of the
initial binding rate of IgII-MBP to immobilized IgI-MBP
versus the concentration of IgII-MBP was performed (Fig.
6c). The initial binding rate of IgII-MBP to immobilized
IgI-MBP in the mixture with IgI P. pastoris was calculated,
plotted versus the concentration of IgI P. pastoris, and the data were fitted to the model (see
"Experimental Procedures") (Fig. 6d), with the resulting
dissociation constant of 5.5 ± 1.6 × 105
M. As expected, this value of dissociation constant was
higher than 1.3 ± 0.3 × 10
5
M.
Since the IgIII domain of chick NCAM has been reported to bind to itself (9, 10), and the IgV, IgIV, IgII, and IgI domains of chick NCAM have been reported to bind to IgI, IgII, IgIV, and IgV, respectively (11), it was of interest to test whether the IgI, IgII, IgIII, IgIV, and IgV domains of mouse NCAM could bind to IgV, IgIV, IgIII, IgII, and IgI domains, respectively, under the chosen conditions. As shown above, soluble IgV-MBP and IgIV-MBP did not bind to immobilized IgI-MBP and IgII-MBP, respectively. When F3I,II-MBP, IgV-MBP, IgIV-MBP, and IgIII-MBP were immobilized, no specific binding of any domain was detected (not shown).
In this study we have investigated the role of the IgI NCAM domain
in NCAM-mediated binding interactions. The functional activity of
IgI-MBP and IgI P. pastoris was tested by studying the
effects of the various NCAM domains on neuronal cell aggregation.
IgI-MBP, IgI P. pastoris, and IgII-MBP inhibited aggregation
of mouse cerebellar neurons, whereas other Ig-like domains of NCAM had
very little, if any, effect, indicating that the IgI and IgII protein
constructs were functionally active and therefore suited for the
binding assays. The binding properties of the IgI NCAM domain were
studied using surface plasmon resonance analysis, and it was
demonstrated that IgI-MBP bound immobilized IgII-MBP, and vice
versa, IgII-MBP bound immobilized IgI-MBP. It was also
demonstrated that IgI P. pastoris inhibited binding of
IgII-MBP to immobilized IgI-MBP. This indicates that the IgI and IgII
domains are directly involved in homophilic interactions of NCAM. Our
results are in agreement with Frei et al. (19), who showed
that the IgI and IgII NCAM domains, synthesized in a bacterial
expression system, when used as immobilized substratum, clearly
promoted cell adhesion of NCAM-expressing cells, whereas other NCAM
domains hardly had any adhesive effects. Our data are also in
accordance with reports that a 25-kDa NH2-terminal NCAM
fragment containing the IgI and IgII NCAM domains is involved in cell
adhesion (14, 30). The fact that IgI-MBP, IgI P. pastoris, and IgII-MBP inhibited aggregation of mouse cerebellar neurons, whereas
other Ig-like domains of NCAM had very little effect, further supports
the notion that the IgI and IgII domains are involved in NCAM-mediated
binding interactions. Our data are also in agreement with our recent
prediction (5), based on the analysis of the three-dimensional
structure of the IgI domain and the predicted three-dimensional
structure of the IgII domain, that an acidic cluster of residues at the
NH2 terminus of the IgI domain (aspartic acid 29, 32, 34, 58, 59, and 84, and glutamic acid 85) and a basic cluster of residues
of the IgII domain (lysine 135, 137, and 185, and arginine 139 and 171)
may be involved in a symmetrical double-reciprocal binding
(schematically depicted in Fig. 7a).
The estimated dissociation constant (KD) of the
interaction of the individual IgI and IgII domains was 5.5 ± 1.6 × 105 M. A rough estimate of the
dissociation constant of the double-reciprocal interaction
(KDdri) can be made. Let
Gst be the standard Gibbs energy change of
the interaction of the individual IgI and IgII domains at constant
temperature and pressure. The standard Gibbs energy change of the
double-reciprocal interaction, then, can be minimally estimated as
2
Gst, when the entropy loss associated
with a more ordered complex is not taken into account, since the model
(Fig. 7a) implies that the double-reciprocal interaction is
symmetrical (5). The true value of the standard Gibbs energy change of
the double-reciprocal interaction might be larger than
2
Gst because of the entropy loss.
Therefore
![]() |
(Eq. 6) |
The homophilic binding site for the chick transmembrane NCAM-B isoform
has been mapped to a decapeptide sequence from Lys-243 to Glu-252 in
the IgIII domain (9, 10), and the counterreceptor for the IgIII domain
has been localized to the same decapeptide sequence (31). Recently, it
has been demonstrated, in aggregation experiments with fluorescent
microspheres, that the IgI, IgII, IgIII, IgIV, and IgV domains of chick
NCAM interact with the IgV, IgIV, IgIII, IgII, and IgI domains,
respectively (11). Our data indicate that there is possibly more than
one mechanism of NCAM homophilic binding. However, in our experiments,
no binding of the IgI, IgII, IgIII, IgIV, and IgV domains of mouse NCAM
to the IgV, IgIV, IgIII, IgII, and IgI domains, respectively, using
surface plasmon resonance analysis could be shown. The absence of
binding may be the result of several factors: inactivation of the
proteins during immobilization, purification, or storage; a very low
affinity of the interaction; incorrect folding since the proteins were produced in bacteria and therefore not glycosylated. However, if five
pairs of NCAM domains are involved in the homophilic binding at the
same time (IgI binds to IgV, IgII to IgIV, IgIII to IgIII, IgIV to
IgII, and IgV to IgI), and if the dissociation constants of the
IgI-IgV, IgII-IgIV, and IgIII-IgIII interactions are approximately the
same, then the KD of the interaction of the
individual domains is approximately the fifth root of the
KD of the homophilic binding of NCAM. Presuming
that the KD of the homophilic binding is as low
as 1010 M (100-1,000 times lower than the
experimental value given in Ref. 8), then the KD
of the interaction of the individual domains is
10
2
M, which cannot be detected by surface plasmon resonance
analysis. Although we cannot demonstrate that IgIII-MBP, IgIV-MBP, and
IgV-MBP were folded correctly, we have several indications that that
was the case for IgI-MBP and IgII-MBP. (i) Polyclonal antibodies of the
first bleeding against IgI-MBP did not recognize a reduced NCAM
preparation, whereas a nonreduced preparation was readily recognized,
indicating that the conformation of the IgI domain in IgI-MBP was close
to the conformation of the nonreduced form of NCAM, and therefore that
the disulfide bridge, which is an essential feature of an Ig-like
domain, was formed. (ii) Both IgI-MBP and IgII-MBP inhibited
aggregation of mouse cerebellar neurons, indicating that these
protein constructs were functionally active. (iii) In contrast to the
IgIII domain, the IgI and IgII domains are not glycosylated in native
NCAM, and therefore, glycosylation is not required for proper folding.
(iv) The binding of IgI-MBP and IgII-MBP to heparin, and of IgI-MBP to
IgII-MBP, indicates that these protein constructs were folded in an
active conformation.
Since the affinity of NCAM homophilic binding increases during development (8), probably because of a decrease in NCAM polysialylation (32, 33), it has been hypothesized that the homophilic binding mechanism is developmentally regulated by the PSA content of NCAM. Because PSA is attached to the IgV NCAM domain (34), the binding mechanism indicated by the model in Fig. 7a may be favored in a situation with a high PSA content, whereas NCAM molecules with a low amount of PSA may be able to slide along each other, favoring the mechanism in which all five Ig-like domains are involved: IgI binds to IgV, IgII to IgIV, IgIII to IgIII, IgIV to IgII, and IgV to IgI (11).
IgI P. pastoris was found to bind heparin with a constant of
dissociation of 9.0 ± 2.0 × 107 M
and association and dissociation rate constants of 1.1 ± 0.3 × 104 M
1 s
1 and
8.4 ± 0.4 × 10
3 s
1,
respectively. No binding could be demonstrated to laminin, collagen type I, or fibronectin. Comparison of the heparin binding of IgI-MBP and IgII-MBP revealed that the IgII domain bound heparin with a higher
affinity than the IgI domain. Heparin binding of the IgI domain may be
considered specific on the following criteria. (i) The IgI domain bound
heparin but did not bind other negatively charged carbohydrates such as
carboxymethylated dextran (the surface of a CM5 sensor chip) and PSA.
(ii) Other extracellular domains of NCAM (with the exception of the
IgII domain) did not bind to heparin. In contrast to the IgII domain,
where a cluster of basic amino acids capable of heparin binding has
been identified, there is no cluster of basic amino acids in the IgI
domain. It is possible that such a cluster may not be required for low
affinity heparin-binding proteins.
It has been demonstrated previously that NCAM binds different types of collagen (12) and that binding of NCAM to collagens type I and V could be inhibited by glycosaminoglycans such as heparin and chondroitin sulfate (14). However, the authors were unable to determine whether the inhibition by glycosaminoglycans was the result of their binding to collagen or to NCAM. Therefore, it is not clear whether NCAM actually can bind collagen directly or via glycosaminoglycans. Neither IgI-MBP nor IgII-MBP bound to collagen type I directly. However, we demonstrated binding of IgI-MBP and IgII-MBP to collagen type I to which heparin was associated. In a competition experiment, heparin inhibited binding of IgI-MBP and IgII-MBP to the collagen-heparin complex, indicating that it was heparin and not collagen type I that bound IgI-MBP and IgII-MBP. Other NCAM domains did not bind to the collagen-heparin complex. The maximal binding of IgII-MBP to heparin alone was approximately 5 times higher than the maximal binding of IgI-MBP, whereas the maximal binding of IgII-MBP to the collagen-heparin complex was only 1.3 times higher than the maximal binding of IgI-MBP, indicating that IgI-MBP bound the collagen-heparin complex almost as well as IgII-MBP. This indicates that the IgI NCAM domain possibly also binds collagen type I although with a very low affinity.
Based on our data, we conclude that heparin can form a "bridge" between collagen type I and NCAM (Fig. 7b). In this model, both the IgI and IgII NCAM domains bind to heparin associated with collagen.
Thus, we present evidence that in mouse the IgI and IgII NCAM domains are directly involved in NCAM-mediated homophilic interaction. Both NCAM domains also bind heparin and, via heparin, collagen type I but probably not collagen type I directly.