From the Medical Research Council Immunochemistry Unit, Department of Biochemistry, University of Oxford, South Parks Road, Oxford OX1 3QU, United Kingdom
Received for publication, January 24, 2001, and in revised form, March 21, 2001
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ABSTRACT |
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Link modules are hyaluronan-binding
domains found in extracellular proteins involved in matrix assembly,
development, and immune cell migration. Previously we have expressed
the Link module from the inflammation-associated protein tumor necrosis
factor-stimulated gene-6 (TSG-6) and determined its tertiary structure
in solution. Here we generated 21 Link module mutants, and these were
analyzed by nuclear magnetic resonance spectroscopy and a
hyaluronan-binding assay. The individual mutation of five amino acids,
which form a cluster on one face of the Link module, caused large
reductions in functional activity but did not affect the Link module
fold. This ligand-binding site in TSG-6 is similar to that determined previously for the hyaluronan receptor, CD44, suggesting that the
location of the interaction surfaces may also be conserved in other
Link module-containing proteins. Analysis of the sequences of TSG-6 and
CD44 indicates that the molecular details of their association with
hyaluronan are likely to be significantly different. This comparison
identifies key sequence positions that may be important in mediating
hyaluronan binding, across the Link module superfamily. The use of
multiple sequence alignment and molecular modeling allowed the
prediction of functional residues in link protein, and this approach
can be extended to all members of the superfamily.
Hyaluronan (HA)1 is a
ubiquitous high molecular weight glycosaminoglycan, composed of
repeating disaccharides of D-glucuronic acid and
N-acetyl-D-glucosamine, which has diverse
biological roles in vertebrates. For instance, this polysaccharide, a
vital structural component of extracellular matrix (e.g.
cartilage, skin, and brain), is required for successful embryonic
development (1), and is involved in cell migration (2, 3). The wide range of functional activities derives from the large number of HA-binding proteins, which can be intracellular, secreted, or on the
cell surface. Many of the extracellular hyaladherins contain a common
domain of ~100 amino acids, termed a Link module, which is involved
in HA binding (4, 51). This domain was first described in link
protein (containing an immunoglobulin module and two contiguous Link
modules (5)), which together with HA and aggrecan forms huge
multimolecular complexes that provide articular cartilage with its load
bearing properties. Aggrecan interacts with HA via its N-terminal G1
domain, and this has the same organization of modules as link protein
(6); it also has another pair of tandem Link modules within its G2
domain, but these do not bind HA (7-9). In the aggrecan G1 domain and
link protein it has been found that both Link modules participate in HA
binding (9, 10).
CD44 is the primary receptor for HA and has a range of functions such
as anchoring the extracellular matrix to the surface of cells
(e.g. in cartilage (11)) and mediating the migration of
activated lymphocytes to sites of inflammation (3). CD44 has a single
Link module that forms part of its HA-binding domain (12), and
functionally important amino acids within this region have been
identified (13, 14).
The inflammation-associated protein TSG-6 (the secreted product of
tumor necrosis factor-stimulated gene-6 (15)) contains a single Link
module. TSG-6 has been implicated in the regulation of leukocyte
migration (16, 17), and its pattern of expression and ligand
specificity indicates that it may be involved in extracellular matrix
remodeling (18-20). Previously, we have expressed the Link module from
human TSG-6 in Escherichia coli (21, 22) and shown that this
material (referred to here as Link_TSG6) interacts with HA using a
microtiter plate assay (18, 19, 23). In addition, nuclear magnetic
resonance (NMR) spectroscopy on Link_TSG6 has revealed that the
Link module is comprised of two Here, we report the production of 21 Link_TSG6 mutants and their
characterization by NMR spectroscopy and a HA- binding assay. Five
amino acids, which are clustered on one face of the TSG-6 Link module,
were identified as having an important role in binding. Comparison of
the HA interaction surfaces in TSG-6 with those determined previously
for CD44 has allowed the prediction of functional residues in link
protein and other members of the Link module superfamily.
Preparation of Link_TSG6 Mutants--
Mutagenesis was carried
out using the Transformer site-directed mutagenesis kit
(CLONTECH), with the plasmid VII-6-1mut8-5 (21) as
the template, according to manufacturer's instructions. VII-6-1mut8-5
contains the coding sequence for the TSG-6 Link module (Link_TSG6) in
the pRK172 vector. Fig. 1 shows the DNA sequences of the 18 oligonucleotides (numbered 1-18) used in the mutagenesis reactions, aligned with wild-type Link_TSG6. Selection primer 1 was used in 16 separate reactions, in combination with each of
the oligonucleotides 3-18, whereas selection/mutagenesis primer 2 was
used on its own. Primers 2, 3, 4, 6, 8-13, and 15-18 were designed to
generate a single amino acid mutation (i.e. Glu-6 Expression, Purification, and Characterization of Wild-type and
Mutant Link_TSG6--
Wild-type and mutant proteins were expressed,
refolded, and purified to homogeneity as described previously (21, 22). Mutants (at 5-7 pmol/µl in 50% (v/v) acetonitrile, 0.2% (v/v) formic acid) were analyzed by electrospray ionization mass spectrometry on a Micromass BioQ II-ZS spectrometer calibrated with horse heart myoglobin (average molecular mass of 16,591.48 Da) and scanned over the mass range 600-1,600 Da.
NMR Spectroscopy--
Lyophilized wild-type and Link_TSG6
mutants were resuspended in 600 µl of 10% (v/v) D2O,
0.02% (w/v) NaN3 and adjusted to pH 6.0 with NaOH to give
concentrations in the range 0.4-1.3 mM. One-dimensional
NMR spectra (128 scans) were recorded at 25 °C on a home-built/GE
Omega spectrometer, operating at a frequency of 500 MHz. The NMR data
were processed using FELIX 2.3 (Biosym Inc.), applying sine bell and
Gauss-Lorentz window functions for resolution enhancement. Proton
chemical shifts were referenced to H2O at 4.74 ppm.
Protein Concentration--
The concentrations of the wild-type
and mutant proteins, used in the NMR analysis, were determined by amino
acid analysis (24) on an Applied Biosystems 420A derivatizer/analyzer
and on-line narrow bore high performance liquid chromatography
system (Applied Biosystems). These "stock solutions" were stored at
4 °C and used subsequently in the HA-binding assays (see below).
Biotinylation of HA--
Rooster comb HA (Sigma) was
biotinylated using a
modification2 of the method
of Yu and Toole (25). Briefly, 20 µl of 250 mM biotin-LC-hydrazide (Pierce and Warriner, Chester, U. K.) in dimethyl sulfoxide was added to 1 ml of 5 mg/ml HA (in 0.1 M MES, pH
5.5) followed by 13 µl of 25 mg/ml EDAC in 0.1 M MES, pH
5.5, and the reaction mixture was stirred at room temperature
overnight. The sample was dialyzed extensively against water and
particulate material removed by centrifugation (12,000 × g for 1 min). The concentrations of HA samples (either
biotinylated or unmodified) were determined using the
metahydroxybiphenyl reaction (26) relative to standards made from
rooster comb HA dried in vacuo over cobalt chloride.
Analysis of HA Binding--
The HA-binding activities of
wild-type and Link_TSG6 mutants were determined colorimetrically using
a microtiter plate assay that measures the binding of biotinylated HA
to protein-coated wells (18, 19, 23). All dilutions, incubations, and
washes were performed in 50 mM sodium acetate, 100 mM NaCl, 0.05% (v/v) Tween 20, pH 6.0, unless otherwise
stated; it has been shown previously that the interaction between
Link_TSG6 and HA is maximal at pH 6.0 (19). Maxisorp F96 plates (Nunc)
were coated overnight with 200 µl/well protein solution (25 pmol/well; protein concentrations were determined for stock solutions
as described above) in 20 mM
Na2CO3, pH 9.6. Control wells were incubated
with buffer alone and then treated as for sample wells. The coating
solution was removed and the plates washed three times. Nonspecific
binding sites were blocked by incubation with 1% (w/v) bovine serum
albumin for 90 min at 37 °C followed by three more washes. A
200-µl solution containing 12.5 ng of biotinylated HA was added to
each well, in the absence or presence of 2,500 ng of unmodified rooster
comb HA and incubated at room temperature for 4 h. Plates were
washed three times, and 200 µl of a 1:10,000 dilution of ExtraAvidin alkaline phosphatase (Sigma) was added and incubated for 30 min. After
three more washes, wells were incubated for 10 min with 200 µl of a 1 mg/ml solution of disodium p-nitrophenyl phosphate (Sigma)
in 100 mM Tris-HCl, 100 mM NaCl, 5 mM MgCl2, pH 9.3. The absorbance at 405 nm was
determined on an MKII Titertek Multiscan Plus plate reader. All
absorbance measurements were corrected by subtracting values from
uncoated control wells. Each interaction was investigated in
quadruplicate in three separate plate assays (i.e.
n = 12).
Isothermal Titration Calorimetry--
The interactions between
six Link_TSG6 mutants and an octasaccharide of HA (HA8)
were investigated on a MicroCal VP-ITC instrument at 25 °C in 5 mM Na-MES, pH 6.0. A 335 µM solution of
HA8, prepared by digestion of human umbilical cord HA with
ovine testicular hyaluronidase and purified by gel filtration and ion
exchange chromatography,3 was
added in 5-µl injections (28 in total) to protein (ranging from 7.3 to 25.7 µM) in the 1.4-ml calorimeter cell. Data were fitted to a one-site model by nonlinear least squares regression with
the Origin software package, after subtracting the heats resulting from
the addition of HA8 into buffer alone as described previously (27).
Link Module Alignment--
A multiple sequence alignment of Link
modules from human proteins was generated as described previously (23).
The Link modules included were from TSG-6 (15), CD44 (28), LYVE-1 (29),
KIA0246 (30), CAB61258 (accession code CAB61258), KIA0527 (31), link
protein repeats 1 and 2 (denoted Lp1 and Lp2, respectively) (32), brain
link protein-1 (BRAL1) repeats 1 and 2 (33), aggrecan repeats 1-4
(34), brevican repeats 1 and 2 (35), versican repeats 1 and 2 (36), and
neurocan repeats 1 and 2 (37).
Homology Modeling--
The three-dimensional structures of the
two Link modules from Lp1 and Lp2 were each modeled using the program
Modeller4 (38) on the basis of the coordinates of the human TSG-6 Link
module (23) and the alignment in Fig. 2.
In each case, 100 independent models were generated, and the model with
the lowest energy (based on the value of the molecular probability
density function) was chosen. XPLOR version 3.8 (39) was used to add
hydrogen atoms and disulfide bonds and to carry out energy minimization
and molecular dynamic simulations with the CHARMm22 force field (40).
Briefly, three rounds of energy minimization were carried out with the backbone fixed. In the first, the electrostatic term was excluded, and
a purely repulsive non-bonded force field was used. In the following
rounds, the full CHARMm22 force field, which included a Lennnard-Jones
potential, was used, but electrostatic interactions were only included
in the third round. This was followed by molecular dynamics where all
atoms of residues 1-71 and 76-99 in Lp1, and amino acids 1-11,
14-39, and 43-96 in Lp2 were fixed so that only regions corresponding
to insertions or deletions, when compared with Link_TSG6 (Fig. 2), were
free to move. A final energy minimization was performed using the full
CHARMm22 force field as in round three above. PROCHECK (41) was used to
determine that the number of sterochemical violations in the final
models were similar to that of the solution structure of TSG-6 Link
module.
Residue Selection and Mutagenesis--
15 sequence positions of
Link_TSG6 were selected for mutagenesis. Eight of these residues
(i.e. Lys-11, Tyr-12, Tyr-59, Lys-72, Asp-77, Tyr-78,
Arg-81, and Glu-86), which form a coherent patch on the Link module
surface, were chosen because they have been predicted previously to be
involved in HA binding (23). Arg-8 was picked because it is adjacent to
this patch, and a basic amino acid at this position is involved in HA
binding in human CD44 (14). Asp-89, which is completely buried in the
hydrophobic core, was chosen because it could be involved in mediating
the unusual pH dependence of HA binding to Link_TSG6 (19, 42). Asn-67,
Phe-70, and Ile-75 are located on the
In total, 21 Link_TSG6 mutant constructs were generated and verified by
DNA sequencing (listed in Table I). All
of the mutants were found to express at levels similar to wild-type
Link_TSG6 (21). Electrospray ionization mass spectrometry revealed that the mutant proteins had molecular masses that differed by less than 1.5 Da from their theoretical masses (data not shown).
Structural Characterization of Link_TSG6
Mutants--
One-dimensional NMR spectroscopy was used to assess the
effect of each of the mutations on the Link module fold. Wild-type Link_TSG6 has a characteristic one-dimensional NMR spectrum (Fig. 3), with well dispersed signals
(e.g. in the amide region ~7.5-9.5 ppm) and the methyl
resonances from Val-57 shifted to high field (-0.5 and -1.1 ppm)
because of their proximity to Trp-51 and Trp-88 in the hydrophobic core
(19, 23). 13 of the mutants (Table I) give NMR spectra (data not shown)
that are essentially identical to that of the wild-type protein
(e.g. Y59F illustrated in Fig. 3). Thus, it can be concluded
that these amino acid substitutions have no effect on the Link_TSG6
fold. However, other mutations give rise either to unfolded protein
(e.g. E86A; Fig. 3) or a Link module that, while folded, is
structurally different from that of wild-type Link_TSG6
(e.g. Y78S; Fig. 3). Therefore, all of the mutants can be
classified as having a wild-type fold, a perturbed fold, or being
unfolded on the basis of their NMR spectra (Table I). Clearly, only the
mutants that have wild-type folds can be used to provide information on
the role of a particular amino acid sequence position in ligand
binding.
HA-Binding Experiments--
The HA-binding activities of wild-type
and mutant Link_TSG6 were analyzed using a microtiter plate assay that
we have described previously (18, 19, 23). For wild-type Link_TSG6,
maximum binding of biotinylated HA (12.5 ng) was seen when protein was coated at 25 pmol/well (data not shown). Amino acid analysis of coating
solutions, following incubation overnight in the microtiter plate,
indicated that greater than 90% of the protein (wild-type and eight
mutants tested) was adsorbed onto the well (data not shown). From Fig.
4, which shows the experimental data for the mutants with wild-type
folds, it can be seen that the binding of biotinylated HA is highly
specific because this is greatly reduced by the presence of unlabeled
HA. Some mutants show a degree of nonspecific (i.e.
non-competable) binding, but in the worst case (N67L) this is less than
15% of the value for wild-type protein determined in absence of
competitor (Fig. 4).
Table I shows the HA binding activities of all of the Link_TSG6 mutants
(as a percentage of wild-type binding). The mutants that have either a
perturbed fold (i.e. N67S, I75A, D77A, Y78S, and D89A) or
are unfolded (i.e. R81A, E86A, and E86S) have a greatly reduced HA binding function (with between 2 and 26% of wild-type binding). Because these mutations affect the Link module structure (see
above) it is impossible to tell whether the loss of activity results
from the residue being involved in binding or from the perturbation of
the interaction surface. Therefore, they provide no information on role
of a particular amino acid in binding.
The binding data for the 13 mutants with wild-type folds are presented
in Fig. 4. From this it can be seen that
E6A, R8A, K13A, N67L, and K72A have functional activities similar to
that of wild-type protein with 93, 108, 89, 79, and 88% of wild-type binding, respectively (Table I). Therefore, it can be concluded that
Glu-6, Arg-8, Lys-13, Asn-67, and Lys-72 are unlikely to be involved in
the interaction of Link_TSG6 with HA.
The mutation of Lys-11, Tyr-12, Tyr-59, Phe-70, or Tyr-78
(i.e. mutants K11Q, Y12F, Y12V, Y59F, Y59S, F70V, Y78F, and
Y78V) each leads to a large reduction in activity (7-30% of wild-type binding; see Table I). Table II shows
that the mutation of these amino acids also leads to a significant
reduction in the affinities of HA binding in solution, whereas K72A
exhibits wild-type activity. This clearly demonstrates that the results
obtained with the microtiter plate assay are reliable and are not an
artifact caused by immobilization of the protein on the plate. These
data indicate that Lys-11, Tyr-12, Tyr-59, Phe-70, and Tyr-78 are
likely to participate directly in HA binding. For example, Lys-11 could
be making an ionic interaction with a carboxyl group of HA; basic amino
acids have been implicated previously in protein-HA interactions (13,
14, 43-45). Recent calorimetry studies indicate that the interaction
of Link_TSG6 with HA8 involves the formation of one or two
salt bridges (46).
The conservative replacement of any of the three tyrosines
(i.e. Tyr-12, Tyr-59, and Tyr-78) with phenylalanine leads
to a large drop in functional activity (Fig. 4), indicating that the hydroxyl groups in these residues make an important contribution to HA
binding. Y12F and Y78F have activities that are similar to those of
Y12V and Y78V, respectively, showing that in Tyr-12 and Tyr-78 the
hydroxyls alone (but not the aromatic rings) are involved in the
interaction. The serine mutant of Tyr-59 (Y59S) has a slightly reduced
binding capacity compared with Y59F, suggesting that the aromatic ring,
in this case, may also take part. Mutation of Phe-70 to Val (F70V)
reduces HA-binding activity significantly, indicating that its aromatic
ring makes an important contact with HA.
The individual mutation of these five amino acids (i.e.
Lys-11, Tyr-12, Tyr-59, Phe-70, and Tyr-78) leads to a large reduction in functional activity, indicating that there is an extensive network
of interactions between the protein and polysaccharide, and loss of any
one of these (such as a hydrogen bond from Tyr-12 to HA) can have a
dramatic effect on HA binding.
Localization of the HA-Binding Site on Link_TSG6--
The
positions of the 15 amino acids mutated here were mapped onto the
structure of the TSG-6 Link module (Fig.
5). The five residues that are implicated
in HA binding (colored red) form a cluster on one face of
the molecule. Therefore, it is likely that this represents the position
of the HA-binding surface on Link_TSG6. This is consistent with recent
NMR studies (27) identifying the residues of Link_TSG6 which exhibit
significant chemical shift changes (for HN, NH,
C
As described above, eight of the amino acids selected for mutagenesis
were predicted to be involved in the interaction with HA (23). Of
these, Lys-11, Tyr-12, Tyr-59, and Tyr-78 have been found to
participate in HA binding, whereas Lys-72 is not involved. Mutation of
Asp-77, Arg-81, and Glu-86 compromises the structural integrity of the
Link module, such that no conclusion can be made regarding their role
in HA binding. However, their involvement cannot be excluded.
Glu-6 and Lys-13, which have been implicated in TGS-6-mediated
inhibition of neutrophil migration (16), are clearly not involved in HA
binding (Fig. 3 and Table I). In this study we mutated both of these
residues to alanine, whereas Wisniewski et al. (16) altered
them to lysine and glutamic acid, respectively. It is possible,
therefore, that these latter mutants (equivalent to E6K and K13E) may
have reduced HA-binding capabilities (e.g. because of having
perturbed structures).
HA-Binding Sites in TSG-6 and CD44 Link Modules--
The residues
of the CD44 Link module which mediate HA binding have been identified
by site-directed mutagenesis (14). Four amino acids (shown in
dark blue on Fig.
6A) are essential for high
affinity binding (i.e. mutation of any one of these greatly reduces functional activity), and five other amino acids (light blue) are involved but not critical. From Fig. 6A it
can be seen that the positions of the HA-binding sites on Link_TSG6 and
CD44 map to the same face of the Link module. In addition, the
essential HA-binding residues Arg-41 and Tyr-42 in CD44 are found at
sequence positions identical to those of Lys-11 and Tyr-12,
respectively, in Link_TSG6 (Fig. 7). This indicates that the location
of the HA-binding surface may be conserved across the Link module
superfamily. Consistent with this, the epitope recognized by a
monoclonal antibody that inhibits HA binding to link protein (47) also
maps to this face of the module.
Further consideration of the HA-binding amino acids in CD44 and TSG-6
suggest that, although the positions of the binding surface are
similar, the molecular details of the interactions are likely to be
significantly different. Apart from the amino acids described above
(i.e. Lys-11 and Tyr-12 in Link_TSG6, and Arg-41 and Tyr-42
in CD44) none of the other HA-binding residues are found at equivalent
sequence position in these proteins. As can be seen from Fig.
7, the critically important residues
Arg-78 and Tyr-79 in CD44 are replaced in TSG-6 by alanines (Ala-48 and Ala-49), which are unable to make ionic or hydrogen bonds to the sugar.
In addition, Lys-38 and Asn-101 in CD44 are both involved in the
interaction with HA, whereas the corresponding residues in Link_TSG6
(i.e. Arg-8 and Lys-72, respectively) have been shown here
not to participate in binding (Fig. 4).
HA-Binding Consensus--
Comparison of the functional residues
determined for TSG-6 and CD44 indicates that at least 12 amino acid
sequence positions of the Link module can be involved in HA binding. As
shown in Fig. 6B (for Link_TSG6) 11 of these form a coherent
surface patch on one face of the module. Only positions 2 and 3 are
utilized in both TSG-6 and CD44, whereas all of the others are either
TSG-6-specific (positions 6, 7, and 11) or CD44-specific (positions 4, 5, 8, 9, and 10). These 11 sequence positions are likely to be of
functional importance in other members of the Link module superfamily;
it is expected that a particular protein will utilize a certain
combination of these "consensus" residues to form its HA-binding
surface. Given the conservation of binding residues at positions 2 and 3 in TSG-6 and CD44 it is probable that these represent key
determinants in the HA interaction, across the superfamily as a whole.
Fig. 7 shows 18 Link modules from 10 different human proteins aligned with TSG-6 and CD44, where the residues (at consensus positions 1-11)
which have the potential to mediate carbohydrate binding (i.e. by making ionic or hydrogen bonds (48, 49)) are
highlighted. For example, Lp1 has potential HA-binding
residues at consensus positions 1, 2, 3, 4, 6, 8, 9, 10, and 11 (Fig.
7). This is also illustrated in Fig. 8,
which shows the locations of these residues on a homology model of Lp1,
generated on the basis of the Link_TSG6 coordinates (see
"Experimental Procedures"). It is possible that some of these
residues may be more likely to participate in the interaction with HA
than others because they correspond to identities or conservative
replacements of functional amino acids in TSG-6 or CD44
(i.e. in Lp1 this corresponds to positions 1, 2, 3, 6, 8, 9, and 11, which are underlined on Fig. 7).
It should be noted that the Link module-containing proteins KIA0246,
CAB61358, and KIA0527 (Fig. 7) have not yet been shown to have an
HA-binding function. As mentioned above, there are likely to be large
networks of interactions required to stabilize HA-protein complexes.
Given the number of conserved residues (compared with CD44 and TSG-6;
underlined in Fig. 7) at the consensus sequence positions
(one in KIA0246 at position 3; three in CAB61358 at positions 3, 6, and
11; none in KIA0527) we predict that KIA0246 and KIA0527 are unlikely
to be HA-binding proteins, although it is possible that CAB61358 may be
functionally active.
All of the HA binding activity of aggrecan is located in its G1 domain,
which contains two contiguous Link modules (aggrecan1 and aggrecan2 in
Fig. 7) (7-9). Both of these Link modules are required for high
affinity HA binding (9). Visual inspection of the nonfunctional Link
modules of the G2 domain (i.e. aggrecan3 and aggrecan4),
show that these have sequences similar to those of aggrecan1 and
aggrecan2 (Fig. 7), respectively. Therefore, it is not obvious why
modules 3 and 4 are inactive. The only significant difference is that
aggrecan4 does not have a basic residue at consensus position 2. However, given the importance of this sequence position in TSG-6 and
CD44 this may be enough to render this module, and hence the G2 domain, inactive.
The analysis described above does not exclude the possibility that
amino acids at other sequence positions are also involved in HA
binding. In this regard, visual inspection of the Lp2 model indicates
that Arg-66, Lys-85, and Tyr-87 (colored green on Figs. 7
and 8) may contribute to the interaction with HA because they are
located in close proximity to the consensus HA-binding residues. From
Fig. 8 it can be seen that Tyr-87 is at a location on the Link module
surface very similar to that usually occupied by consensus position 3 (which is a leucine in Lp2 rather than a tyrosine). It is also
interesting to note that all of the Link modules that have a leucine at
position 3 (Lp2, BRAL1-2, brevican2, and neurocan2) have an arginine in
an equivalent sequence site to Arg-66 in Lp2. Therefore, careful
analysis of the Link module alignment (Fig. 7) in conjunction with
individual Link module models (such as those for Lp2 in Fig. 8) allows
the identification of amino acids that have a reasonable probability of
being involved in HA binding. Clearly, not all of the residues
identified in this manner will be involved in binding, but as such
these provide excellent candidates for programs of site-directed mutagenesis.
Conclusions--
Site-directed mutagenesis has identified five
amino acids in the Link module of human TSG-6 which contribute to HA
binding. Comparison of this ligand-interaction surface with that
determined previously for CD44 has led to a prediction of the
HA-binding residues in other members of the Link module superfamily by
using a combination of sequence alignment and molecular modeling.
INTRODUCTION
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ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS AND DISCUSSION
REFERENCES
-helices and two triple-stranded
-sheets arranged around a large hydrophobic core (23).
EXPERIMENTAL PROCEDURES
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ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS AND DISCUSSION
REFERENCES
Ala,
Arg-8
Ala, Lys-11
Gln, Lys-13
Ala, Asn-67
Leu, Asn-67
Ser, Phe-70
Val, Lys-72
Ala, Ile-75
Ala, Asp-77
Ala, Arg-81
Ala, Glu-86
Ala, Glu-86
Ser, and Asp-89
Ala, respectively), whereas the degenerate primers 5, 7, and 14 had the
potential to alter Tyr-12, Tyr-59, or Tyr-78, respectively, to each of
four different residues (e.g. oligonucleotide 5 could change
Tyr-12 to Ala, Phe, Ser, or Val). DNA sequencing was used to identify
plasmids with the desired mutations, and these were used to transform
the E. coli expression strain BL21(DE3)pLysS (Novagen,
Madison, WI).
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Fig. 1.
DNA and translated protein sequences of the
VII-6-1mut8-5 plasmid used in site-directed mutagenesis. The amino
acids of Link_TSG6 targeted for mutagenesis are indicated in
bold; residues are numbered according to the sequence of the
expressed Link module (21). The sequences of oligonucleotides, denoted
1-18, used in the mutagenesis reactions are shown in
italics, aligned below the wild-type sequence, with the
altered nucleotides in bold. Oligonucleotides 1 and 2 are
selection primers that change the wild-type NcoI restriction
site (CCATGG) to NdeI (CATATG).
Oligonucleotide 1 is used in conjunction with the mutagenesis primers
3-18, whereas oligonucleotide 2 is a combined selection and
mutagenesis primer. Oligonucleotides 5, 7, and 14 are degenerate
primers, each with the potential to produce four different mutant
sequences.
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Fig. 2.
Alignment of Link_TSG6 with Lp1 and Lp2.
Lp1 and Lp2, which correspond to residues 159-257 and 259-354,
respectively, in human link protein (32), are aligned with residues
1-97 of Link_TSG6 (23). This alignment (23) was used for molecular
modeling of Lp1 and Lp2 on the basis of the Link_TSG6
coordinates.
RESULTS AND DISCUSSION
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ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS AND DISCUSSION
REFERENCES
4/
5 loop and have been
demonstrated to be perturbed significantly on binding to HA8 (27). Glu-6 and Lys-13 were selected because they have
been implicated in TGS-6-mediated inhibition of neutrophil migration in
an in vivo model of inflammation (16).
HA-binding activities of Link_TSG6 mutants and the effect of
mutagenesis on protein structure
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Fig. 3.
One-dimensional 1H-NMR spectra of
wild-type and mutant proteins. The wild-type Link_TSG6
(WT) has a well dispersed spectrum with the methyl
resonances of Val-57 (V57), which forms part of the stable
hydrophobic core, being shifted to high field. Y59F has a NMR spectrum
that is essentially identical to the wild-type and therefore can be
classified as having a wild-type fold. E86A has poorly dispersed
resonances, with no high field-shifted Val-57 methyls, and the spectrum
is characteristic of that of an unfolded protein. The spectrum of Y78S,
although having some features of a folded protein (i.e. with
high field shifted methyls), is significantly different from that of
wild-type. This mutant therefore can be classified as having a
perturbed fold.
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Fig. 4.
Comparison of the HA-binding activities of
Link module mutants with wild-type Link_TSG6. The binding of
biotinylated HA to wild-type (WT) or mutant proteins was
determined using a colorimetric assay in the absence or presence of
competing unlabeled HA (200-fold molar excess). Values are plotted as
the mean absorbance (n = 12) at 405 nm after a 10-min
development time ± the S.E. The mutants shown here are those that
have wild-type folds (Table I).
Binding constants for the interaction of selected mutants with
HA8
, and C
atoms) on binding to HA, which
includes Lys-11, Tyr-59, Phe-70, and Tyr-78. Other amino acids, located
on the
4/
5 loop (residues 61-74), which were found here not to
be involved in HA binding, also experienced large shift perturbations
(i.e. Asn-67 and Lys-72 (27)), indicating that this region
of the Link module undergoes a ligand-induced conformational change.
This structural alteration may be mediated, in part, by the interaction
of the aromatic ring of Phe-70 with HA.
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Fig. 5.
Position of the HA-binding site on
Link_TSG6. The TSG-6 Link module structure (23) is shown as a
space-filling representation (generated using the program RasMol (50))
in four orientations. Mutated amino acids are color-coded according to
the effect of the amino acid substitution on HA-binding activity or the
structural integrity of the Link module fold. Residues in which all of
the mutations made lead to a perturbed/unfolded structure are shown in
pink; no conclusions can be made about their role in ligand
binding. Amino acids that are important for HA binding (i.e.
the mutation leads to a large reduction in functional activity) are
colored red; those that are not involved are denoted in
green.
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Fig. 6.
Comparison of HA-binding sites on TSG-6 and
CD44. In A, the Link modules from TSG-6 and CD44
(modeled on the Link_TSG6 coordinates (4)) are shown in similar
orientations on the basis of their secondary structural elements.
Residues that are involved in HA binding in Link_TSG6 (determined here)
are colored red; amino acids of CD44 which are critical or
important for interaction with HA are shown in dark blue or
light blue, respectively. The functional residues of CD44
were identified by site-directed mutagenesis as described in Bajorath
et al. (14) and are numbered accordingly. All of the
HA-binding residues on the CD44 Link module are visible apart from
Lys-68, which is on the opposite face of the protein. B, the
Link_TSG6 structure (as in A) showing the 11 sequence
positions that can contribute to HA binding in TSG-6 and/or CD44 and
form a coherent patch on one face of the Link module surface. These are
color-coded, as described below, and numbered 1-11.
Sequence positions that are involved in HA binding in either TSG-6 or
CD44 alone are colored as in A (i.e.
red or blue, respectively). Positions 2 and 3, which mediate HA binding in both TSG-6 and CD44, are depicted in
purple.
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Fig. 7.
Alignment of Link module sequences.
Residues of TSG-6 and CD44 which have been demonstrated by mutagenesis
to interact with HA are colored as in Fig. 6A; amino acids
that are not involved in HA binding are shown in lowercase.
Asterisks denote sequence positions that can contribute to
HA binding in TSG-6 and/or CD44 (numbered 1-11 as in Fig.
6). These are colored (as in Fig. 6B) to indicate whether
the sequence position is functionally TSG-6-specific, CD44-specific, or
utilized by both proteins. This color coding is also used to indicate
whether an amino acid capable of making an interaction with HA
(i.e. salt bridges or hydrogen bonds) is found at these
positions in the Link modules from other members of the Link module
superfamily. Residues are underlined if they are identical
to, or a conservative replacement of, functional amino acids in TSG-6
or CD44. Residues shown in green in Lp2 may also be involved
in HA binding (see Fig. 8 and "Results and
Discussion").
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Fig. 8.
Putative HA-binding site on human link
protein. Lp1 and Lp2 were modeled on the basis of the Link_TSG6
coordinates. The models are shown in the same orientation (on the basis
of secondary structure elements) as for Link_TSG6 and CD44 in Fig. 6.
Amino acids that could participate in HA binding are colored (as in
Fig. 6B and Fig. 7) to indicate whether the sequence
position at which they are found is TSG-6-like (red),
CD44-like (dark or light blue), or common
(purple). In Lp2, additional amino acids can be identified
(green), which could contribute to HA binding and are in
close proximity to the consensus residues.
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ACKNOWLEDGEMENTS |
---|
We thank Iain D. Campbell for helpful advice; Robin T. Aplin for performing mass spectrometry; Valerie Cooper for synthesis of oligonucleotides; Antony C. Willis for amino acid analysis; and Jan D. Kahmann, Jennifer R. Potts, and Peter Teriete for assistance with NMR spectroscopy, which was performed at the Oxford Center for Molecular Sciences, funded by the Biotechnology and Biological Sciences Research Council, the Engineering and Physical Sciences Research Council, and the Medical Research Council. We are also grateful to Caroline M. Milner for critical review of the manuscript.
![]() |
FOOTNOTES |
---|
* This work was supported in part by Arthritis Research Campaign Grants D0540 and D0569 and by the Medical Research Council.The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
Recipient of a Biotechnology and Biological Sciences Research
Council studentship.
§ To whom correspondence should be addressed: MRC Immunochemistry Unit, Dept. of Biochemistry, University of Oxford, South Parks Rd., Oxford OX1 3QU, U. K. Tel.: 44-1865-275-349; Fax: 44-1865-275-729; E-mail: ajday@bioch.ox.ac.uk.
Published, JBC Papers in Press, April 3, 2001, DOI 10.1074/jbc.M100666200
2 S. Banerji, personal communication.
3 D. J. Mahoney, R. T. Aplin, A. Calabro, V. C. Hascall, A. J. Day, manuscript in preparation.
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ABBREVIATIONS |
---|
The abbreviations used are: HA, hyaluronan; HA8, an octasaccharide of HA; TSG-6, tumor necrosis factor-stimulated gene-6; Link_TSG6, the recombinant Link module from human TSG-6; EDAC, 1- ethyl-3-(3-dimethylaminopropyl)carbodiimide; MES, 2-[N-morpholino]ethane-sulfonic acid.
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