(Received for publication, November 29, 1994; and in revised form, December 22, 1994)
From the
Soluble CD14 (sCD14) is a 55-kDa serum protein that binds
lipopolysaccharide (LPS) and mediates LPSdependent responses in a
variety of cells. Using recombinant sCD14 expressed in Chinese hamster
ovary (CHO) cells, we examined the structural characteristics of sCD14
and sCD14LPS complexes. The circular dichroism and fluorescence
spectra of the sCD14 indicate that it contains substantial
-sheet
(40%) and a well-defined tertiary structure with the tryptophan
residues located in environments with different degrees of
hydrophobicity and solvent exposure. The spectra of the sCD14
LPS
complex are identical within experimental error to the uncomplexed
sCD14. Changes in surface accessibility upon LPS binding were examined
using limited proteolysis with endoproteinase Asp-N. This analysis
revealed that aspartic acid residues at amino acids 57, 59, and 65 are
susceptible to cleavage by Asp-N, while the same residues are protected
from proteolytic cleavage in the sCD14
LPS complex. These results
suggest that a region including amino acids 57 to 64 is involved in LPS
binding by sCD14.
Soluble CD14 (sCD14) ()is a 55-kDa protein found in
serum at concentrations of 2-6 µg/ml(1, 2) .
Recent data have shown that sCD14 mediates the lipopolysaccharide
(LPS)-dependent activation of endothelial cells, epithelial cells, and
polymorphonuclear
leukocytes(3, 4, 5, 6, 7, 8) .
Complexes of LPS and sCD14 thus appear to be a key intermediate in the
cellular responses to LPS that underlie septic shock.
Recently, we
showed that sCD14 binds LPS stoichiometrically and that the resulting
sCD14LPS complexes remain stable over time(9) .
Furthermore, we demonstrated that formation of sCD14
LPS complexes
was a prerequisite for the responses of polymorphonuclear leukocytes
and U373 cells to LPS(9, 10) . In order to understand
how sCD14
LPS complexes activate cells, we initiated a series of
experiments detailing the physical characteristics associated with LPS
binding to sCD14.
In this report, we utilized the techniques of circular dichroism (CD) and intrinsic fluorescence to analyze the secondary structure of sCD14 in the presence or absence of LPS. We also used limited proteolysis to determine whether the binding of LPS to sCD14 alters the susceptibility of sCD14 to proteolytic digestion. These experiments reveal that LPS does not significantly change the overall structure or stability of sCD14 but does protect a specific site in sCD14 from digestion by proteases.
Figure 1:
Native PAGE of
CD14 samples visualized by silver staining. Lane 1, 8 µg
each of reference protein; lane 2, sCD14 (5 µg); lane
3, sCD14LPS complex (5 µg); lane 4, dgsCD14 (5
µg); lane 5, dgsCD14
LPS complex (5
µg).
Figure 2:
A,
far-UV spectra of sCD14 (-) and sCD14LPS
complexes(- - -). B, near-UV spectra of
sCD14 (-) and sCD14
LPS complexes(- -
-). The analysis was performed in PBS as described under
``Materials and Methods.''
Near-UV CD spectra arise from the
location of the aromatic amino acids and disulfide bonds in an
asymmetric environment and can be used as a probe of tertiary
structure. The near-UV CD spectra of the sCD14 and sCD14LPS
complex are also shown (Fig. 2B). The spectra are
characterized by two predominant maxima (at 294 and 285 nm) which are
attributable to the presence of tryptophan and tyrosine in a rigid
environment. The spectra for sCD14 and sCD14
LPS complex are
identical within experimental error and indicate that there is no
change in the environment of the aromatic amino acids in sCD14 upon LPS
binding.
We also utilized fluorescence spectroscopy as a probe of
tertiary structure. This technique yields a sensitive measure of the
environment surrounding tryptophan residues. The intensity and
wavelength of fluorescence reflect the hydrophobicity of the tryptophan
environment. The fluorescence spectra of uncomplexed sCD14 and
sCD14LPS complex (Fig. 3) show a broad peak from 330 to
346 nm. This indicates that tryptophans in sCD14 are located in
different environments varying from the interior of the protein, in a
fairly hydrophobic, solvent-protected environment (330 nm), to a much
less hydrophobic, solvent-exposed environment closer to the surface of
the sCD14 (346 nm). The fluorescence spectra are also identical within
experimental error and are consistent with the CD results, i.e. no conformational change and no involvement of tryptophan residues
in LPS binding.
Figure 3:
Fluorescence emission spectra of sCD14
(-) and sCD14LPS(- - -) complexes.
The spectra were taken at 0.22 mg/ml protein, with excitation at 280
nm, in PBS as described under ``Materials and
Methods.''
The conformational stability of sCD14 and
sCD14LPS were also assessed by following changes in ellipticity
at 215 nm as the temperature was raised. Both samples melted in a
cooperative transition with a midpoint transition temperature of 56
°C (data not shown). This melting temperature is in the range seen
for many proteins and indicates that sCD14 is folded with a reasonable
conformational stability. Binding of LPS did not affect the stability
of sCD14 to temperature.
Pilot
experiments were performed to determine which proteases were most
suitable for limited proteolysis of sCD14. Selected proteases were
mixed with sCD14 or sCD14LPS complex and incubated at 4 °C,
room temperature, or 37 °C. Samples were removed at several time
points and analyzed by SDS-PAGE and reverse phase HPLC to determine the
degree of digestion. Free or complexed sCD14 was relatively resistant
to digestion by trypsin and endoproteinase Glu-C, but was extremely
sensitive to subtilisin protease. Chymotrypsin and AspN gave
intermediate results. AspN slowly digested sCD14 until a limit digest
was reached at 10 h, after which very little change in the peptide map
occurred. The rate of digestion of sCD14
LPS was slower than that
of sCD14 alone and resulted in a limit digest of fewer peptides.
Chymotrypsin was more efficient in digesting sCD14 and
sCD14LPS complex than AspN but nonetheless resulted in several
moderate-sized peptides which could be reproducibly generated. The
sCD14
LPS complex was digested significantly more slowly than
sCD14 alone, again with fewer peptides generated. Therefore, AspN and
chymotrypsin were both used in the further analysis of sCD14 and
sCD14
LPS complexes.
Figure 4:
Putative AspN protease and N-glycosylation sites on sCD14. AspN protease sites are
indicated by an arrow (), and putative N-glycosylation
sites are indicated by an asterisk (*).
Figure 5:
SDS-PAGE of time course of AspN
proteolysis. sCD14 (A) and sCD14/LPS complexes (B) were incubated with AspN at 37 °C for 0 h (lane
1), 1 h (lane 2), 3 h (lane 3), 6 h (lane
4), and 24 h (lane 5) and analyzed on a 10-20%
Tricine gel. Proteins were visualized with Coomassie stain. Molecular
weight standards are as indicated in lane
6.
Figure 6:
A,
HPLC chromatogram of the endoproteinase Asp-N digest of sCD14. B, HPLC chromatogram of the endoproteinase Asp-N digest of
sCD14LPS complex. Digestion was carried out for 16 h at 37 °C
with a 1:1000 enzyme to substrate ratio. Arrows indicate
peptides subjected to amino-terminal sequencing and mass spectrometry
as listed in Table 1.
The
sCD14LPS complex was much less susceptible to digestion than
sCD14. Under conditions identical with those used above, no cleavage
was observed before aspartic acid residues which were susceptible to
cleavage in uncomplexed sCD14 ( Fig. 5and Fig. 6). The
cleavage observed in the sCD14
LPS complex at amino acid 327
results from Asn-Ser rearrangement. The resulting peptide is not seen
in the sCD14 and is likely to occur during the incubation of sCD14 with
LPS. In experiments where more vigorous proteolysis conditions were
used (i.e. longer incubation or more enzyme), we observed
cleavage before aspartic acid residues 284, 297, and 308 but not before
aspartic acid residues 57, 59, and 65 in the sCD14
LPS complex
(data not shown). These results suggest that the binding of LPS to
sCD14 results in the protection of a region spanning amino acids
57-64 from proteolytic digestion.
dgsCD14 was tested for its ability to bind LPS.
Native PAGE showed that dgsCD14 retained the ability to form stable
complexes with LPS (Fig. 1). This observation indicates that N-linked carbohydrates are not necessary for binding of LPS to
sCD14. dgsCD14 and dgsCD14LPS complexes were subjected to limited
proteolysis by AspN, and the resulting peptides were resolved by HPLC.
The results of this analysis (data not shown) are similar to that seen
for glycosylated sCD14 and sCD14
LPS complexes in that very little
digestion was observed in the dgsCD14
LPS complexes. Some low
level peaks were observed in the dgsCD14 AspN digest that were not
observed in the sCD14 AspN digest. Amino-terminal sequence analysis of
these peptides indicates that these peaks are due to the deamidation of
Asn
to Asp
during the removal of
carbohydrate by N-glycanase. This process yields a new
potential AspN cleavage site.
From these results we conclude that N-linked carbohydrate does not protect putative AspN cleavage sites located in the amino-terminal 50 amino acids of sCD14 from digestion. This suggests that sites which are resistant to cleavage by AspN in this region may be located in a tightly folded domain that is inaccessible to the protease. We also conclude that N-linked carbohydrate is not required for LPS binding to sCD14 nor does it affect the ability of LPS to protect a region spanning amino acids 57-64 from proteolytic digestion.
Using CD and fluorescence spectroscopy, we found that sCD14
did not undergo a major conformational change upon LPS binding. If
conformational changes did occur, they likely would be confined to the
amino-terminal 152 amino acids of sCD14 (sCD14)
as we have shown that sCD14
binds LPS and
activates cells normally(8) . There are four tryptophan
residues among amino acids 1-152 and only one tryptophan residue
in amino acids 153-348. Since intrinsic fluorescence reports the
environment of tryptophan residues, our analysis focuses on this
precise portion of the sCD14 molecule. Fluorescence was unaffected by
LPS binding, indicating that there is no change in the local
environment caused by binding of LPS. We cannot rule out the
possibility, however, that LPS did induce small, local changes in sCD14
that were not detected in our analysis of the overall conformation of
sCD14, but any such change in conformation would have to affect an
extremely small region of sCD14.
Recent reports have demonstrated
that sCD14LPS complexes cause strong cellular responses in a
variety of cells. However, the mechanism by which cellular activation
is achieved by these complexes is not understood. One hypothesis which
has been proposed is that sCD14
LPS complexes activate cells
through an as yet unidentified cellular
receptor(3, 6) . Since uncomplexed sCD14 is unable to
activate cells, this model would predict that LPS induces a
conformational change in sCD14, thus enabling sCD14 to interact with a
receptor. Data in this report argue against such a model of cellular
activation by sCD14
LPS complexes.
An alternative hypothesis to
explain how sCD14LPS complexes activate cells is that sCD14 acts
to facilitate the transfer of LPS into cell membranes. In this model,
there is no requirement for any change in the conformation of sCD14 to
occur upon LPS binding. In support of this model, we have recently
shown that sCD14 acts as a lipid transfer protein, moving LPS into high
density lipoprotein particles. (
)The role of this LPS
transfer activity in mediating cellular responses to LPS is currently
under study.
In this report, we also demonstrate that AspN protease
cleaves sCD14 before aspartic acid residues 57, 59, and 65.
Furthermore, AspN cleavage at these residues is greatly reduced in
sCD14LPS complexes. These data, combined with the observation
that little AspN cleavage is observed at the other 13 potential
cleavage sites in sCD14, suggest that sCD14 is comprised of two
tightly-folded domains which are connected by a solvent-accessible
bridge spanning amino acids 57-64. Proteolysis with chymotrypsin
verified these results. Based on protection of this region from limited
proteolysis, we would predict that this bridge binds LPS. In our
accompanying paper(10) , we test this hypothesis by performing
site-directed mutagenesis in the putative bridge region.
sCD14 that
was enzymatically deglycosylated at N-linked sites was still
able to form complexes with LPS, as determined by shifts in mobility on
native gels (Fig. 1). The dgsCD14 could also activate cells in vitro (data not shown). This demonstrates that the N-linked sugars are not involved in the binding of LPS by
sCD14, nor in the activation of cells by the sCD14LPS complex.
The AspN digestion pattern of the dgsCD14 was also similar to that of
the native protein. In particular, the amino-terminal 56 amino acids
remained fairly intact, indicating that the protection of AspN sites
from proteolysis which we see with native sCD14 is due to the structure
of the protein itself and not to the presence of carbohydrates on the
surface. These results suggest that N-linked carbohydrates
have little effect on the structure or function of sCD14 and may serve
only to modulate the longevity of sCD14 in the circulation or at the
cell surface.