©1995 by The American Society for Biochemistry and Molecular Biology, Inc.
CD14: Physical Properties and Identification of an Exposed Site That Is Protected by Lipopolysaccharide (*)

(Received for publication, November 29, 1994; and in revised form, December 22, 1994)

Michael D. McGinley (§) Linda O. Narhi Michael J. Kelley Elyse Davy John Robinson Michael F. Rohde Samuel D. Wright (1)(¶) Henri S. Lichenstein

From the From Amgen Inc., Amgen Center, Thousand Oaks, California 91320 and the Laboratory of Cellular Physiology and Immunology, The Rockefeller University, New York, New York 10021

ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
FOOTNOTES
ACKNOWLEDGEMENTS
REFERENCES

ABSTRACT

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 sCD14bulletLPS complexes. The circular dichroism and fluorescence spectra of the sCD14 indicate that it contains substantial beta-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 sCD14bulletLPS 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 sCD14bulletLPS complex. These results suggest that a region including amino acids 57 to 64 is involved in LPS binding by sCD14.


INTRODUCTION

Soluble CD14 (sCD14) (^1)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 sCD14bulletLPS complexes remain stable over time(9) . Furthermore, we demonstrated that formation of sCD14bulletLPS complexes was a prerequisite for the responses of polymorphonuclear leukocytes and U373 cells to LPS(9, 10) . In order to understand how sCD14bulletLPS 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.


MATERIALS AND METHODS

Purification of sCD14

Recombinant human sCD14 was affinity-purified from CHO cell-conditioned medium as described previously(9) . In experiments utilizing deglycosylated sCD14 (dgsCD14), 0.2 mg/ml sCD14 was treated with 75 units/ml N-glycanase (Genzyme), 0.02% sodium azide, 10 mM Tris-HCl, pH 7.5, at 37 °C for 40 h. Neuraminidase (Calbiochem) was then added to 3.7 units/ml and incubated at 37 °C for an additional 8 h.

Isolation of sCD14bulletLPS Complexes

Complexes were formed as described (9) with some modifications. One ml of sCD14 or dgsCD14 (1 mg/ml protein) was incubated at 37 °C in PBS with 300 µl of 5 mg/ml LPS (Salmonella minnesota strain R595[Re], List Biological Laboratories) for 16 h at 37 °C. To remove unbound LPS, the mixture was passed over a 1.5 times 50 cm Sephacryl 200 (Pharmacia Biotech Inc.) column that had been equilibrated in PBS, pH 7.4. Fractions containing the sCD14bulletLPS complex were then concentrated in a Microcon 10 concentrator (Amicon, Inc.). Complex formation was confirmed by silver staining samples run on 10% native polyacrylamide gels (Novex) in 25 mM Tris, 160 mM glycine, pH 8.8, at 75 mA as described(9) .

Circular Dichroism Spectra

Circular dichroism (CD) analysis was performed on a Jasco J-720 spectropolarimeter controlled by a DOS-based computer using a cuvette with a path length of 1 cm for the near-UV region (340-240 nm) and 0.02 cm for the far-UV region (250-190 nm). Protein concentrations were determined from the absorbance at 280 nm assuming an of 0.89 for a 0.1% protein solution at ambient temperature (theoretical calculation), and ellipticity calculations were made assuming a mean residue weight of 107. The spectra shown represent the average of two different samples.

Fluorescence Spectroscopy

The intrinsic fluorescence of sCD14 was determined using an SLM-Aminco 500S spectrofluorimeter controlled by a DOS-based computer. Slit widths were set at 5 nm, and the emission spectra were obtained by exciting 0.2 mg/ml protein at 280 nm, using a cuvette with a path length of 0.5 cm.

Limited Proteolysis

All proteases used were of sequencing grade (Boehringer Mannheim). sCD14 and sCD14bulletLPS complex, prepared as described above, were diluted to 1 mg/ml in PBS. This solution was then made 20 mM in Tris-HCl at pH 8.0 and incubated at 4 °C, 23 °C, or 37 °C with a 1 µg/ml concentration of the selected protease. For time course experiments, samples of 5 µg of protein were removed at the desired times, mixed with Novex SDS Tricine sample buffer, and stored at 4 °C. Before or after boiling, the SDS unfolded the AspN and proteolysis stopped immediately; boiling did not change the gel profile. Samples were then analyzed by SDS-PAGE on 10-20% gels in Tris-Tricine (Novex) and run at 75 mA/gel.

HPLC

Trifluoroacetic acid (Pierce) was added to the sCD14 digests to a final concentration of 0.1% trifluoroacetic acid. The acidified sCD14 digests were then injected on a Hewlett Packard Model 1090 high performance liquid chromatograph (HPLC) equipped with a 4.6 times 250 cm C4 reverse phase column (Vydac) and a diode array detector. The column was previously equilibrated with 100% buffer A (0.1% trifluoroacetic acid in water (v/v)) (Burdick Jackson). Elution was performed at a flow rate of 0.75 ml/min using a linear gradient of 0 to 60% buffer B (90% acetonitrile (Burdick Jackson) in water with 0.09% trifluoroacetic acid) increasing at a rate of 0.75%/min. The elution of peptides was monitored at 214 nm, and fractions were collected manually. Amino-terminal sequence analysis and mass spectrometry were performed on selected fractions.

Amino-terminal Sequence Analysis

Amino-terminal sequence analysis was performed by loading aliquots of sCD14 and sCD14bulletLPS peptides on the reverse phase portion of biphasic sequencing columns. The columns were then sequenced on a Hewlett Packard G1000A protein sequencer with on-line phenylthiohydantoin amino acid analysis performed by an in-line Hewlett Packard 1090 HPLC. Quantitation of peptides observed are based on either direct calculation from an observed sequence level with correction for amount loaded and initial sequence yields, or by external standard area calculations based on direct sequence recoveries from identical maps.

Mass Spectrometry

Mass spectra of selected HPLC fractions were obtained by spotting fractions and matrix (either alpha-cyano-4-hydroxycinnamic acid or 2,5-dihydrobenzoic acid (Kratos, Manchester, UK, MALDI grade)) on a sample slide and running the slide on the Kratos MALDI III time of flight matrix-assisted laser desorption mass spectrometer (LD/MS). Mass spectra of selected fractions were also obtained by flow injection on the Sciex API III electrospray mass spectrometer (ESI/MS).


RESULTS

Isolation of sCD14bulletLPS Complexes

We have shown (9) that addition of LPS to sCD14 at a ratio of 1:1.5 (w/w) results in the formation of biologically active complexes which exhibit a shift in mobility on native polyacrylamide gels. To further characterize sCD14bulletLPS interactions, it was necessary to isolate preparative amounts of this complex. Therefore, sCD14 and LPS were incubated at a ratio of 1:1.5 (w/w), and complexes were isolated from free LPS by gel filtration chromatography. Fig. 1shows that all sCD14 prepared by this method exhibited the shifted mobility characteristics of LPSbulletsCD14 complexes. Free LPS appears as a silver-stained band near the top of native gels(9) . The absence of such a band in our preparations (Fig. 1) confirms the effectiveness of our purification procedure.


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, sCD14bulletLPS complex (5 µg); lane 4, dgsCD14 (5 µg); lane 5, dgsCD14bulletLPS complex (5 µg).



Solution Structure of sCD14 and sCD14bulletLPS Complexes

CD and fluorescence spectroscopies were used to probe the solution structure of sCD14 and sCD14bulletLPS complexes. The far-UV CD spectra of both the sCD14 and the sCD14bulletLPS complex are shown (Fig. 2A). Far-UV CD spectra arise from peptide bonds located in an asymmetric environment such as that found in a regular secondary structure (see, for example, (11) ). The secondary structure of a new protein can be assessed by comparing its spectra to those of peptides and proteins of known structure. The spectra of sCD14 and the sCD14bulletLPS complex are very similar and consist of a single trough with a minimum at 215 nm and a maximum at 190 nm. This is the spectrum of a classic beta-sheet protein, with no alpha-helix present(11) .


Figure 2: A, far-UV spectra of sCD14 (-) and sCD14bulletLPS complexes(- - -). B, near-UV spectra of sCD14 (-) and sCD14bulletLPS 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 sCD14bulletLPS 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 sCD14bulletLPS 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 sCD14bulletLPS 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 sCD14bulletLPS(- - -) 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 sCD14bulletLPS 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.

Susceptibility of sCD14 or sCD14bulletLPS Complexes to Proteolysis

Since LPS did not significantly alter the secondary or tertiary structure of sCD14, the technique of limited proteolysis was used to probe changes in the accessibility of sCD14 or sCD14bulletLPS surfaces to proteases. In this technique, native protein is incubated with a protease, and the number, size, and amino acid sequence of the generated peptides are determined. Only peptide bonds that are accessible to the protease will be cleaved, while susceptible peptide bonds that are inaccessible to the protein (either by being located in the interior of the protein core or by being protected by ligand) will not be cleaved(12, 13, 14) . Since we cannot detect structural changes in sCD14 upon LPS binding, any difference in peptide maps between sCD14 and sCD14bulletLPS may signify protection of that surface or region by the bound LPS.

Pilot experiments were performed to determine which proteases were most suitable for limited proteolysis of sCD14. Selected proteases were mixed with sCD14 or sCD14bulletLPS 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 sCD14bulletLPS was slower than that of sCD14 alone and resulted in a limit digest of fewer peptides.

Chymotrypsin was more efficient in digesting sCD14 and sCD14bulletLPS complex than AspN but nonetheless resulted in several moderate-sized peptides which could be reproducibly generated. The sCD14bulletLPS 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 sCD14bulletLPS complexes.

Limited Proteolysis of sCD14 and sCD14bulletLPS Complexes by AspN

The CD14 sequence contains 16 potential AspN cleavage sites distributed throughout the molecule (Fig. 4). Nevertheless, AspN digestion yielded a single dominant species on SDS gels (Fig. 5). To map the location of the dominant cleavage site, individual peptides derived by AspN digestion of sCD14 were isolated by HPLC (Fig. 6) and subjected to amino-terminal sequence analysis and mass spectrometry. The results of this analysis (Table 1) indicate that the most susceptible AspN cleavage sites in CD14 are located before aspartic acid residues 57, 59, and 65. Cleavage at these sites results in release of peptides of amino acids 1-56, 1-58, 56-64, 58-64, and 65-348. We also observed some cleavage before aspartic residues 284, 297, and 308; comparison of the amounts of these peptides generated indicate that cleavage at these sites occurs with greatly decreased frequency when compared to the region between amino acids 57 and 64.


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 sCD14bullet/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 sCD14bulletLPS 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 sCD14bulletLPS 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 sCD14bulletLPS 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 sCD14bulletLPS 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.

Limited Proteolysis of dgsCD14 and dgsCD14bulletLPS Complexes by AspN

The lack of digestion at various AspN sites in sCD14 or sCD14bulletLPS complexes could be due to protection of susceptible bonds by the structure of the folded protein or protection by carbohydrate at putative N-linked glycosylation sites (Fig. 4) on the protein surface. To address these possibilities, we prepared dgsCD14 by incubating sCD14 with Nglycanase and neuraminidase. After enzymatic deglycosylation, dgsCD14 had an apparent M(r) of 42,000 when analyzed by reducing SDS-PAGE (data not shown). Since fully deglycosylated sCD14 would be expected to have an M(r) of 37,216 based on its amino acid sequence, it is possible that dgsCD14 bears O-linked carbohydrates.

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 dgsCD14bulletLPS 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 sCD14bulletLPS complexes in that very little digestion was observed in the dgsCD14bulletLPS 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.

Limited Proteolysis with Chymotrypsin

In order to corroborate the results obtained with AspN, we used chymotrypsin, which cleaves in hydrophobic regions, in limited proteolysis studies. The digestion with chymotrypsin was more complicated than with AspN because it never reached a limit digest. One of the most abundant peaks in the sCD14 peptide map, however, was a peptide from amino acid 51 to 63 (1300 pmol). Chymotrypsin digestion of the sCD14bulletLPS complex released significantly lower levels of this peptide (less than 100 pmol, data not shown). Moreover, no peptides could be observed in this digest with either an amino or carboxyl terminus within this region. The digestion with chymotrypsin thus verifies the AspN results. In both cases, the region from residues 51 to 64 appears to be protected by LPS binding.


DISCUSSION

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 sCD14bulletLPS 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 sCD14bulletLPS 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 sCD14bulletLPS complexes.

An alternative hypothesis to explain how sCD14bulletLPS 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. (^2)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 sCD14bulletLPS 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 sCD14bulletLPS 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.


FOOTNOTES

*
The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore by hereby marked ``advertisement'' in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

§
To whom correspondence and reprint requests should be addressed. Tel.: 805-447-4076; Fax: 805-499-7464.

Supported by United States Public Health Service Grant AI-30556.

(^1)
The abbreviations used are: sCD14, soluble CD14; AspN, endoproteinase Asp-N; CHO, Chinese hamster ovary; dg, deglycosylated; HPLC, high performance liquid chromatography; LPS, lipopolysaccharide; PBS, phosphate-buffered saline; PAGE, polyacrylamide gel electrophoresis.

(^2)
sCD14 acts as a shuttle in the neutralization of LPS by lipopolysaccharide-binding protein and high density lipoprotein (Wurfel, M. W., Hailman, E., and Wright, S. D.(1995) J. Exp. Med., in press).


ACKNOWLEDGEMENTS

We thank Joan Bennett for preparing the manuscript, Jennifer Keysor for preparing the figures, and Mark Zukowski and Tsutomu Arakawa for reviewing the manuscript.


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©1995 by The American Society for Biochemistry and Molecular Biology, Inc.