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J. Biol. Chem., Vol. 279, Issue 51, 53282-53287, December 17, 2004

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Identification of a Region in the Vitamin D-binding Protein that Mediates Its C5a Chemotactic Cofactor Function^*

Jianhua Zhang and Richard R. Kew

From the Department of Pathology, Stony Brook University School of Medicine, Stony Brook, New York 11794-8691

Received for publication, October 7, 2004

	ABSTRACT

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The vitamin D-binding protein (DBP), also known as group-specific component or Gc-globulin, is a multifunctional plasma protein that can significantly enhance the leukocyte chemotactic activity to C5a and C5a des-Arg. DBP is a member of the albumin gene family and has a triple domain modular structure with extensive disulfide bonding that is characteristic of this protein family. The goal of this study was to identify a region in DBP that mediates the chemotactic cofactor function for C5a. Full-length and truncated versions of DBP (Gc-2 allele) were expressed in Escherichia coli using a glutathione S-transferase fusion protein expression system. The structure of the expressed proteins was confirmed by SDS-PAGE and immunoblotting, whereas protein function was verified by quantitating the binding of [³H]vitamin D. Dibutyryl cAMP-differentiated HL-60 cells were utilized to test purified natural DBP and recombinant expressed DBP (reDBP) for their ability to enhance chemotaxis and intracellular Ca²⁺ flux to C5a. Natural and full-length reDBP (458 amino acid residues) as well as truncated reDBPs that contained the N-terminal domain I (domains I and II, residues 1–378; domain I, residues 1–191) significantly enhanced both cell movement and intracellular Ca²⁺ concentrations in response to C5a. Progressive truncation of DBP domain I localized the chemotactic enhancing region between residues 126–175. Overlapping peptides corresponding to this region were synthesized, and results indicate that a 20-amino-acid sequence (residues 130–149, 5'-EAFRKDPKEYANQFMWEYST-3') in domain I of DBP is essential for its C5a chemotactic cofactor function.

	INTRODUCTION

TOP ABSTRACT INTRODUCTION MATERIALS AND METHODS RESULTS DISCUSSION REFERENCES

 
Vitamin D-binding protein (DBP)¹ is a multifunctional and highly polymorphic plasma protein synthesized primarily in the liver (1, 2). DBP is expressed as a single polypeptide chain with a molecular mass of 56 kDa and circulates in plasma at 6–7 µM (1, 2). Due to its extensive polymorphisms, DBP initially was named the group-specific component of serum, later shortened to Gc-globulin (3). DBP is a member of the albumin (ALB), -fetoprotein (AFP), and -albumin/afamin (AFM) gene family and thus has the characteristic multiple disulfide-bonded, triple domain modular structure (1). Besides functioning as a circulating vitamin D transport protein, it has been demonstrated that plasma DBP effectively scavenges G-actin released at sites of necrotic cell death and prevents polymerization of actin in the circulation (1, 2). Distinct binding regions within the 458-amino-acid sequence of DBP have been identified: a vitamin D sterol binding segment in the N-terminal domain (amino acids 35–49) and a G-actin binding region in the C-terminal domain (amino acids 373–403) (4, 5). More recent work on the crystal structure of DBP (bound to either vitamin D₃ or actin) has confirmed the vitamin D sterol binding site but has demonstrated that actin interacts with distinct amino acid sequences in all three DBP domains (6–9).

Complement C5a is a 74-amino-acid peptide generated by limited proteolytic cleavage of C5 during complement activation (10). C5a is a very potent chemotactic factor for all leukocytes as well as several other cell types, and the peptide has several other proinflammatory functions as well (10). C5a exerts these activities by binding to its high affinity receptor (C5aR or CD88) on the plasma membrane of target cells (11). Several groups have demonstrated that purified DBP can significantly enhance the neutrophil chemotactic activity (i.e. co-chemotatic activity) of C5a and its stable breakdown product C5a des-Arg (12–17). In addition to neutrophils, DBP can also augment the C5a chemotactic activity for monocytes and fibroblasts (18, 19). The chemotactic enhancing properties of DBP appear to be restricted to C5a/C5a des-Arg since this protein cannot enhance the chemotactic activity of formylated peptides, IL-8, leukotriene B₄, or platelet-activating factor (12, 13, 16, 18). The mechanisms by which DBP acts as a chemotactic cofactor for C5a are not known. The goal of this study was to determine whether a cochemotactic region could be located within DBP. The results demonstrate for the first time that DBP enhances both C5a-mediated chemotaxis and Ca²⁺ influx in differentiated HL-60 cells. In addition, a 20-amino-acid sequence within the N-terminal domain I of DBP (residues 130–149) was found to possess the C5a chemotactic cofactor activity.

	MATERIALS AND METHODS

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Reagents—Recombinant C5a was purchased from Sigma. Purified human DBP was obtained from Biodesign International (Kennebunkport, ME). Full-length human DBP cDNA (Gc-2 allele), clone number CS0DM004YF02, was purchased from Invitrogen. DNA restriction and modification enzymes were purchased from New England Biolabs (Beverley, MA). Oligonucleotides were synthesized by Invitrogen. Anti-human DBP was purchased from DiaSorin, (Stillwater, MN). pGEX-4T-2 expression vector was purchased from Amersham Biosciences. Dibutyryl-cAMP (Bt₂cAMP), Me₂SO, and protease inhibitor mixture were obtained from Sigma. Complement-activated serum using yeast cell walls (zymosan A) was prepared as described previously (20). DBP-derived peptides, peptide 1 (residues 130–152, EAFRKDPKEYANQFMWEYSTNYG) and peptide 2 (residues 150–172, NYGQAPLSLLVSYTKSYLSMVGS), were synthesized and purified (>95% purity) by the American Peptide Company, Inc. (Sunnyvale, CA).

Cells and Cell Culture—Human promyelocytic cell line HL-60 was obtained from ATCC (Rockville, MD) and cultured in RPMI 1640 medium supplemented with 10% fetal bovine serum. Differentiation of HL-60 cells was initiated with 250 µM Bt₂cAMP for 48 h.

Construction of Full-length and Truncated Recombinant Expressed DBP (reDBP)—To construct GST fusion proteins, DNA sequences corresponding to the indicated regions of full-length human DBP or DBP domains were amplified by PCR, designed to generate products with 5' BamHI and the 3' XhoI restriction site. The Escherichia coli-expressed proteins were designated as follows: full-length reDBP (residues 1–458), DBP domains I and II (residues 1–378), DBP domains II and III (residues 192–458), DBP domain I (residues 1–191), DBP domain II (residues 192–378), DBP domain III (residues 379–458) were cloned into the corresponding site using pGEX-4T-2 expression plasmid as GST fusion partners in E. coli (sequences of all primers available from the authors upon request). E. coli strain XL-1 Blue (Stratagene, CA) served as host for DNA manipulation, and the E. coli strain BL-21 served the host for protein expression. Truncated versions of domain I (residues 1–175, 1–150, 1–125, and 1–112) also were expressed in E. coli as described above. Each construct was confirmed by DNA sequencing.

Expression and Purification of Full-length and Truncated reDBP— DBP was expressed in E. coli according to the protocol described by Swamy et al. (21). BL21 cells carrying pGEX-4T-2 plasmids expressed fusion protein GST linked to the DBP constructs. E. coli were grown at room temperature (22–24 °C) in Luria broth (LB) containing 100 µg/ml ampicillin and 50 µg/ml chloramphenicol until absorbance at 600 nm (A₆₀₀) was 0.4–0.6. The expression of fusion proteins was induced by the addition of 0.5–1.0 mM isopropyl-1-thio--D-galactopyranoside for 4–8 h. Cells were collected by centrifugation at 5000 x g. The E. coli pellets (from 1 liter) were resuspended in 40 ml of Tris-buffered saline (50 mM Tris-HCl, pH 8.3, 150 mM NaCl) containing 0.2 mg/ml lysozyme, 0.1% Triton X-100, and 2 ml of a protease inhibitor mixture. The cells were disrupted by sonication, and insoluble material was removed by centrifugation at 12,000 x g for 20 min. The detergent-soluble cell lysate containing the expressed fusion protein was mixed with 1 ml of a 50% slurry of glutathione-agarose, preequilibrated in Tris-buffered saline. After several washes with Tris-buffered saline, the bound GST·DBP was eluted with 20 mM oxidized glutathione in 100 mM Tris buffer, pH 8.3. Fusion protein was cleaved with thrombin and dialyzed against PBS to remove oxidized glutathione, and GST was separated from reDBP using a glutathione-Sepharose affinity column. Sequencing fidelities of the reDBPs were confirmed by N-terminal sequencing.

Vitamin D₃ Binding Assay—Competitive binding assays of natural DBP full-length and truncated reDBP with [³H]25-OH-D₃ were carried out according to a published procedure with minor modifications (5). In a typical experiment, solutions containing natural DBP, reDBP (200 ng), [³H]25-OH-D₃ (0.1 pmol), 25-OH-D₃ (0.5–64 pmol) in 10 µl with 490 µl of assay buffer (50 mM Tris-HCl, pH 8.3, 150 mM sodium chloride, 1.5 mM EDTA, 0.1% Triton X-100) were incubated at 4 °C for 20 h followed by treatment with 100 µl of 2.5% ice-cold dextran-coated charcoal and centrifugation at 5000 x g at 4 °C. Clear supernatants from the centrifuged samples were mixed with the scintillation mixture and counted for radioactivity. Each sample was assayed in triplicate.

Ligand-induced Calcium Mobilization—Calcium mobilization studies were performed using the Fluo-3 AM probe as described previously (22). Differentiated HL-60 cells (1 x 10⁷ cells/ml) were resuspended in HBSS, 1% BSA containing 2 µM Fluo-3 AM (Molecular Probes, Eugene, OR) and incubated at 37 °C for 40 min. Cells incubated without the dye were used as a control to measure autofluorescence (F_min). Following the dye uptake, cells were washed twice and then suspended at 5 x 10⁶ cells/ml in HBSS containing 1% BSA. Half of the cell suspension was treated with 50 nM DBP for 30 min at room temperature, and the other half served as the untreated control. For each measurement, 400 µl of cell suspension (with or without DBP) was added to a cuvette and stimulated with various concentrations of purified C5a at room temperature (22–24 °C). Fluorescence was then measured immediately using a PerkinElmer Life Sciences LS-5 fluorometer at 505-nm excitation, 526-nm emission for Fluo-3 AM. F_max was measured by treating labeled cells with 60 µM digitonin. Intracellular free calcium concentrations were calculated using the following formula: (Ca²⁺) = K_d (F – F_min)/(F_max – F), where K_d = 325 nM for Fluo-3 according to the manufacturer (Molecular Probes).

Chemotaxis Assay—Chemotaxis was performed as described previously (23). In brief, 35 µl of C5a (0.01–1 nM) in the chemotaxis buffer (HBSS with 1% BSA, 10 mM HEPES) was placed in duplicate in the lower chamber of a 48-well chamber (Neuroprobe, Gaithersburg, MD). The lower compartments were covered with a 5-µm pore cellulose nitrate filter, and then 50 µl of the cell suspension (2.5 x 10⁵/well) was pipetted into the upper compartments. The chemotactic chamber was incubated for 1 h at 37° in a humidified incubator with 5% CO₂. Following incubation, the filter was fixed, stained, and mounted on a microscope slide. Cell movement was measured as the distance in microns that the leading front of the cells had migrated into the filter (24).

Data Analysis and Statistics—Results of several experiments were analyzed for significant differences among group means using analysis of variance followed by Newman-Keul's multiple comparisons post-test utilizing the statistical software program InStat (GraphPad Software, San Diego, CA).

	RESULTS

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DBP Enhances C5a-induced Chemotaxis and Ca²⁺ Influx in Differentiated HL-60 Cells—Previous published work in our laboratory has demonstrated that neutrophils co-incubated with DBP display significant increased movement to suboptimal concentrations (10–100 pM) of C5a, i.e. the cochemotactic effect (12, 23, 25). However, neutrophils are short-lived, terminally differentiated cells and cannot be genetically manipulated. A cell culture model for neutrophils is the promyelocytic cell line HL-60 that can be induced to differentiate into neutrophil-like cells using agents such as Me₂SO or Bt₂cAMP. Differentiation of HL-60 cells for 48 h using 250 µM Bt₂cAMP induced expression of the C5a receptor and both permitted chemotaxis to C5a alone and significantly enhanced movement to C5a in the presence of purified natural DBP (Fig. 1A). These results are consistent with our previous reports using neutrophils and indicate that differentiated HL-60 cells will serve as a good cell culture model to investigate the cochemotactic function of DBP.

View larger version (19K): [in this window] [in a new window]   FIG. 1. Effect of DBP on C5a-mediated responses in differentiated HL-60 cells. HL-60 cells were differentiated using 250 µM bt2cAMP for 48 h. A, cells (6 x 106/ml) were resuspended in chemotaxis buffer allowed to respond to 25, 50, 100, or 200 pM C5a with or without 50 nM DBP for 60 min at 37 °C. Numbers represent net cell movement in 60 min, mean ± S.E. of 3–5 experiments. B, cells loaded with Fluo-3 AM dye were resuspended in HBSS + 1% BSA at 5 x 106/ml with or without 50 nM DBP and then incubated at room temperature for 30 min. The increase in intracellular calcium after stimulation with 25, 50, 100, or 200 pM C5a was measured at excitation of 505 nm and emission of 525 nm. Data are expressed mean ± S.E. of the nM change in intracellular Ca2+ level of 3–5 separate experiments. Asterisks denote that the indicated sample is significantly greater (p < 0.01) than the corresponding control value.

Analysis of E. coli-expressed DBP by SDS-PAGE and Western Blotting—The commercially available (Invitrogen) 1374-nucleotide DBP cDNA, Gc2 allele, was expressed in E. coli BL-21 in the plasmid vector pGEX-4T-2 fused to GST. Upon isopropyl-1-thio--D-galactopyranoside induction, the GST·DBP fusion protein was expressed, as judged by SDS-PAGE (Fig. 2A). SDS-PAGE of the elution protein revealed an 80-kDa band corresponding to the molecular mass of full-length DBP fused to GST. The bands at 65, 55, and 45 kDa correspond to the molecular mass of GST with truncated forms of DBP. All lanes have a small amount of a 30-kDa band. This probably represents a degradation product of GST or the unfinished product of the protein synthesis. Thrombin cleavage of the purified fusion protein resulted in separation of GST from DBP. The SDS-PAGE and immunoblotting after cleavage and separation of GST revealed a single protein band of 56 kDa for full-length DBP, 40 kDa for domains I and II, 30 kDa for domains II and III, and 20 kDa for domain I or domain II (Fig. 2B).

View larger version (39K): [in this window] [in a new window]   FIG. 2. Structural analysis of full-length and truncated recombinant DBP. A, SDS-PAGE analysis. Lane 1 from left, GST with full-length reDBP-(1–458), GST with domain I and domain II-(1–378), GST with domain II and domain III-(192–458), GST with domain I-(1–191), and GST with domain II-(192–378). GST alone is 28 kDa, Positions of molecular mass markers are shown on the right of panel. Mol wt. marker indicates molecular mass markers. B, Western blot analysis of expressed recombinant DBP (after thrombin cleavage to remove the GST). 250 ng of each recombinant DBP was separated on 10% SDS-PAGE and immunoblotted using polyclonal anti-DBP.

View larger version (22K): [in this window] [in a new window]   FIG. 3. Analysis of vitamin D sterol binding to reDBP. A competitive [3H]25(OH)D3 binding assay of natural DBP and reDBPs was performed. Solutions containing natural DBP or reDBPs (200 ng), [3H]25-OH-D3 (0.1 pmol), or unlabeled 25-OH-D3 total (0.5–64 pmol) in a volume of 10 µl were added to 490 µl of assay buffer (50 mM Tris-HCl, pH 8.3, 150 mM NaCl, 1.5 mM EDTA, 0.1% Triton X-100) and incubated at 4 °C for 20 h. Samples were then treated with 100 µl of ice-cold 2.5% dextran-coated charcoal to remove free vitamin D and then were separated by centrifugation at 5000 x g at 4 °C. Supernatants were mixed with scintillation mixture and counted for radioactivity. Data are presented as the ratio of radiolabel bound with unlabeled competitor (B) divided by radiolabel bound without competitor (B0). Results are representative of 3 separate experiments.

View larger version (28K): [in this window] [in a new window]   FIG. 4. Effect of reDBP on C5a-mediated responses in differentiated HL-60 cells. HL-60 cells were differentiated using 250 µM bt2cAMP for 48 h. A, cells (6 x 106/ml) were resuspended in chemotaxis buffer and allowed to respond to 100 pM C5a alone (Control) or C5a plus 50 nM each indicated DBP for 60 min at 37 °C. Numbers represent net cell movement in 60 min, mean ± S.E. of 3–5 experiments. B, cells loaded with Fluo-3 AM dye were resuspended in HBSS + 1% BSA at 5 x 106/ml with or without 50 nM indicated DBP and then incubated at room temperature for 30 min. The increase in intracellular calcium after stimulation with 100 pM C5a was then measured. Data are expressed as the maximal nM change in the intracellular Ca2+ level. Results are the mean ± S.E. of 3 separate experiments. Asterisks denote that the sample is significantly greater (p < 0.01) than control value.

View larger version (37K): [in this window] [in a new window]   FIG. 5. Structural analysis of truncated versions of DBP domain I. A, SDS-PAGE analysis, lanes 1–4 from left: GST with DI-(1–112), GST with DI-(1–125), GST with DI-(1–150), GST with DI-(1–175). Positions of molecular mass markers are shown on the right of the panel. Mol wt. marker indicates molecular mass markers. B, Western blot analysis of expressed recombinant DBP (after thrombin cleavage to remove the GST). 250 ng of domain I truncations was separated on 10% SDS-PAGE and immunoblotted using polyclonal anti-DBP.

View larger version (29K): [in this window] [in a new window]   FIG. 6. Effect of truncated reDBP of domain I on C5a-mediated responses in differentiated HL-60 cells. HL-60 cells were differentiated using 250 µM bt2cAMP for 48 h. A, cells (6 x 106/ml) were resuspended in chemotaxis buffer allowed to respond to 100 pM C5a alone (Control) or C5a plus 50 nM each indicated DBP for 60 min at 37 °C. Numbers represent net cell movement in 60 min, mean ± S.E. of 3–5 experiments. B, cells loaded with Fluo-3 AM dye were resuspended in HBSS + 1% BSA at 5 x 106/ml with or without 50 nM indicated DBP and then incubated at room temperature for 30 min. The increase in intracellular calcium after stimulation with 100 pM C5a was then measured. Data are expressed as the maximal nM change in the intracellular Ca2+ level. Results are the mean ± S.E. of 3 separate experiments. Asterisks denote that the sample is significantly greater (p < 0.01) than control value.

View larger version (31K): [in this window] [in a new window]   FIG. 7. Effect of DBP domain I peptides on C5a-mediated chemotaxis. Cells (6 x 106/ml) were resuspended in chemotaxis buffer and treated with chemotaxis buffer (Control), 1 µM peptide 1-(130–152), or 1 µM peptide 2-(150–172) for 30 min at room temperature. The chemotactic response of differentiated HL-60 cells to 100 pM C5a alone, C5a plus 50 nM full-length natural DBP, or 1% zymosan-activated serum (ZAS) was measured for 60 min at 37 °C. Numbers represent net cell movement in 60 min, mean ± S.E. of 3 experiments. Asterisks denote that the indicated sample is significantly less (p < 0.01) than the corresponding control value.

	DISCUSSION

TOP ABSTRACT INTRODUCTION MATERIALS AND METHODS RESULTS DISCUSSION REFERENCES

DBP contains 14 disulfide bonds and, therefore, presents a challenge for expressing the protein in a functional form. Swamy et al. (21) previously reported expression of functional recombinant DBP in E. coli using GST as a fusion partner. Using their approach, we generated a series of functional truncated proteins that localized the C5a cochemotactic function to a 50-amino-acid region (residues 126–175) in the C-terminal portion of domain I. Two overlapping peptides covering this region were synthesized and clearly demonstrated that the cochemotactic function was confined to the 20-amino-acid sequence 5'-EAFRKDPKEYANQFMWEYST-3' (residues 130–149). This sequence is identical among the three major allelic forms of human DBP (Gc-1F, Gc-1S, Gc-2) and correlates with the fact that there is no difference in the C5a cochemotactic function between these DBP isoforms (16). BLAST search of this sequence produced no other match, besides DBP, in any eukaryote, indicating that this region is unique to DBP and that it probably does not function by mimicking a similar sequence in a signaling molecule. In addition, alignment of this human peptide with the corresponding sequences in rat (26), mouse (27), and rabbit DBP (28), the only other mammalian sequences currently described, show a very high degree of amino acid similarity; 78% of the residues are identical, and the remainder are conservative or homologous substitutions. Thus, it is reasonable to speculate that this region in domain I of DBP functions as the cochemotactic sequence in all mammals.

Several recent reports have described the crystal structure of DBP, either unligated or bound to G-actin or vitamin D (6–9). Analysis of the structure has revealed that the protein is comprised of a series of -helices, much like albumin (29), but in contrast to albumin, DBP folds into a hook-like structure that serves as the G-actin binding site (30). The cochemotactic sequence in DBP (residues 130–149) is located partly in -helix number 7 (residues 125–134) but mostly in -helix number 8 (residues 136–150) in domain I (6–9). Three-dimensional analysis of DBP crystal structure using the NIH NCBI software program Cn3D (version 4.1) indicates that this region is not blocked when DBP binds G-actin or vitamin D sterols and is accessible to interact with cells. This structural analysis correlates well with our recent functional studies that demonstrated that ligation of DBP with G-actin, 25-OH vitamin D₃, or both did not alter the C5a cochemotactic activity of DBP.² Therefore, this cochemotactic peptide is a distinct functional sequence and constitutes the forth such region (the others are G-actin, vitamin D, and the polysaccharide structure of DBP-MAF (macrophage-activating factor) to be defined in the plieotropic DBP.

The binding of DBP to cells is required for the protein to mediate its numerous functions: a chemotactic cofactor for C5a, a macrophage or osteoclast-activating factor, clearance of DBP-actin complexes by the liver, and delivery of vitamin D sterols and free fatty acids to cells (1, 2). The cell surface DBP binding site is only partially characterized, but this essential link in DBP physiology is poorly understood. DBP appears to bind with relatively low affinity to multiple cell surface ligands including chondroitin sulfate proteoglycans (31), low density lipoprotein receptor family members megalin and cubulin (32, 33), and possibly CD36 (34). Recently, we have shown that the platelet-derived adhesive glycoprotein thrombospondin-1 (a known ligand for CD36) is required for the full C5a cochemotactic activity of DBP and may be involved in the formation or regulation of the putative DBP binding site complex (20). The results presented in this report do not answer the question of whether there is a distinct cell binding and cochemotactic region in DBP or whether the sequence identified (residues 130–149) performs both functions. DBP has been shown to bind to chondroitin sulfate proteoglycans, and this protein has several glycosaminoglycan consensus binding sequences (K/R-rich regions) in domains II and III. In particular, an 11-amino-acid sequence (403–413) in domain III (5'-KKKLAERLKAK-3') is a very basic region that may be a putative glycosaminoglycan binding site. However, the cell binding site on DBP remains to be identified. The results also do not preclude the possibility that there are other sequences in DBP that may be involved in its cochemotactic function. The cochemotactic sequence 5'-EAFRKDPKEYANQFMWEYST-3' (residues 130–149) can effectively block the capacity of full-length natural DBP to enhance chemotaxis to C5a, but the sequence alone cannot augment chemotaxis, possibly suggesting that other regions of DBP may be involved in this function. Nevertheless, the results reported herein provide strong evidence that amino acids 130–149 play an essential role in the C5a chemotactic cofactor function of DBP and, furthermore, could signify that compounds derived from this sequence may have therapeutic potential to limit C5a-mediated tissue injury.

	FOOTNOTES

 
^* This work was supported by National Institutes of Health Grant GM 63769 (to R. R. K.). The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

To whom correspondence should be addressed. Tel.: 631-444-3941; Fax: 631-444-3424; E-mail: rkew{at}notes.cc.sunysb.edu.

¹ The abbreviations used are: DBP, vitamin D-binding protein; reDBP, recombinant expressed DBP; GST, glutathione S-transferase; HBSS, Hank's buffered saline solution; BSA, bovine serum albumin.

² A. B. Shah, S. J. Dimartino, G. Trujillo, and R. R. Kew, manuscript submitted.

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TOP ABSTRACT INTRODUCTION MATERIALS AND METHODS RESULTS DISCUSSION REFERENCES

 

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