©1995 by The American Society for Biochemistry and Molecular Biology, Inc.
Trp-145 Is Essential for the Binding of 25-Hydroxyvitamin D to Human Serum Vitamin D-binding Protein (*)

(Received for publication, July 8, 1994; and in revised form, October 17, 1994)

Narasimha Swamy Marni Brisson Rahul Ray (§)

From the Department of Bioorganic and Protein Chemistry, Vitamin D Laboratory, Boston University School of Medicine, Boston, Massachusetts 02118

ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS AND DISCUSSION
FOOTNOTES
REFERENCES

ABSTRACT

Chemical modification of specific amino acid residues in a protein has been a valuable tool in identifying amino acid residues that are responsible for ligand binding of a protein. In the present investigation, we targeted Trp and His residues in human serum vitamin D-binding protein (hDBP) by modifying them with specific chemical modifiers. We also evaluated the results of these modifications in the binding of 25-hydroxy[26(27)-^3H]vitamin D(3) ([^3H]25-OH-D(3)) to hDBP. We observed a dose-dependent loss of binding activity by N-bromosuccinimide (specific for Trp). Similar results were observed with diethylpyrocarbonate (specific for His). Furthermore, loss of [^3H]25-OH-D(3)-binding was protected by preincubation of hDBP samples with an excess of 25-hydroxyvitamin D(3). These results strongly emphasized the importance of Trp (single residue at position 145) and 1 His residue (out of a total of 6) in the vitamin D sterol-binding by vitamin D-binding protein.


INTRODUCTION

Vitamin D-binding protein (DBP), (^1)also known as group specific component (Gc) is a multifunctional protein. Its most well-studied function is the transport of vitamin D and its metabolites to target organs and tissues(1) . Although all of the major metabolites of vitamin D are known to bind strongly to DBP, the strongest binding is for 25-hydroxyvitamin D(3) (25-OH-D(3)) (K = 10M). Another interesting property of DBP is its ability to bind and sequester monomers of actin during cell injury or lysis. Thus, DBP, in conjunction with plasma gelsolin, prevents the formation of harmful actin polymers in the bloodstream(2, 3) . The third function of DBP, i.e. its association with the membranes of IgG-related B-lymphocytes and T-lymphocytes with IgG Fc receptor, indicates a possible trans-membrane signaling role of DBP(4, 5) . Recently, Yamamoto and Homma (6) have provided strong evidence to suggest that DBP is a precursor to macrophage activating factor.

It is an open question whether DBP binds to its diverse ligands through a common binding pocket. This is an important issue, particularly in the light of the fact that DBP is present in a large excess over that necessary for vitamin D sterol transport, when compared to the normal concentrations of vitamin D sterols in serum(7) . During the past several years, we have developed photoaffinity and affinity labeling techniques to identify the region of DBP which is responsible for vitamin D sterol binding(8, 9, 10) . (^2)In the present investigation, we have employed methods involving specific modification of amino acid residues, a biochemical equivalent of point mutation studies, to identify the amino acid residues that are important for the binding of 25-OH-D(3) to human serum DBP (hDBP).


MATERIALS AND METHODS

Human DBP, as a mixture of isoforms, was purchased from Calbiochem, San Diego, CA. 25-Hydroxy[26(27)-^3H]vitamin D(3) (specific activity 25-30 Ci/mM) was obtained from Amersham Corp. N-Bromosuccinimide (NBS) and diethylpyrocarbonate (DEPC) were purchased from Sigma. Hydroxylapetite (Bio-Gel HTP) was a product of Bio-Rad. Functional activity of hDBP samples was determined by competitive binding assays with [^3H]25-OH-D(3)(10) . Fluorescence and absorption spectra of the samples were recorded on Hitachi F-2000 Fluorescence and Hitachi U-200 Spectrophotometers, respectively. For fluorescence spectral studies, the samples were excited at 285 nm, and emission spectra were recorded. Analysis of all the samples were run in triplicate.

Determination of the Role of Trp in the 25-OH-D(3) Binding: NBS Oxidation

The role of Trp residue in hDBP was probed by using the NBS oxidation procedure of Spande et al.(12) . hDBP was treated with 0.5-10 mol of freshly recrystallized NBS/mol of hDBP in 20 mM sodium acetate buffer, pH 4.0, at 25 °C. After 10 min, excess of NBS was quenched with 10 mM tryptophan solution in the same buffer. The samples were analyzed for 25-OH-D(3) binding activity by binding assays with [^3H]25-OH-D(3)(10) .

The fluorescence spectra of the hDBP samples were recorded before and after NBS oxidation (4-fold molar excess), using a 0.5-cm path length quartz cuvette in 20 mM sodium acetate buffer, pH 4.0, at 25 °C.

The protection of the putative Trp at the binding site from NBS oxidation was carried out by oxidizing hDBP in the presence of an excess of 25-OH-D(3). Thus, a sample of hDBP was preincubated with 12.5-fold molar excess of 25-OH-D(3) for 12 h at 4 °C, followed by treatment with 4-fold molar excess of NBS. After quenching the excess of NBS, the sample was loaded on to a mini-hydroxylapatite column, which was washed with 10 mM phosphate buffer (10 ml), pH 7.0. The bound protein was eluted with 1 ml of phosphate buffer (1 M), pH 7.0, and the binding activity was analyzed. Fluorescence spectra of the samples were recorded after and before the addition of NBS and prior to mini-hydroxylapetite chromatography.

Determination of the Role of His in the 25-OH-D(3) Binding: Reaction with DEPC

Histidyl residues of hDBP were targeted with DEPC, a His-specific chemical modifier. The carbethoxylation of His was carried out according to the procedure of Miles(13) . hDBP was treated with 0.1-4.0 mM DEPC (stock solution in acetonitrile) in 100 mM potassium phosphate buffer, pH 6.0, at 25 °C for 30 min. The excess of DEPC was quenched with 0.1 M imidazolebulletHCl buffer, pH 7.4. The samples were analyzed for [^3H]25-OH-D(3) binding activity.

Protection of the putative His at the binding site, from being modified by DEPC, was carried out by preincubating hDBP samples with 12.5-fold molar excess of 25-OH-D(3) for 12 h at 4 °C and then treated with one mM of DEPC. After quenching the excess of DEPC, the bound-25-OH-D(3) was stripped by passing through a mini-hydroxylapatite column, and the [^3H]25-OH-D(3) binding activity was analyzed.

The number of histidyl residues modified by DEPC was calculated from the absorbance of the samples at 240 nm (Delta = 3200 cmM) by the method of Ovadi and Keleti(14) . In the protection experiments with 25-OH-D(3)-treated samples, the spectrophotometric method was ineffective due to the strong absorption of 25-OH-D(3) at 240 nm.


RESULTS AND DISCUSSION

We explored the probable role of Trp and His present in hDBP toward 25-OH-D(3)-binding by specific chemical modifications of these amino acid residues under controlled conditions. For example, NBS oxidizes the indole chromophobe of Trp to oxindole which has diminished fluorescence yield at 320 nm when exited at 285 nm(12) . The reaction of NBS is usually restricted to Trp residues under acidic conditions with carefully controlled amounts of NBS.

In the case of hDBP, addition of increasing amount of NBS progressively decreased the binding of [^3H]25-OH-D(3) to hDBP. Approximately 95% of binding activity was destroyed by the addition of 4 mol/mol of NBS to hDBP. Further increase in the NBS concentration brought about only marginal changes. These results are shown in Fig. 1.


Figure 1: Samples of hDBP were treated with 0.5-10 mol of NBS/mol of hDBP in 20 mM sodium acetate buffer, pH 4.0, at 25 °C. After 10 min, excess of NBS was quenched with 10 mM tryptophan solution in the same buffer. The protein samples, after passage through a mini-hydroxylapatite column, were analyzed for [^3H]25-OH-D(3) binding activities.



The loss in binding activity following NBS treatment was, however, protected almost completely (96%) in the presence of an excess of 25-OH-D(3) (Fig. 2). In contrast only 8% of the control binding activity was retained in the absence of added 25-OH-D(3). The loss of binding activity following NBS treatment, and protection of this loss in the presence of an excess of 25-OH-D(3), clearly emphasizes the role of Trp in 25-OH-D(3)-binding.


Figure 2: Protection assay against NBS oxidation of Trp. hDBP samples were preincubated with a 12.5-fold molar excess of 25-OH-D(3) for 12 h at 4 °C, followed by treatment with 4-fold molar excess of NBS. After quenching the excess of NBS, the sample was chromatographed on a mini-hydroxylapatite column to remove 25-OH-D(3), and the protein samples were assayed for [^3H]25-OH-D(3) binding activities.



Modification of Trp and its role in 25-OH-D(3)-binding was further substantiated by fluorescence studies of the native and NBS-oxidized hDBP as well as samples which were preincubated with 25-OH-D(3). As shown in Fig. 3fluorescence emission (I) of the native protein diminished sharply following the NBS treatment (II). Interestingly, when a sample of hDBP was incubated with 25-OH-D(3), intensity of fluorescence diminished by approximately 6% (III) compared with the apoprotein (I), indicating possible a change in conformation of hDBP upon binding to 25-OH-D(3). Addition of NBS to this sample (25-OH-D(3)-incubated) caused the fluorescence to decrease by approximately 20% (IV), which indicated that 25-OH-D(3) largely protected Trp from reacting with NBS. It is noteworthy that hDBP contains a single Trp at position 145(11, 15, 16) ; and our results, described above, strongly implicate this amino acid residue toward binding of 25-OH-D(3), the most important ligand for hDBP.


Figure 3: Fluorescence spectral studies of hDBP to determine the effects of NBS treatment on native hDBP and hDBP, preincubated with 25-OH-D(3). I, native hDBP; II, hDBP, treated with 4-fold molar excess of NBS; III, hDBP- incubated with 12.5-fold molar excess of 25-OH-D(3); and IV, hDBP-incubated with 12.5-fold molar excess of 25-OH-D(3) followed by treatment with 4-fold molar excess of NBS.



DEPC is a specific His modifier in a protein. In the case of hDBP, increase in DEPC concentration resulted in the decrease in [^3H]25-OH-D(3) binding by hDBP. As shown in Fig. 4, decrease in the original binding activities were approximately 85 and 96% with 0.5 mM and 1 mM of DEPC, respectively.


Figure 4: Samples of hDBP were treated with 0.1-4.0 mM DEPC in 100 mM potassium phosphate buffer, pH 6.0, at 25 °C for 30 min. The excess of DEPC was quenched with 0.1 M imidazolebulletHCl buffer, pH 7.4. The samples were analyzed for [^3H]25-OH-D(3) binding activity.



The loss of binding activity upon DEPC treatment was, however, protected by an excess of 25-OH-D(3). As seen in Fig. 5, when hDBP was treated with 0.5 mM DEPC in the presence of an excess of 25-OH-D(3), approximately 90% of the binding activity was protected, as compared with only 10% in the absence of the added 25-OH-D(3). The loss of binding activity by DEPC treatment, and the protection of this loss by an excess of 25-OH-D(3) strongly indicates that His residue/residues play an important role in the binding of 25-OH-D(3) to hDBP.


Figure 5: Protection assay against modification of His by DEPC. Samples of hDBP were preincubated with 12.5-fold molar excess of 25-OH-D(3) for 12 h at 4 °C and then treated with one mM of DEPC. After quenching the excess of DEPC, the bound-25-OH-D(3) was stripped by passing through a mini-hydroxylapatite column, and [^3H]25-OH-D(3) binding activities were analyzed.



To determine the average number of DEPC-modifiable His residues in hDBP, out of a total of 6, we used a spectrophotometric method by Ovadi and Keleti(14) . As shown in Table 1, an average of 0.8 His residue was modified by 0.1 mM of DEPC with an 18% loss of corresponding binding activity. On the other hand, 1.9 His residues were modified by 0.5 mM of DEPC with 85.5% loss of binding activity. Modification of other residues by higher concentration of DEPC (e.g. 1.0 mM) did not show any significant change in the loss of binding activity (96%, 5 His modified). These results strongly indicate that 2 His residues were initially modified by DEPC. While the first, and the most accessible residue, was not involved in binding, modification of the second residue led to almost complete destruction of binding activity.



Our results also indicate that the second His is relatively inaccessible (buried) for modification, so that a large amount (0.5 mM) of DEPC was required to reduce the binding activity to 14.5%. This view is also supported by a 59% loss of activity by 0.2 mM of DEPC, where the first His was completely modified and the second one reacted only partially with DEPC.

During the past few years, we have undertaken a structure-functional approach to study the interaction between DBP and vitamin D sterols, which is the most well studied and best understood among various functions of the protein. In a recent publication(8) , we have demonstrated that the putative 25-OH-D(3)-binding domain in hDBP is located in the N-terminal part(1-107) of the protein molecule. Results of the studies described in this article strongly implicate Trp-145, located in the same area of the protein, as being essential for [^3H]25-OH-D(3)-binding. These studies also suggest that a single His (of unknown position) is crucial for the binding interaction between hDBP and 25-OH-D(3).

It is well documented that hDBP, a member of the albumin gene family, displays remarkable structural homology with human albumin(11, 15, 16) . Certain striking differences, however, exist, which include lack of any Cys in hDBP and absence of Trp-145 in human albumin. Since our results strongly demonstrate that Trp-145 is critical in 25-OH-D(3)-binding, it leaves one wondering whether, during evolution, mutation at position 145 set human albumin and hDBP apart so that hDBP became a specific carrier of vitamin D sterols.

Physiological significance of the multiple functions of DBP has remained largely unclear to date. For example, it has been suggested that the vitamin D sterol binding by DBP serves as a protective mechanism against vitamin D intoxication either as a result of dietary excess or overabundant synthesis in the skin. This view is supported by the low saturation (5%) of DBP with vitamin D sterols, and the fact that DBP acts as an inhibitor of renal 25-hydroxyvitamin D(3)-1alpha-hydroxylase(7) . If this hypothesis is true, absence of DBP would lead to infantile hypercalcemia. However, no support of this hypothesis currently exists. Interestingly, extensive screening of the human population for DBP/Gc electrophoretic polymorphism has failed to detect any deletion or gross alteration of the DBP gene lending support to the idea that such mutations may be lethal(1) . Structure-function studies, as described above, would provide information crucial for a better understanding of the probable physiologic role of this multifunctional serum protein.


FOOTNOTES

*
This work was supported by Grants DK 44337 and 47418 from the National Institute of Diabetes and Digestive and Kidney Diseases of the National Institutes of Health. 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 should be addressed: Boston University School of Medicine, 80 E. Concord St., Boston, MA 02118. Tel.: 617-638-8199; Fax: 617-638-8882.

(^1)
The abbreviations used are: DBP, vitamin D-binding protein; DEPC, diethylpyrocarbonate; NBS, N-bromosuccinimide; 25-OH-D(3), 25-hydroxyvitamin D(3); [^3H]25-OH-D(3), 25-hydroxy[26(27)-^3H]vitamin D(3).

(^2)
N. Swamy and R. Ray, manuscript submitted for publication.


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




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