(Received for publication, July 8, 1994; and in revised form, October 17, 1994)
From the
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)-H]vitamin D
([
H]25-OH-D
) 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 [
H]25-OH-D
-binding was
protected by preincubation of hDBP samples with an excess of
25-hydroxyvitamin D
. 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.
Vitamin D-binding protein (DBP), ()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
(25-OH-D
) (K
=
10
M
). 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) . ()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
to human serum DBP
(hDBP).
Human DBP, as a mixture of isoforms, was purchased from
Calbiochem, San Diego, CA.
25-Hydroxy[26(27)-H]vitamin D
(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
[
H]25-OH-D
(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.
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. Thus, a sample of hDBP was
preincubated with 12.5-fold molar excess of 25-OH-D
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.
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 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
was stripped by passing through a mini-hydroxylapatite column,
and the [
H]25-OH-D
binding activity
was analyzed.
The number of histidyl residues modified by DEPC was
calculated from the absorbance of the samples at 240 nm
( = 3200 cm
M
) by the method of Ovadi and
Keleti(14) . In the protection experiments with
25-OH-D
-treated samples, the spectrophotometric method was
ineffective due to the strong absorption of 25-OH-D
at 240
nm.
We explored the probable role of Trp and His present in hDBP
toward 25-OH-D-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
[H]25-OH-D
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
[H]25-OH-D
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 (Fig. 2). In contrast only 8% of
the control binding activity was retained in the absence of added
25-OH-D
. The loss of binding activity following NBS
treatment, and protection of this loss in the presence of an excess of
25-OH-D
, clearly emphasizes the role of Trp in
25-OH-D
-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 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
, and the protein samples were assayed for
[
H]25-OH-D
binding
activities.
Modification of Trp and its role in
25-OH-D-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
. 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
, 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
. Addition of NBS to this
sample (25-OH-D
-incubated) caused the fluorescence to
decrease by approximately 20% (IV), which indicated
that 25-OH-D
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
, 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. 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
; and IV, hDBP-incubated with 12.5-fold
molar excess of 25-OH-D
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
[H]25-OH-D
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 imidazoleHCl buffer, pH 7.4. The
samples were analyzed for [
H]25-OH-D
binding activity.
The loss of binding activity upon DEPC treatment
was, however, protected by an excess of 25-OH-D. As seen in Fig. 5, when hDBP was treated with 0.5 mM DEPC
in the presence of an excess of 25-OH-D
, approximately 90%
of the binding activity was protected, as compared with only 10% in the
absence of the added 25-OH-D
. The loss of binding activity
by DEPC treatment, and the protection of this loss by an excess of
25-OH-D
strongly indicates that His residue/residues play
an important role in the binding of 25-OH-D
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 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
was stripped by passing through a
mini-hydroxylapatite column, and [
H]25-OH-D
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-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
[
H]25-OH-D
-binding. These studies
also suggest that a single His (of unknown position) is crucial for the
binding interaction between hDBP and 25-OH-D
.
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-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-1
-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.
HOME | HELP | FEEDBACK | SUBSCRIPTIONS | ARCHIVE | SEARCH | TABLE OF CONTENTS |
All ASBMB Journals | Molecular and Cellular Proteomics |
Journal of Lipid Research | Biochemistry and Molecular Biology Education |