Fatty acid-binding site environments of serum vitamin D-binding protein and albumin are different

Article abstract of DOI:10.1016/j.bioorg.2008.02.002

Vitamin D-binding protein (DBP) and albumin (ALB) are abundant serum proteins and both possess high-affinity binding for saturated and unsaturated fatty acids. However, certain differences exist. We surmised that in cases where serum albumin level is low, DBP presumably can act as a transporter of fatty acids. To explore this possibility we synthesized several alkylating derivatives of ¹⁴C-palmitic acid to probe the fatty acid-binding pockets of DBP and ALB. We observed that N-ethyl-5-phenylisooxazolium-3′-sulfonate-ester (WRK-ester) of ¹⁴C-palmitic acid specifically labeled DBP; but p-nitrophenyl- and N-hydroxysuccinimidyl-esters failed to do so. However, p-nitrophenyl ester of ¹⁴C-palmitic acid specifically labeled bovine ALB, indicating that the micro-environment of the fatty acid-binding domains of DBP and ALB may be different; and DBP may not replace ALB as a transporter of fatty acids.

Full text of DOI:10.1016/j.bioorg.2008.02.002

Available online at www.sciencedirect.com
Bioorganic Chemistry 36 (2008) 165–168
www.elsevier.com/locate/bioorg
Preliminary Communication
Fatty acid-binding site environments of serum vitamin
D-binding protein and albumin are different
Narasimha Swamy , Rahul Ray *
Bioorganic Chemistry & Structural Biology, Department of Medicine, Boston University School of Medicine, Boston, MA, USA
Received 23 January 2008
Available online 28 March 2008
Abstract
Vitamin D-binding protein (DBP) and albumin (ALB) are abundant serum proteins and both possess high-affinity binding for sat-
urated and unsaturated fatty acids. However, certain differences exist. We surmised that in cases where serum albumin level is low,
DBP presumably can act as a transporter of fatty acids. To explore this possibility we synthesized several alkylating derivatives of
14C-palmitic acid to probe the fatty acid-binding pockets of DBP and ALB. We observed that N-ethyl-5-phenylisooxazolium-30-sulfo-
nate-ester (WRK-ester) of 14C-palmitic acid specifically labeled DBP; but p-nitrophenyl- and N-hydroxysuccinimidyl-esters failed to
do so. However, p-nitrophenyl ester of 14C-palmitic acid specifically labeled bovine ALB, indicating that the micro-environment of
the fatty acid-binding domains of DBP and ALB may be different; and DBP may not replace ALB as a transporter of fatty acids.
Ó 2008 Elsevier Inc. All rights reserved.
Keywords: Fatty acid-binding by vitamin D-binding protein (DBP) and albumin (ALB); Serum transport of fatty acids; Affinity labeling analogs of
palmitic acid; Affinity labeling of fatty acid-binding sites of DBP and ALB
1. Introduction
these small molecules to organs and cells wherever they
are required. In addition DBP plays an integral role in
the circulating actin-scavenging system in plasma. Plasma
gelsolin severe filaments of F-actin, and DBP binds to actin
monomer (G-actin) with high affinity, thus preventing G-
actin to polymerize and clog arteries during cell-injury
and lysis [5,6]. Presence of actin–DBP complex in the sera
of human and animals sustaining injuries/inflammation,
e.g. trophoblastic emboli, severe hepatitis, acute lung
injury, etc. positively implicates DBP in thrombosis and
heart attack [7]. DBP also binds chemotactic agents such
as C5a and C5a des Arg, thus enhancing complement acti-
vation on neutrophil chemotaxis [8,9]. Furthermore, a
post-translationally modified form of DBP (DBP-macro-
phage activating factor, DBP-maf) has been shown to have
strong macrophage- and osteoclast-activating [10–14] and
anti-angiogenic and anti-tumor properties [15,16].
Group specific component (Gc) or vitamin D-binding
protein (DBP) is a sparsely glycosylated and polymorphic
serum protein. The two major phenotypes are Gc1 and
Gc2, differing from each other by four (4) amino acids in
the primary structure as well as structure of attached poly-
saccharide. Gc1 is further divided into two subtypes differ-
ing in primary structure as well as structure of the attached
carbohydrates [1–3].
DBP is a multi-functional protein [4]. Its binding of vita-
min D and its metabolites has been studied extensively
leading to the understanding that DBP is responsible for
the stepwise activation of vitamin D3 to 25-hydroxyvitamin
D3 (25-OH-D3) and finally to its physiologically
most active metabolite, 1a,25-dihydroxyvitamin D3
(1,25(OH)2D3). It is also involved in the transportation of
In addition to above properties of DBP and its deriva-
tive (DBP-maf), DBP binds saturated and unsaturated
fatty acids with high affinity (Kd = 105–106 Mꢀ1), similar
to plasma ALB [17,18]. However, certain differences do
*
Corresponding author. Fax: +1 617 638 8194.
E-mail address: bapi@bu.edu (R. Ray).
Deceased.
0045-2068/$ - see front matter Ó 2008 Elsevier Inc. All rights reserved.
doi:10.1016/j.bioorg.2008.02.002

166
N. Swamy, R. Ray / Bioorganic Chemistry 36 (2008) 165–168
exist. For example, Ena et al. demonstrated that molar
ratio of fatty acids, bound to human DBP to DBP is 0.4
compared with 1.8 for human ALB [19]. Furthermore,
majority of DBP-bound fatty acids are mono-unsaturated
or saturated, and abundance of poly-unsaturated fatty
acids is less than 5% (of the total bound fatty acids) [19].
Another interesting observation includes competition
between vitamin D sterols and fatty acids in terms of bind-
ing to DBP. For example, it was reported that poly-unsat-
urated fatty acids, such as arachidonic or linoleic acid,
strongly compete with 25-OH-D3 and 1,25(OH)2D3 for
binding to DBP, in sharp contrast with saturated fatty
acids e.g. palmitic acid, which offer no significant competi-
tion [19,20]. Furthermore, Bouillon et al. observed that
addition of human ALB in a physiological ALB:DBP ratio
did not impair the inhibitory effect of linoleic acid towards
DBP-25-OH-D3-binding [20].
2.2. Affinity labeling studies of bovine serum ALB and DBP
with N-hydroxy-succinimido-14C-palmitate (A), p-nitrophenyl-
14C-palmitate (B), and WRK-14C-palmitate (C)
Twenty-microgram samples each of BSA and DBP in
20 ll of TEST buffer (50 mM Tris–HCL, 150 mM NaCl,
1.5 mM EDTA, 0.1% Triton X-100, pH 8.8) were treated
with N-hydroxysuccinimdo-14C-palmitate (A), p-nitro-
phenyl-14C-palmitate (B), or WRK-14C-palmitate (C) (each
20,000 cpm) at 25 °C for 20 h. Parallel samples of BSA and
DBP containing additional sodium palmitate (1 lg in 10 ll
of buffer) were also treated the same way. At the end of the
experiment all the samples were analyzed on a 7.5% SDS–
polycarylamide gel, followed by drying the gel and scan-
ning of radioactivity in a Biosan phosphorimager.
3. Results and discussion
We hypothesized that this apparent anomaly between
DBP and ALB in terms of fatty acid-binding might be
related to the actual binding process between these proteins
and fatty acids, which, in turn, might be related to the
micro-environment of the fatty acid-binding pockets of
these proteins. In order to evaluate this possibility we syn-
thesized several reactive esters of 14C-palmitic acid as
potential affinity labeling reagents for DBP and ALB.
Results of these studies and their probable physiological
implications are discussed in this report.
There is a remarkable structural homology among ALB,
DBP, a-feto protein (AFP) and afamin, members of the
albumin gene family. All these proteins have modular
structures with three domains (domains I–III) and high
cysteine-content [22]. In the case of DBP all the Cys resi-
dues (total 28) are oxidized to form 14 disulfide bonds.
In contrast, ALB contains several free sulphydryl groups
in its primary structure. Furthermore, DBP has a shorter
domain III than ALB. These structural differences may
explain gross functional differences between DBP and
ALB. For instance, vitamin D sterols- and G-actin binding
and related functions are unique to DBP. On the other
hand, DBP possesses relatively weaker binding for fatty
acids compared with ALB. Furthermore, DBP contains a
single high-affinity fatty acid-binding site compared to
ALB which contains several low- and high-affinity binding
sites [18]. In ALB these binding sites are distributed among
various domains of the protein, although high-affinity-
binding sites are located in domain III [23]. Moreover, as
described earlier, DBP, in contrast with ALB, discriminates
between saturated and unsaturated fatty acids in terms of
binding.
2. Materials and methods
Purified human DBP was obtained from commercially
available pooled human serum (American Red Cross, Ded-
ham, MA) by a ligand affinity chromatographic method
developed in our laboratory [21]. Defatted bovine serum
ALB (BSA) and all chemicals were purchased from
Sigma–Aldrich, Milwaukee, WI, except 1-14C-palmitic acid
(specific activity 56 mCi/mmol) which was a product of
NEN-DuPont, Boston, MA.
2.1. Synthesis (Fig. 1)
All the above observations point to difference in the nat-
ure of binding between ALB and DBP and fatty acids,
which in turn may be related to the fatty acid-binding
pocket structure of these proteins. Affinity and photo-
affinity labeling techniques have been used widely to probe
binding pockets and catalytic active sites of receptors and
enzymes, respectively [24]. Our laboratory has used these
techniques, and others to probe the vitamin D and actin-
binding domain structures of DBP, leading to crystal struc-
ture of the DBP–actin complex [25–35].
In the current study we synthesized radiolabeled ver-
sions of three reactive esters of palmitic acid to probe
the fatty acid-binding pockets of DBP and ALB. We
chose palmitic acid, a saturated fatty acid as model
because DBP has a propensity to bind saturated and
mono-unsaturated fatty acids stronger that poly-unsatu-
rated fatty acids [19,20].
The N-hydroxysuccinimido- and p-nitrophenyl-esters of
palmitic acid were synthesized by dicyclohexylcarbodiimide
(DCC)-coupling of palmitic acid with N-hydroxy-
succinimide, or p-nitrophenol in the presence of a catalytic
amount of N,N0-dimethylaminopryridine (DMAP) in
anhydrous dichloromethane. Synthesis of WRK-palmitate
was carried out by treating palmitic acid with N-ethyl-5-
phenyl-isooxazolium-30-sulfonate (Woodward’s reagent
K) and triethylamine in acetonitrile. Product from each
reaction was purified by preparative chromatography on
silica plates (Analtech, Vineland, NJ), and each product
was characterized by NMR. Radioactive synthesis was car-
ried out exactly the same way except palmitic acid was
replaced with 14C-palmitic acid. Products from the radioac-
tive reaction were isolated by TLC matching with corre-
sponding unlabeled compounds.

N. Swamy, R. Ray / Bioorganic Chemistry 36 (2008) 165–168
167
O
N
14
CH (CH ) C O
3
2 14
O
O
A
14
C
P-Nitrophenol/DCC/DMAP/CH2Cl2
14
C
CH (CH )
NO
2
O
3
2 14
CH (CH )
OH
3
2 14
O
O
B
SO3
SO
3
O
N
14
O
CH (CH )
C
3
2 14
Et
+
O
O
Et-N
H2
C
Fig. 1. Scheme for the synthesis of N-hydroxy-succinimido-14C-palmitate (A), p-nitrophenyl-14C-palmitate (B), and WRK-14C-palmitate (C).
Reed employed WRK-14C-palmitate (C) to affinity
label the fatty acid-binding pocket/s of bovine serum
ALB [36]. In our case, incubation of a sample of human
serum DBP (hDBP) with WRK-14C-palmitate (C) cova-
lently labeled the protein as determined by autoradiogra-
phy (Fig. 2, lane 1). When the incubation was carried
out in the presence of an excess of sodium palmitate, label-
ing was completely obliterated (Fig. 2, lane 2). These
results strongly indicated that WRK-14C-palmitate (C)
specifically labeled the palmitic acid-binding pocket in
hDBP. These results also suggested that structure and
chemical environment of the fatty acid-binding pockets
of DBP and ALB are similar.
Surprisingly other activated esters of palmitic acid i.e.
N-hydroxysuccinimidyl-14C-palmitate (A) and p-nitro-
phenyl-14C-palmitate (B) failed to label DBP in the pres-
ence or in the absence of an excess of sodium palmitate.
In the case of BSA, N-hydroxysuccinimidyl-14C-palmitate
(A) failed to label this protein. But, p-nitrophenyl-14C-
palmitate (B) labeled BSA, and labeling was significantly
reduced in the presence of an excess of palmitic acid, denot-
ing specific labeling of the fatty acid-binding pocket (results
not shown).
1
2
200
116
97
66
Collectively the above results suggest that chemical/elec-
tronic environments of the fatty acid-binding pockets of
DBP and ALB are different, so that ALB can tolerate a
hydrophobic (p-nitrophenyl) as well as a hydrophilic
(Woodward K reagent) head group at the carboxy terminal
of palmitic acid. But, fatty acid-binding site of DBP can
only accommodate a polar and Zwitterionic head group
(Woodward K reagent).
44
31
Analbuminemia is a rare hereditary disease in which the
afflicted individuals have very low or negligible amount of
circulating serum ALB [37–39]. We surmised that since
both ALB and DBP bind fatty acids with high affinity,
DBP may replace ALB in carrying fatty acids, particularly
saturated and mono-unsaturated fatty acids in the cases of
low or negligible amount of circulating ALB. However,
Fig. 2. Affinity labeling of hDBP with WRK-14C-palmitate (C): samples
of hDBP were incubated at 25 °C with WRK-14C-palmitate (C) alone
(lane 1), or in the presence of an excess of sodium palmitate (lane 2). The
samples were electroporesed on a SDS gel and exposed to a phosphor-
imager. Positions of the standard molecular weight markers are denoted
on the right.

168
N. Swamy, R. Ray / Bioorganic Chemistry 36 (2008) 165–168
[16] O. Kisker, S. Onizuka, C.M. Becker, M. Fannon, E. Flynn, R.
D’Amato, B. Zetter, J. Folkman, R. Ray, N. Swamy, S. Pirie-
Shepherd, Neoplasia 5 (2003) 32–40.
[17] M.H. Williams, E.L. Van Alstyne, R.M. Galbraith, Biochem.
Biophys. Res. Commun. 153 (1988) 1019–1024.
[18] M. Calvo, J.M. Ena, Biophys. Res. Commun. 163 (1989) 14–17.
[19] J.M. Ena, C. Esteban, M.D. Perez, J. Uriel, M. Calvo, Biochem. Int.
19 (1989) 1–7.
results of the study delineated in this communication sug-
gest that chemical and electronic environment of the fatty
acid-binding pockets of DBP and ALB might be different.
As a result binding and transportation of various fatty
acids might be different. Thus, DBP may not replace
ALB in terms of fatty acid scavenging and transportation.
[20] R. Bouillon, D.Z. Xiang, R. Convents, H. Van Baelen, J. Steroid
Biochem. Mol. Biol. 42 (1992) 855–861.
Acknowledgment
[21] N. Swamy, A. Roy, R. Chang, M. Brisson, R. Ray, Protein Expr.
Purif. 6 (1995) 185–188.
[22] J. Svasti, A. Kurosky, A. Bennett, B.H. Bowman, Biochemistry 18
(1979) 1611–1617.
[23] A.A. Spector, in: A.M. Scanu, A.A. Spector (Eds.), Biochemistry Ad
Biology of Plasma Lipoproteins, Marcel Dekkar Inc., NY, 1986, pp.
247–279.
This work was supported in part by a grant from Na-
tional Institute of Diabetes, Digestive and Kidney Diseases
(RO1 DK 44337).
References
[24] F.W. Sweet, G.L. Murdock, Endo. Rev. 8 (1987) 154–184.
[25] R. Ray, S.A. Holick, N. Hanafin, M.F. Holick, Biochemistry 25
(1986) 4729–4733.
[26] R. Ray, R. Bouillon, H.G. Van Baelen, M.F. Holick, Biochemistry 36
(1991) 4809–4813.
[1] H.S. Lichenstein, D.E. Lyons, M.M. Wurfel, D.A. Johnson, M.D.
McGinley, J.C. Leidli, D.B. Trollinger, J.P. Mayer, S.D. Wright,
M.M.S.D. Zukowski, J. Biol. Chem 269 (1994) 18149–18154.
[2] R. Ray, Proc. Soc. Exp. Biol. Med. 212 (1996) 305–312.
[3] N.E. Cooke, E.V. David, J. Clin. Invest. 76 (1985) 2420–2424.
[4] J.G. Haddad, J. Steroid Biochem. Mol. Biol. 53 (1995) 579–582.
[5] P.J. Goldschmidtt-Clermont, H. van Baelen, R. Bouillon, T.E.
Shook, M.H. Williams, A.E. Nel, R.M. Galbraith, J. Clin. Invest.
81 (1988) 1519–1527.
[27] R. Ray, R. Bouillon, H.G. Van Baelen, M.F. Holick, Biochemistry 30
(1991) 7638–7642.
[28] N. Swamy, R. Ray, Arch. Biochem. Biophys. 319 (1995) 504–507.
[29] N. Swamy, R. Ray, Arch. Biochem. Biophys. 333 (1996) 139–144.
[30] J.K. Addo, R. Ray, Steroids 63 (1998) 218–223.
[31] J.K. Addo, N. Swamy, R. Ray, Steroids 64 (1999) 273–282.
[32] N. Swamy, J. Addo, M.R. Uskokovic, R. Ray, Arch. Biochem.
Biophys. 373 (2000) 471–478.
[6] W.M. Lee, R.M. Galbraith, N. Engl. J. Med. 326 (1992) 1335–1341.
[7] S.E. Lind, D.B. Smith, P.A. Janmey, T.P. Stossel, J. Clin. Invest. 78
(1986) 736–742.
[8] A.B. Shah, S.J. DiMartino, G. Trujillo, R.R. Kew, Mol. Immunol. 43
(2006) 1109–1115.
[33] A. Ray, N. Swamy, R. Ray, Biochem. Biophys. Res. Commun. 365
(2007) 746–750.
[9] J. Zhang, R.R. Kew, J. Biol. Chem. 279 (2004) 53282–53287.
[10] N. Yamamoto, S. Homma, Proc. Natl. Acad. Sci. USA 88 (1991)
8539–8543.
[11] N. Yamamoto, R. Kumashiro, J. Immunol. 151 (1993) 2794–2802.
[12] N. Yamamoto, D.D. Lindsay, V.R. Naraparaju, R.A. Ireland, S.N.
Popoff, J. Immunol. 152 (1994) 5100–5107.
[13] G.B. Schneider, K.A. Benis, N.W. Flay, R.A. Ireland, S.N. Popoff,
Bone 16 (1995) 657–662.
[14] N. Swamy, S. Ghosh, G.B. Schneider, R. Ray, J. Cell. Biochem. 81
(2001) 535–546.
[34] N. Swamy, J.F. Head, D. Weitz, R. Ray, Arch. Biochem. Biophys.
402 (2002) 14–23.
[35] J.F. Head, N. Swamy, R. Ray, Biochemistry 41 (2002) 9015–9020.
[36] R.G. Reed, J. Biol. Chem. 261 (1986) 15619–15624.
[37] S. Watkins, J. Madison, M. Galliano, L. Minchiotti, F.W. Putnam,
Proc. Natl. Acad. Sci. USA 91 (1994) 9417–9421.
[38] F. Campagna, F. Fioretti, M. Burattin, S. Romeo, F. Sentinelli, M.
Bifolco, M.I. Sirinian, M. Del Ben, F. Angelico, M. Arca, Clin.
Chem. 51 (2005) 1256–1258.
[39] L. Docini, G. Caridi, M. Dagnino, A. Sala, G. Gokce, S. Sokucu, M.
Campagnoli, M. Galliano, L. Minchiotti, Clin. Chem. 53 (2007) 1549–
1552.
[15] Y. Koga, V.R. Naraparaju, N. Yamamoto, Proc. Soc. Exp. Biol.
Med. 220 (1999) 20–26.