From the
A phospholipid transfer protein was purified from chicken liver
which, in addition to phosphatidylinositol (PI) and phosphatidylcholine
(PC), carries sphingomyelin (SM) between membranes. For comparison, the
PI-transfer protein from chicken liver only carries PI and PC.
Specificity was established by use of phospholipids that carry a
pyrene-labeled acyl chain. Based on the N-terminal sequence and Western
blot analysis we conclude that this protein is an isoform of the
PI-transfer protein. At increasing length of the pyrene-labeled acyl
chain, the isoform expresses a high activity toward SM, a low activity
toward PI, and virtually no activity toward PC.
Phospholipid transfer proteins have been purified from mammals,
plants, fungi, and bacteria (Wirtz, 1991). The best characterized ones
are the phosphatidylcholine (PC)
Recently, the agonist-controlled metabolism of sphingomyelin (SM)
has raised considerable interest because of its crucial role in
regulating cellular function (Hannun, 1994). Given this novel role of
SM, we thought it of interest to investigate whether mammalian tissues
have a SM-transferring protein. A nsL-TP with a pronounced activity
toward SM was purified from rat hepatoma (Dyatlovitskaya et
al., 1978). Distinct SM-transfer activity was identified in
chicken liver (Koumanov et al., 1982). Here we report the
purification to homogeneity of the SM-transferring protein from this
latter tissue. While this study was in progress, a cDNA encoding an
isoform of PI-TP from rat brain was identified (Tanaka and Hosaka,
1994). Evidence will be provided that the SM-transferring protein is
very similar, if not identical, to this isoform.
Q-Sepharose, Sephacryl S-100, heparin-Sepharose CL-6B, Mono
P, and Polybuffer 74 were obtained from Pharmacia (Uppsala, Sweden).
Hydroxylapatite (Bio-Gel, HTP) was from Bio-Rad. Egg yolk PC, PA, and
TNP-PE were from Sigma. Pyr(x)SM, Pyr(x)PI, and
Pyr(x)PC were synthesized as described (Kasurinen and
Somerharju, 1992). High bonding microtiter plates were from Greiner
(Alphen a/d Rijn, The Netherlands). Goat anti-rabbit IgG conjugated
with peroxidase (GAR-PO) was obtained from Nordic Immunological
Laboratories (Tilburg, The Netherlands) and goat anti-rabbit IgG
conjugated with alkaline phosphatase (GAR-AP) from Sigma.
Nitrocellulose membranes were from Schleicher and Schuell (Dassel,
Germany). The anti-PI-TP antibody was raised in rabbits against
predicted epitopes of rat brain PI-TP (Snoek et al., 1992).
All steps were performed at 4 °C.
The assay is based on the increase of the pyrene monomer
fluorescence intensity as a result of the transfer of
pyrenylacyl(Pyr(x))-labeled phospholipid from quenched donor
vesicles (2 nmol of total phospholipid) to a 25-fold excess of
unquenched acceptor vesicles (Van Paridon et al., 1988). The
donor vesicles consisted of Pyr(x)SM or Pyr(x)PC,
TNP-PE, PA, and egg PC (10:10:10:70 mol %) and Pyr(x)PI,
TNP-PE, and egg PC (10:10:80 mol %) and the acceptor vesicles of PA and
egg PC (5:95 mol %). The reaction was carried out in 2 ml of 20 mM Tris-HCl, 5 mM EDTA, 200 mM NaCl (pH 7.4)
containing 0.1 mg of BSA at 37 °C. The reaction was initiated by
the addition of fractions (10-50 µl) containing phospholipid
transfer activity. The initial slope of the progress curve was taken as
an arbitrary unit of transfer activity. Measurements were performed on
a SLM-Aminco SPF-500C fluorimeter equipped with a thermostated cuvette
holder and a stirring device.
The ELISA for the qualitative detection of PI-TP was
performed using high bonding microtiter plates. Aliquots of the column
fractions were added to the wells and incubated for 1 h at 37 °C.
After washing the wells six times with PBS-0 (phosphate-buffered
saline: 137 mM NaCl, 2.7 mM KCl, 6.5 mM Na
Samples were analyzed by electrophoresis on a 15%
SDS-polyacrylamide gel (Laemmli, 1970). Proteins were
electrophoretically transferred from the gel to nitrocellulose
membranes at 1 mA/cm
The N-terminal amino acid sequence of the purified
phospholipid transfer proteins was determined by automated Edman
degradation using the 476A protein sequencer (Applied Biosystems).
Under identical conditions of transfer, the preference of
PI-TP for molecular species was determined (Fig. 4B).
The activity profiles were quite similar to what was observed for
bovine brain PI-TP (Van Paridon et al., 1988). For the species
measured, the protein transferred PI 5-7 times faster than PC.
Transfer of Pyr(x)SM species by PI-TP was negligible. Another
striking point is that PI-TP has a 4-5-fold lower activity toward
both PI and PC, compared with PI/SM-TP (Fig. 4A).
It remains
to be established whether in analogy with the involvement of PI-TP in
the phosphoinositide cycle, the isoform of PI-TP plays a role in the
sphingomyelin cycle.
We thank F. van der Lecq and Dr. A. J. Aarsman of the
Sequence Centre Utrecht for performing the sequence analyses.
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS AND DISCUSSION
FOOTNOTES
ACKNOWLEDGEMENTS
REFERENCES
(
)-transfer
protein from bovine liver (Kamp et al., 1973), the
phosphatidylinositol (PI)-transfer protein (PI-TP) from bovine and rat
brain (Helmkamp et al., 1974; Venuti and Helmkamp, 1988) and
yeast (Aitken et al., 1990), and the nonspecific lipid
transfer protein (nsL-TP) from bovine, rat, and human liver (Crain and
Zilversmit, 1980; Bloj and Zilversmit, 1977; Van Amerongen et
al., 1987) and plants (Kader, 1975). Recently, PI-TP has raised
considerable interest because of its proposed roles in the
phosphoinositide cycle (Thomas et al., 1993) and the
ATP-dependent, Ca
-activated secretory process (Hay
and Martin, 1993). Furthermore, PI-TP from yeast is identical to the
protein SEC14p, which is an essential factor in the secretory vesicle
flow from the trans-Golgi network to the plasma membrane (Bankaitis et al., 1989, 1990). Extensive studies have been carried out
to establish that the lipid transfer activity of SEC14p is related to
its function in protein secretion (Cleves et al., 1991;
Skinner et al., 1993; McGee et al., 1994).
Materials
Purification of Phospholipid Transfer Proteins
Step 1
A 33% (w/v) homogenate of fresh chicken liver
(2000 g) was prepared in 0.9% NaCl, 20 mM Tris-HCl, pH 7.4, 1 mM EDTA, 10 mM NaF, 1
mM sodium pyrophosphate, 0.17 mg of trypsin inhibitor/liter,
and 1 mM phenylmethylsulfonyl fluoride. After centrifugation
at 14,000
g for 1 h, the supernatant was collected.
Step 2
A 40-90% ammonium sulfate precipitate
of the supernatant was prepared and sedimented by centrifugation at
14,000 g for 60 min. The pellet was dissolved in 20
mM Tris-HCl, pH 7.4, 1 mM EDTA, 10 mM
-mercaptoethanol (TEM buffer). After dialysis overnight
against TEM buffer, the solution was centrifuged for 1 h at 100,000
g to remove insoluble material.
Step 3
The dialysate was applied to a column (11.5
21 cm) of Q-Sepharose equilibrated with TEM buffer. After
rinsing with TEM buffer (2 liters), the protein was eluted with a
linear gradient of TEM buffer and 500 mM NaCl/TEM buffer (2
2.5 liters) (flow rate of 400 ml/h; 20-ml fractions).
SM-transfer activity eluted between 350 and 400 mM NaCl.
Step 4
The active fractions were applied to a
Sephacryl S-100 column (5 94 cm) equilibrated with 10
mM potassium phosphate, pH 6.8, 10 mM
-mercaptoethanol. The column was rinsed with the same buffer (flow
rate of 180 ml/h; 18-ml fractions). SM-transfer activity eluted at
about half the column volume.
Step 5
The active fractions were applied to a
hydroxylapatite column (1.4 40 cm) equilibrated with 10 mM potassium phosphate, pH 6.8, 10 mM
-mercaptoethanol.
The column was rinsed with a linear gradient of 10-50 mM potassium phosphate, pH 6.8, 10 mM
-mercaptoethanol
(2
300 ml) (flow rate of 25 ml/h; 5-ml fractions). SM-transfer
activity eluted at 20 mM phosphate. This activity co-eluted
with PI-transfer activity. A second peak of PI-transfer activity devoid
of SM-transfer activity eluted at 35 mM phosphate (see Fig. 1).
Figure 1:
Separation of PI/SM-TP and PI-TP by
chromatography on hydroxyl-apatite. Column fractions were assayed for
PI-transfer activity () and SM-transfer activity
(
).
Step 6
The PI/SM- and PI-transfer activity peaks
were pooled separately, and each active pool was dialyzed against 20
mM Tris-HCl, pH 7.2, 50 mM NaCl, 5 mM -mercaptoethanol and then applied to a heparin-Sepharose
CL-6B column (2.3
24 cm). By isocratic elution SM/PI-transfer
activity was recovered between 120 and 200 ml and PI-transfer activity
between 200 and 360 ml of elution buffer. The active fractions were
pooled and dialyzed against 25 mM methylpiperazine pH 5.7, 10
mM
-mercaptoethanol.
Step 7
Each active pool was applied to a Mono P
column (0.5 20 cm). The column was eluted with 25 ml of 10%
Polybuffer 74-HCl, pH 4, 10 mM
-mercaptoethanol (flow
rate of 0.8 ml/min, 1-ml fractions). Both PI/SM-TP and PI-TP activity
eluted from the column in two peaks between pH 4 and 5.
Phospholipid Transfer Assay
ELISA Procedure
HPO
, and 1.5 mM KH
PO
), the wells were incubated for 1 h at
37 °C with PBS-0 containing 1% (w/v) BSA as blocking agent. Further
manipulations were carried out at room temperature. After washing the
wells with PBS/Tween (PBS-0 containing 0.05% (v/v) Tween 20), the wells
were incubated for l h with anti-PI-TP antibody in PBS/Tween/BSA (1%,
w/v). The wells were washed with PBS/Tween and incubated with GAR-PO
(0.10 ml; 1:1000 diluted in PBS/Tween/BSA) for 1 h. After washing the
wells with PBS/Tween, the substrate solution was added and the
absorbance determined at 492 nm.
Gel Electrophoresis and Blotting
for 75 min. 3% gelatin (w/v) in
Tris-buffered saline (20 mM Tris-HCl, 0.5 M NaCl, pH
7.4) was used as a blocking agent. PI-TP was identified by incubating
the nitrocellulose membranes with antibody in Tris-buffered saline
containing 1% (w/v) gelatin followed by an incubation with GAR-AP and
color development.
Amino Acid Sequence Determination
Purification
Based on the evidence that the
membrane-free supernatant of chicken liver contains SM-transfer
activity (Koumanov et al., 1982), we have selected this tissue
for the purification of the active protein (see ``Experimental
Procedures''). Fractionation on both Q-Sepharose (see ``Step
2'') and Sephacryl S-100 (see ``Step 3'') yielded a
single SM-transfer activity peak. Initially, this activity was detected
by measuring the transfer of bovine
[N-methyl-C]SM from
unilamellar vesicles to bovine heart mitochondria in an assay similar
to that described for the transfer of
[
C]phosphatidylethanolamine (Poorthuis and
Wirtz, 1983). Subsequently, transfer activities were routinely measured
by making use of pyrene-labeled phospholipid analogs (see
``Experimental Procedures''). Since in a previous study PI-TP
purified from bovine brain was shown to have a limited capacity to
transfer SM between membranes (DiCorletto et al., 1979), the
column fractions were tested also for PI-TP by use of a specific ELISA.
With both Q-Sepharose and Sephacryl S-100 one immunoreactive PI-TP peak
was detected that coincided with the SM-transfer activity peak. Hence,
in the subsequent purification steps the column fractions were tested
for both SM- and PI-transfer activity. Fractionation on hydroxylapatite
(see ``Step 4'') yielded two peaks with PI-transfer activity,
the first peak of which only contained the SM-transfer activity (Fig. 1). Further purification on a heparin-affinity column (see
``Step 5'') and Mono P (see ``Step 6'')
demonstrated convincingly that, in addition to PI-TP, a protein exists
with both PI- and SM-transfer activity (denoted as PI/SM-TP).
Fractionation on the isoelectrofocusing column (Mono P) showed that
PI-TP consists of two forms (PI-TP I and II), which differ slightly in
isoelectric point. In agreement with previous observations on bovine
brain PI-TP, this charge difference is probably due to one form
containing an endogenously bound PI molecule and the other form a PC
molecule (Van Paridon et al., 1987). Possibly for similar
reasons, two forms of PI/SM-TP (I and II) were collected from the Mono
P column. Analysis by SDS-polyacrylamide gel electrophoresis (Fig. 2A) indicated that by fractionation on Mono P both
PI-TP I and II (lanes3 and 4) and PI/SM-TP
I (lane5) were purified to homogeneity. PI/SM-TP II (lane6) was contaminated with one major protein and
was not further purified. Starting from 2000 g of liver the yields were
0.2 mg of PI-TP I, 0.15 mg of PI-TP II, and 0.4 mg of PI/SM-TP I.
Figure 2:
SDS-polyacrylamide gel electrophoresis (A) and Western blot analysis (B) of
phospholipid-transfer proteins purified from chicken liver. Lane1, bovine brain PI-TP (control); lane2, marker proteins; lane3, PI-TP I, lane4, PI-TP II; lane5, PI/SM-TP
I; lane6, PI/SM-TP II.
Homology
Analysis by Western blotting (Fig. 2B) using the antibody elicited against epitope
segments of rat brain PI-TP showed that the antibody was cross-reactive
with both forms of PI-TP (lanes3 and 4) and
PI/SM-TP (lanes5 and 6). This strongly
suggests an extensive similarity between chicken liver and rat brain
PI-TP, as well as between PI-TP and PI/SM-TP. On the other hand, one
may infer from this analysis that the molecular mass of PI-TP (35 kDa)
is different from that of PI/SM-TP (39 kDa). To further corroborate the
similarity between these proteins, the N-terminal amino acid sequence
was analyzed by automated Edman degradation. As shown in Fig. 3,
the first 21 amino acid residues of PI/SM-TP I were identical to that
of the isoform of rat brain PI-TP (designated -PI-TP), the
sequence of which was deduced from the cDNA nucleotide sequence (Tanaka
and Hosaka, 1994). The N-terminal sequence (first 21 amino acid
residues) of PI-TP I was identical to that of PI-TP II and nearly
homologous with that of rat brain PI-TP with conservative replacements
at position 3 (Ile for Leu) and at position 15 (Glu for Asp). PI/SM-TP
was highly homologous with PI-TP I and II with conservative
replacements at position 6 (Phe for Tyr) and position 9 (Val for Ile).
The residue at position 12, which is presumed to be Cys, was replaced
for Val and Gln at position 15 for Glu. A similar high homology was
observed for the N-terminal sequence of
-PI-TP and PI-TP from rat
brain (Fig. 3) (Tanaka and Hosaka, 1994). In the latter study it
was reported that the total amino acid sequence of
-PI-TP showed
77% identity to that of PI-TP and about 94% similarity.
Figure 3:
N-terminal amino acid sequences of chicken
liver PI/SM-TP I and PI-TP I and II. For comparison, the sequence of
the isoform of rat brain PI-TP (-PI-TP) deduced from the cDNA
nucleotide sequence (Tanaka and Hosaka, 1994) and rat brain PI-TP
sequence (Dickeson et al., 1989) are shown. The sequence
method used is not suitable to identify Cys (see PI/SM-TP I, position
12).
The amino
acid sequences of mouse, rat, and human PI-TP share about 99% identity,
which makes this protein exceedingly conserved (Dickeson et
al., 1989, 1994; Geijtenbeek et al., 1994). Recently, we
established that, in addition to chicken liver PI/SM-TP, the N-terminal
amino acid sequence of bovine brain PI/SM-TP is also identical to that
of rat brain -PI-TP.
(
)This strongly
suggests that, similar to PI-TP, PI/SM-TP is highly conserved among
species.
Specificity
During the purification of PI/SM-TP
and PI-TP, the transfer activities were determined in a continuous
fluorescent transfer assay using as substrates
1-palmitoyl-2-pyrenyldecanoyl-PI (Pyr(10)PI) and N-pyrenylmyristoyl-SM (Pyr(14)SM). Previously it was found
that by varying the acyl chain length at the sn-1- and sn-2-position, PI-TP from bovine brain had a preference for
certain molecular species of PI (Van Paridon et al., 1988). In
order to establish whether this was also the case for PI/SM-TP, the
pyrenylacyl chain length was varied at the sn-2-position of
1-palmitoyl-2-Pyr(x)PI and at the amine moiety of
Pyr(x)SM. As shown in Fig. 4A, the protein
expressed the highest transfer activity with Pyr(10)PI and, relative to
this species, a 5-fold lower activity with Pyr(14)PI. Very
characteristically, PI/SM-TP expressed a very low activity with
Pyr(10)SM and, relative to this species, a 9-10-fold higher
activity with Pyr(12)SM and Pyr(14)SM. With the Pyr(14) species the
protein expressed a more than 3-fold higher activity with SM than with
PI. Under conditions where PI/SM-TP is incubated with the donor
vesicles only, this protein binds Pyr(x)PI and
Pyr(x)SM species with a preference similar to that observed
for transfer (data not shown). Given the occurrence of long chain fatty
acids (>18 carbon atoms) in natural PI and SM, we predict that in situ PI/SM-TP has a distinct preference for SM.
Figure 4:
Effect
of acyl chain length on the transfer of pyrenyl acyl-labeled
phospholipid species by PI/SM-TP (A) and PI-TP (B).
, PI;
, SM;
, PC. x indicates the number
of carbon atoms of the pyrenyl-labeled acyl
chain.
As was
previously observed for bovine brain PI-TP (Van Paridon et
al., 1988; Kasurinen et al., 1990) PI/SM-TP may also
transfer PC, albeit at a relatively low rate (Fig. 4A).
It is quite remarkable that with both PC and SM having the same polar
headgroup, the protein expressed virtually no activity with Pyr(12)PC
and Pyr(14)PC. This strongly suggests that, provided the correct chain
length is present, PI/SM-TP recognizes molecular features of SM that
are alien to PC like the sphingosine moiety. On the other hand, the
polar headgroup is required for activity as the protein failed to
transfer the analogous pyrene-labeled ceramide species (data not
shown).
Conclusion
A novel phospholipid transfer protein
has been identified with a distinct affinity for SM. This protein is an
isoform of PI-TP with the remarkable feature that it expresses a high
activity toward long chain SM, concomitant with a low activity toward
PI and PC. This protein also occurs in mammalian tissues.
©1995 by The American Society for Biochemistry and Molecular Biology, Inc.