(Received for publication, July 17, 1995; and in revised form, August 23, 1995)
From the
Liposomes recovered from the blood of liposome-treated CD1 mice
were previously reported to have a complex protein profile associated
with their membranes (Chonn, A., Semple, S. C., and Cullis, P. R.(1992) J. Biol. Chem. 267, 18759-18765). In this study, we have
further characterized and identified the major proteins associated with
very rapidly cleared large unilamellar vesicles. These liposomes
contained phosphatidylcholine, cholesterol, and anionic phospholipids
(phosphatidylserine, phosphatidic acid, or cardiolipin) that
dramatically enhance the clearance rate of liposomes from the
circulation. These anionic phospholipids are normally found exclusively
in the interior of cells but become expressed when cells undergo
apoptosis or programmed cell death, and thus, they are believed to be
markers of cell senescence. Analysis of the proteins associated with
these liposomes by SDS-polyacrylamide gel electrophoresis revealed that
two of the major proteins associated with the liposome membranes are
proteins with electrophoretic mobilities corresponding to M of 66,000 and 50,000-55,000. The 66-kDa
protein was identified to be serum albumin by immunoblot analysis.
Using various biochemical and immunological methods, we have identified
the 50-55-kDa protein as the murine equivalent of human
2-glycoprotein I.
2-glycoprotein I has a strong affinity for
phosphatidylserine, phosphatidic acid, and cardiolipin inasmuch as the
levels of
2-glycoprotein I associated with these anionic liposomes
approach or even exceed those of serum albumin, which is present in
serum at a concentration 200-fold greater than
2-glycoprotein I.
Further, we demonstrate that the amount of
2-glycoprotein I
associated with liposomes, as quantitated by an enzyme-linked
immunosorbent assay, is correlated with their clearance rates;
moreover, the circulation residency time of cardiolipin-containing
liposomes is extended in mice pretreated with anti-
2-glycoprotein
I antibodies. These findings strongly suggest that
2-glycoprotein
I plays a primary role in mediating the clearance of liposomes and, by
extension, senescent cells and foreign particles.
The clearance of liposomes from the circulation, primarily by
phagocytic cells of the reticuloendothelial system, is markedly
affected by the lipid composition of the liposomes(1) . Thus,
incorporation of normally intracellular occurring phospholipids such as
cardiolipin (CL), ()phosphatidic acid (PA), or
phosphatidylserine (PS) markedly enhances the clearance rate of the
liposomes. The biochemical and immunological basis for this clearance
phenomenon is for the most part poorly understood. However, this
clearance is widely believed to involve blood proteins that associate
with liposome membranes. To date, few in vivo studies have
been reported providing evidence that this may in fact be the case. For
example, rat perfusion studies have indicated that in order for
significant liposome uptake by liver macrophages (Kupffer cells) to
occur, the liposome perfusate must contain plasma proteins (2, 3) . Further, we have recently shown using an in vivo mouse animal model that the ability of liposomes to
interact with blood proteins is indeed related to their clearance rate
from the circulation (4) . Liposomes capable of binding the
most blood proteins are cleared very rapidly from blood, whereas
liposomes that exhibit reduced blood protein binding abilities are
cleared at significantly slower rates.
By analyzing the proteins
that associate with the liposomes in blood, we and others (4, 5, 6, 7, 8) have shown
that a myriad of blood proteins associate with rapidly cleared
liposomes. We have demonstrated by using immunoblot analysis that
liposomes binding high levels of blood proteins are enriched with those
blood proteins that function as opsonins, namely complement component
C3 fragments and IgG(5) . The complexity of the protein
profiles associated with the liposome membranes upon exposure to blood
would suggest that other proteins may play important roles in the
immune recognition of foreign particles. Here, we report the
identification of a protein that appears to have a high affinity for
liposomes containing CL, PA, or PS. We employed various biochemical and
immunological techniques to show that this protein is the murine
equivalent of human 2-glycoprotein I (
2 gpI). The
predominance of
2 gpI on rapidly cleared membranes strongly
suggests that
2 gpI may be a key blood protein involved in the
immunological detection of non-self or apoptotic membranes.
Previously, we have demonstrated that LUVs composed of CL, PA, or PS
(20 mol % negative charge) bind blood proteins in amounts exceeding 40
g of protein/mol of lipid immediately upon intravenous administration
in mice and are cleared very rapidly from the circulation (half-lives
of less than 10 min)(4) . This suggests that some of the
proteins associated with the liposome membranes enhance immune
recognition and clearance. Fig. 1depicts the major proteins
associated with liposomes recovered from the blood of CD1 mice 2 min
post-injection. It is immediately clear from Fig. 1that there
are at least two major proteins, having electrophoretic mobilities
corresponding to M of approximately 66,000 and
50,000-55,000 that are associated with these anionic membranes.
Identification of these proteins was carried out. Based on the apparent M
and the fact that albumin is the most abundant
protein in plasma (concentration approximately 40 mg/ml), we suspected
immediately that the 66,000-Da protein band corresponded to serum
albumin. This was confirmed by immunoblot analysis (results not shown).
Figure 1:
Major
proteins associated with LUVs recovered from the circulation of mice.
The proteins associated with recovered LUVs (400 nmol total lipid) were
separated on a 4-20% SDS-PAGE gel under non-reducing conditions
and visualized by Coomassie staining. Lane 1 represents
molecular weight standards (Bio-Rad; myosin, 200,000; phosphorylase B,
97,400; albumin, 66,200; ovalbumin, 45,000; carbonic anhydrase, 31,000;
trypsin inhibitor, 21,500; hen egg lysozyme, 14,400); lane 2,
10 µl of normal mouse serum (1:25 dilution); lane 3, 1
µg of purified mouse 2-glycoprotein I. Lanes 4-7 represent the proteins associated with recovered PC:CH (55:45),
PC:CH:DOPS (35:45:20), PC:CH:DOPA (35:45:20), and PC:CH:CL (35:45:10)
LUVs, respectively.
We were not able to identify this protein by immunoblot analysis
using antisera to several of the major blood proteins (the so-called
``Big Twelve'' group)(14) . However, by comparing the
observed properties of this protein to the reported properties of minor
protein components of blood, one possibility for the identity of this
protein was 2 gpI. Immunoblot analysis of the proteins associated
with the circulating LUVs indicated that this protein cross-reacted
with an antibody to human
2 gpI (Fig. 2) and suggested that
the 50-55-kDa protein was the murine homologue of human
2
gpI.
Figure 2:
Immunoblot detection of
2-glycoprotein I associated with rapidly cleared LUVs. The
proteins associated with LUVs (25 nmol total lipid) recovered from mice
were separated on a 4-20% SDS-PAGE gel under non-reducing
conditions and were subsequently immunoblotted for
2-glycoprotein
I using commercially available rabbit antisera to human
2-glycoprotein I. Lane 1, 10 µl of normal mouse serum
(1:25 dilution); lane 2, 1 µg of purified mouse
2-glycoprotein I. Lanes 3-6 represent the proteins
associated with recovered PC:CH (55:45), PC:CH:DOPS (35:45:20),
PC:CH:DOPA (35:45:20), and PC:CH:CL (35:45:10) LUVs,
respectively.
In subsequent studies, we determined whether this
50-55-kDa protein shared similar biochemical properties to human
or rat 2 gpI. Human and rat
2 gpI have been previously
reported to be heparin-binding
proteins(11, 15, 16, 17, 18) .
To test whether the murine 50-55-kDa protein shared this
property, the proteins associated with PC:CH:CL LUVs were solubilized
with octylglucoside and chromatographed on a heparin-agarose column. As
demonstrated by retention on heparin-agarose (Fig. 3), the
murine 50-55-kDa protein also has an affinity for heparin. The
50-55-kDa protein from mouse serum was found to be soluble in
1.0-3.0% perchloric acid, which is in agreement with previous
findings for human and rat
2
gpI(11, 15, 16, 17, 18) .
Figure 3:
Heparin affinity of the 50-55-kDa
protein associated with rapidly cleared LUVs. PC:CH:CL LUVs were
incubated with normal mouse serum and isolated from unbound components
on a 2.5 90-cm BioGel A15, 100-200 mesh column as
described under ``Experimental Procedures.'' The isolated
liposomes were solubilized in 2% octylglucoside and applied onto a 1.5
7-cm heparin-agarose affinity column (A). The column
was eluted with a step gradient of NaCl in 50 mM Tris, pH 7.4,
and 2-ml fractions were collected. Eluted proteins were analyzed by
dot-blot analysis for
2-glycoprotein I (B).
Finally, to determine conclusively whether 50-55-kDa protein
cross-reacting with the anti-human 2 gpI antibody was the murine
homologue of human
2 gpI, the murine 50-55-kDa protein was
purified using an anti-human
2 gpI affinity column (Fig. 4)
and subjected to N-terminal region protein sequence analysis. As shown
in Fig. 5, the amino acid sequence in the N-terminal region of
the murine 50-55-kDa protein shares a high degree of homology
with that of human
2 gpI and furthermore is identical to the
recently published amino acid sequence of murine
2 gpI.
Figure 4:
Anti-human 2-glycoprotein I affinity
chromatography. A, column elution profile of subfractionated
murine 50-55-kDa protein (see ``Experimental
Procedures'' for details). The bound protein was eluted from the
column with buffer B (50 mM Tris, pH 7.5, 2.5 M
MgCl
). B, dot-blot analysis of column
fractions.
Figure 5:
Partial N-terminal region sequence
analysis of immunoaffinity-purified 50-55-kDa protein. The
obtained sequence was compared to the previously reported amino acid
sequences of human (35) and murine (36) 2-glycoprotein I.
Figure 6:
Plasma recovery of CL-containing LUVs from
anti-2 gpI antibody-treated mice. CD1 mice were pretreated with
two different doses of anti-
2 gpI antibody or 0.9% sodium chloride
solution at 6 and 2 h prior to administering a dose of
100 mg/kg
of PC:CH:CL (35:45:10) LUVs as described under ``Experimental
Procedures.'' The bars represent the average recoveries
(plus standard deviation) from eight mice.
LUVs represent ideal model systems to study the blood
proteins that mediate the clearance of foreign particles. First, the
clearance properties of liposomes are markedly dependent on their lipid
composition. Second, the lipid compositions of the liposomes are
readily altered and defined; thus, factors such as surface charge,
lipid head group specificity, and membrane fluidity can be considered.
Third, the extrusion procedure through 100-nm pore-sized filters
generates stable vesicles that are essentially unilamellar, allowing
the quantitation of the amount of protein associated with the vesicles (4, 5, 9) . Fourth, recent advances in the
isolation of LUVs from the blood components have made possible the
rapid isolation of LUVs from the blood of liposome-treated mice in the
absence of coagulation inhibitors; thus, the stable blood
protein-liposome interactions that occur in vivo can be
analyzed(5) . Finally, analysis of the proteins that associate
with the LUVs is greatly simplified due to the absence of interfering
membrane proteins that exist for systems such as bacteria or senescent
cells. An analysis, therefore, of the various proteins that associate
with very rapidly cleared liposomes compared to circulation-stable
compositions should yield clues as to which blood proteins play a role
in mediating the clearance of foreign particles. In this study, we have
employed various biochemical and immunological methods to identify one
of the major proteins associated with very rapidly cleared liposomes,
and not with slowly cleared liposomes, as being 2-glycoprotein I.
As shown qualitatively in Fig. 1, the levels of 2 gpI
binding to CL-, PA-, or PS-containing PC:CH LUVs corresponds to similar
or even greater levels than those for albumin. This is significant
inasmuch as the reported values for the concentration of
2 gpI in
rats and humans is approximately 0.2 mg/ml plasma (60% is found in the
lipoprotein-free
> 1.21 g/ml bottom fraction after
ultracentrifugation, and the remaining 40% is associated with
triglyceride-rich lipoproteins)(19, 20) . By direct
comparison, the reported values for the concentration of albumin in
rats and humans is approximately 40 mg/ml, 200-fold greater than the
plasma concentration of
2 gpI. If one assumes that the association
of albumin to these vesicles is nonspecific(14) , then this
finding would indicate that
2 gpI is greatly concentrated on these
anionic membranes, suggesting that
2 gpI has a high affinity for
CL, PA, and PS. Interestingly, apolipoprotein J, which shares some
structural similarities to
2 gpI (also known as apolipoprotein H),
has no affinity for these anionic LUVs as determined by immunoblot
analysis using antibodies to apolipoprotein J. (
)This
indicates that not all apolipoproteins have an affinity for CL-, PA-,
or PS-containing LUVs.
As suggested by these findings, 2 gpI
may play a significant role in the clearance of foreign membranes by
phagocytic cells of the reticuloendothelial system. To this effect,
there appears to be a correlation between the amount of
2 gpI
associated with liposomes and their clearance rate.
2 gpI is
associated in relatively high amounts with CL-, PA-, or PS-containing
liposomes, all of which possess very rapid clearance kinetics; low
amounts with phosphatidylglycerol- or phosphatidylinositol-containing
or PC:CH (55:45) LUVs, all of which possess moderately slow clearance
kinetics; or in very low levels with ganglioside
G
-containing liposomes, which are capable of extended
circulation lifetimes(4) . Further substantiating a possible
role of
2 gpI in vesicle clearance is the finding that
2 gpI
exerts a significant effect on triglyceride clearance in
rats(21) . Perhaps the most suggestive evidence that supports a
role of
2 gpI in the clearance of liposomes is our preliminary
observation showing that we are able to significantly prolong the
circulation half-life of CL-containing LUVs in mice that were
pretreated with anti-human
2 gpI antibodies to depress the
circulating levels of
2 gpI.
Our finding that 2 gpI has an
affinity for negatively charged phospholipids is consistent with a
previous report on the lipid specificity of
2 gpI(16) . We
have extended these studies here to show that
2 gpI binds to
higher levels to liposomes containing specifically CL, PA, or PS.
Inasmuch as these phospholipids are not normally expressed on the
exterior surfaces of cells, it is interesting to speculate that
2
gpI plays a role in the detection of these ``foreign''
phospholipids, which are expressed when cells undergo apoptosis or
senescence. A recent study has indeed demonstrated a direct relation
among PS exposure in the outer leaflet of human red blood cells, cell
age, and the propensity for clearance by mononuclear
cells(22) . As well, PS expression on B cells undergoing
apoptosis is enhanced due to loss of membrane phospholipid
asymmetry(23) . Several investigators have suggested that PS
expression is a direct signal for macrophage adhesion and/or
internalization via PS or scavenger
receptors(24, 25, 26, 27, 28) .
Recognition via the PS receptors has been proposed to be an important
phagocyte recognition system of cells undergoing
apoptosis(29, 30) .
A recent study, however, has
challenged the role of scavenger receptors in the phagocytic uptake of
PS-containing liposomes (31) and implied that another, yet
unidentified, mechanism was involved. Here, we propose that inasmuch as
2 gpI is one of the major proteins coating PS-containing LUVs,
perhaps
2 gpI mediates the phagocytic uptake of these vesicles by
macrophages, either directly via
2 gpI receptors or via a
multimeric complex with other blood proteins similar to the C1-IgG
complex of the initiation complex of the classical activation pathway
of complement. With regard to the latter possibility,
2 gpI has
recently been described to function as a cofactor for the binding of
anti-phospholipid antibodies to membranes containing anionic
phospholipids(17, 18) . Anti-phospholipid antibodies
have been shown to be expressed in high titres in autoimmune diseased
states, human immunodeficiency-infected serum, as well as in some
normal serum (32, 33, 34) . The exact role of
2 gpI in the formation of the anti-phospholipid-anionic
phospholipid complex is not known. Whether this anionic
phospholipid(s)-
2 gpI-anti-phospholipid antibody complex leads to
the activation of the classical pathway of complement is not known. The
role of
2 gpI in immune clearance warrants further investigation.