(Received for publication, September 9, 1994; and in revised form, January 4, 1995)
From the
Microsomal triglyceride transfer protein (MTP) is a lipid transfer protein that is required for the assembly and secretion of very low density lipoproteins by the liver and chylomicrons by the intestine. To further elucidate the nature of the lipid molecule binding and transport site on MTP, we have studied the relative rates at which MTP transports different lipid species. Assay conditions were chosen in which there were minimal changes in the physical properties of the substrate membranes so that transfer rates would reflect MTP-lipid interactions at a membrane surface. Lipid transport rates decreased in order of triglyceride > cholesteryl ester > diglyceride > cholesterol > phosphatidylcholine. Changes in the hydrophobic nature of a lipid molecule by the addition of a fatty acid, modulated the ability of MTP to transport it. Addition of one acyl chain from diglyceride to triglyceride, lysophosphatidylcholine to phosphatidylcholine, or cholesterol to cholesteryl ester increased the rate of MTP-mediated transport 10-fold. In contrast, the lipid transport rate was insensitive to the changes in the structure or charge of the polar head group on phospholipid substrates. Zwitterionic, net negative, or net positive charged phospholipid molecules were all transported at a comparable rate. The ability of MTP to transport lipids is strongly correlated to the binding of these lipids to MTP. Thus, MTP has a specific preference for binding and transporting nonpolar lipid compared with phospholipids, and within a class of lipid molecules, a decrease in polarity increases its tendency to be transported.
Microsomal triglyceride transfer protein (MTP) ()catalyzes the transfer of triglyceride (TG), cholesteryl
ester (CE), and phosphatidylcholine (PC) between membranes (Wetterau
and Zilversmit, 1985). The transfer activity is found within the lumen
of microsomes isolated from the liver and intestinal mucosa. MTP has a
unique structure compared with other characterized lipid transfer
proteins. It is a heterodimer containing subunits of molecular mass 58
and 97 kDa (Wetterau et al., 1991a). Other intracellular
mammalian lipid transfer proteins characterized to date are single
polypeptides with molecular masses ranging from 11 to 36 kDa (Wirtz,
1991). The 58-kDa component of MTP has been identified as the
multifunctional protein, protein disulfide isomerase (Wetterau et
al., 1990). The large 97-kDa subunit possesses the lipid transfer
activity or confers lipid transfer activity on the complex. The large
subunit of MTP was not detectable in four subjects with the disease
abetalipoproteinemia (Wetterau et al., 1992), a rare human
genetic disease characterized by a defect in very low density
lipoprotein (VLDL) and chylomicron assembly (reviewed in Gregg and
Wetterau(1994)). The discovery of mutations in the gene encoding the
large subunit of MTP of subjects with abetalipoproteinemia, which would
result in an absence of functional MTP, demonstrates that an absence of
MTP causes abetalipoproteinemia and that MTP is required for
lipoprotein assembly or secretion (Sharp et al., 1993;
Shoulders et al., 1993).
The general scheme for lipoprotein assembly and secretion is well accepted (reviewed in Vance and Vance(1990), Gibbons(1990), Boren et al.(1991), Davis(1993), and Yao and McLeod(1994)). Initial assembly occurs in the endoplasmic reticulum (ER) where apolipoproteins, cholesterol, phospholipid, and triacylglyceride are synthesized and incorporated into lipoprotein particles. The particles are subsequently transported to Golgi and secreted. However, the sequence of events leading to formation of mature lipoprotein particles in ER lumen is still under investigation. Several reports have suggested that plasma VLDL is assembled by sequential addition of lipids to nascent apolipoproteins (Jenero and Lane, 1983; Higgins and Hutson, 1984; Boström et al., 1988). Although MTP is required for lipoprotein assembly, its exact role in the assembly process is not known. Because MTP transports lipid by a shuttle mechanism (Ping Pong Bi Bi kinetics) (Atzel and Wetterau, 1993a), it has been hypothesized that MTP acts as carrier of lipids from their site of synthesis to nascent lipoproteins within the ER.
To further our understanding of the lipid transport properties of MTP and the relationship between lipid structure and MTP-mediated lipid transport, the initial rates of transfer of different classes of neutral lipid and phospholipid from donor to acceptor small unilamellar vesicles (SUV) were measured and compared with the binding of these lipid molecules to MTP. The experiments were performed under conditions where the physiochemical properties of the donor membranes were minimally affected. Therefore, the rate of lipid transfer should reflect the interaction of the lipid molecules with MTP at the membrane surface. In this study, we demonstrated that MTP has a preference for transporting neutral lipids and that the size of the hydrophobic moiety, regardless of the lipid class investigated, plays an important role in the MTP-mediated transfer. In contrast, MTP did not discriminate between different phospholipids. These results were used to further refine our model for the lipid molecule binding and transport site on MTP and provide additional insight into the role of MTP in lipoprotein assembly.
1,2-Dioleoyl-L-3-phosphatidyl-L-[3-C]serine,
1,2-di[1-
C]oleoyl-L-3-phosphatidylcholine,
1,2-dipalmitoyl-L-3-phosphatidyl[N-methyl-
H]
choline,
1,2-dioleoyl-L-3-phosphatidyl[2-
C]ethanolamine,
[4-
C]cholesterol, cholesteryl
[1-
C]oleate, and L-3-phosphatidyl[U-
C]inositol were
obtained from Amersham Corp., and [
H]squalene was
obtained from DuPont NEN. All lipids and lipolytic enzymes were
obtained from Sigma. Lipids were stored under N
gas in
chloroform at -20 °C. MTP was purified from bovine liver by a
modification of an earlier published method (Wetterau et al.,
1991b) which will be published elsewhere. The purified protein had an
activity of 3.0% TG transfer/min/µg of protein in our assay and
showed only two bands of apparent molecular masses 88 and 58 kDa on 8%
SDS-polyacrylamide gel electrophoresis (Laemmli and Favre, 1973).
To prepare monoglyceride (MG), C-labeled PC was incubated with both phospholipase A
and phospholipase C (2 units each) and extracted as described
above. MG was separated from DG, lyso-PC, and PC on silica gel plates
initially developed in solvent B until the solvent front ascended half
way up the plate and then in solvent A to the top of the plate.
Radioactive MG was identified and extracted as described above. The
identity of labeled substrates was confirmed by thin-layer
chromatography.
The lipid transfer assay mixture contained donor membranes (40 nmol of egg PC, 7.5 mol % cardiolipin, and 0.25 mol % radiolabeled substrate), acceptor membranes (240 nmol of egg PC), and 5.0 mg of bovine serum albumin in a total volume of 0.68 ml of assay buffer. The reaction was started by the addition of MTP (100-3000 ng for different substrates) in 20 µl. The transfer reaction was carried out at two different concentrations of MTP for each substrate. After 60 min, the reaction was terminated by the addition of 0.5 ml of DE52-cellulose (Whatman, Maidstone, United Kingdom) preequilibrated in 15 mM Tris-HCl, pH 7.4, 1.0 mM EDTA and 0.02% sodium azide (1:1, v/v). The mixture was agitated for 5 min and centrifuged at maximum speed in a Biofuge B centrifuge (Baxter Scientific, McGaw Park, IL) for 3 min to pellet the DE52 containing bound donor vesicles. The recovery of acceptor vesicles after the sedimentation of donor membranes with DE52-cellulose was determined by measuring the concentration of phospholipid using a phosphate assay (Rouser et al., 1966). The recovery of acceptor vesicles from the reaction mixture ranged from 92 to 105%, with a mean of 98 ± 6%. To quantitate lipid transfer, radioactivity in 0.5 ml of supernatant was measured.
First order kinetics were used to calculate the lipid
transfer rate using the equation [S] =
[S]e
(Wetterau and
Zilversmit, 1985), where [S]
and [S]
are the fraction of the available labeled lipid in the donor membrane
at times 0 and t, respectively, and k is the fraction
of the available labeled lipid transferred per unit time. This
calculation corrects for the depletion of labeled lipid in donor
vesicles which occurs with time. Background transfer of neutral or
phospholipid in the absence of MTP were 1-2% or 5-7%/h,
respectively. Background transfer in the absence of MTP was subtracted
from total transfer to calculate MTP-mediated transfer. To calculate
phospholipid transfer, we only used total transfer values which were at
least twice that of background. The transfer rates with all substrates
were linear up to 20-30% transfer.
To validate the
lipid binding assay, MTP (320 pmol) was incubated with donor vesicles
containing 0.25 mol % [C]TG as described above
and then the MTP was isolated from vesicles on a Superose 12 HR (10/30)
column using an fast protein liquid chromatography system (Pharmacia,
Upsala, Sweden). On this column, vesicles elute in the void volume (7.5
ml), whereas MTP elutes with a elution buffer of 11 ml. The amount of
TG bound to MTP was quantitated by measuring radioactivity and protein
in each eluted fractions. The amount of TG bound to MTP obtained by
this method agreed with the results obtained from the
ultracentrifugation method (see results).
Figure 1:
Transport of
triglyceride and phosphatidylcholine by MTP. The lipid transfer from
donor vesicles containing 0.25 mol % [C]TG or
[
C]PC to acceptor vesicles was measured as
described under ``Materials and Methods.'' The assay
contained 0-50 µg of MTP, and the reaction was carried out
for 1 h at 37 °C.
The conditions established for TG and PC were used for estimating the available pool sizes of other substrates in the donor membranes. Lipid transfer was measured in the presence of a large excess of MTP (50 µg) at 37 °C for 1 h. The results summarized in Table 1show that with the exception to MG, which was not transferred by MTP, 77-95% of all neutral lipid examined (TG, DG, CE, or squalene) was available for MTP-catalyzed lipid transfer, whereas 54-68% of the phospholipids tested was available for transfer. These results show that most if not all of the neutral lipid in the donor membranes was accessible for transfer by MTP, whereas it appears that phospholipid was evenly distributed between the monolayers of the bilayer and that only the phospholipid distributed on the vesicle surface was available for MTP-mediated transfer. Thus, when measuring the initial transfer rates, the available labeled lipid used for calculating lipid transfer rates (see ``Materials and Methods'') was 66% of the total amount present for phospholipid and 100% for neutral lipid.
Figure 2: Relative rate of MTP-mediated lipid transfer for different classes of lipid substrates. The relative rates of MTP-mediated lipid transfer were measured using donor vesicles containing 0.25 mol % lipid substrate in PC vesicles as described under ``Materials and Methods.'' The transfer rates were measured at two different concentrations of MTP for 1 h at 37 °C and calculated as percent transfer per mg of MTP. Results are expressed as the transfer rate relative to that of TG transfer rate (for TG, 100% = 3.5 pmol/min/µg). Each value is the average of at least four different measurements ± S.D.
To further test for specific charge-charge interactions between MTP and a transported phospholipid molecule, MTP-mediated transport of a positively charged phospholipid molecule was investigated. A PC analog containing a net positive charge (ethyl-PC) was synthesized by alkylating the phosphate of zwitterionic PC with an ethyl group (Table 2). This addition neutralizes the negative charge on the phosphate. Similar to the results with other phospholipid species tested, the rate of transfer was not affected significantly by a net positive charge in the phospholipid head group (Table 2). These findings indicate that the lipid transport is insensitive to the changes in the structure or charge of the polar moiety of a phospholipid substrate.
Figure 3: Relative rate of MTP-mediated lipid transfer for lipid molecules with decreasing number of fatty acyl chains. The relative rates of MTP-mediated lipid transfer were measured using donor vesicles containing 0.25 mol % lipid substrate in PC vesicles as described under ``Materials and Methods.'' The transfer rates were measured at two different concentrations of MTP for 1 h at 37 °C and calculated as percent transfer per mg of MTP. Results are expressed as the transfer rate relative to that of TG (left panel), PC (middle panel), or CE (right panel) transfer rates. For TG, PC, or CE, 100% transfer was equal to 3.5, 0.09, or 2.3 pmol/min/µg of protein, respectively. Each value is the average of three to ten measurements ± S.D.
TG was bound to MTP at 0.044 mol/mol of MTP (Table 3). The amount of TG bound, as determined by ultracentrifugation, was similar to that obtained when MTP was reisolated by column chromatography (0.039 mol/mol of MTP) (see ``Materials and Methods''). This value is also comparable, although somewhat higher than that reported by Atzel and Wetterau (1993b) for similar TG binding experiments performed at 23 °C. The amount of lipid bound to MTP decreased in the order of TG > CE > DG > squalene > PC which correlated well with their decreasing rates of transfer. The amount of MG bound to MTP did not correlate with its transfer. A small amount of MG was bound to MTP ( that of TG), but MTP did not transfer this lipid from donor to acceptor membranes. Using the data from Fig. 2and Table 3, the ratio of lipid bound per MTP molecule was plotted against the relative transfer rates of the different lipid substrates (Fig. 4). A linear relationship between substrate binding to MTP and relative transfer rates was observed with a correlation coefficient of 0.99. This indicates that the ability of MTP to bind different classes of lipid molecules at the membrane surface governs the relative rates at which these lipids are transported between the membranes.
Figure 4: A linear correlation between the binding of different lipid substrates to MTP and their transfer rates. The lipid transfer activity from Fig. 2was plotted against amounts of lipid bound to MTP from Table 3.
The binding of different
phospholipids to MTP was also investigated by incubating donor vesicles
containing 0.25 mol % [C]phospholipid substrate
and a trace of [
H]dipalmitoyl PC.
[
H]dipalmitoyl PC binding to MTP was measured as
a control. The values for the amount of lipid bound to MTP obtained for
the different phospholipid species (see Table 4) were all very
similar, analogous to what was found for their transfer rates (Table 2). These results indicate that changes in the structure
or charge of the headgroup of a phospholipid molecule do not affect the
binding of the phospholipid to MTP.
Using lipid binding assays and kinetic analysis, it has been demonstrated previously that MTP binds and shuttles lipid molecules between membranes (Atzel and Wetterau, 1993a). To further understand the nature of the site or sites on MTP which bind and transport lipid molecules, the rates of MTP-mediated transport of lipid molecules of varying structure were compared. For the transfer reactions used in this study, donor membranes contained PC, 7.5 mol % cardiolipin, and a trace (0.25 mol %) of the lipid studied. Acceptor membranes contained PC. Trace quantities of the lipid studied were used so that the effect of the lipid on the physical properties of the membranes was minimal. Thus, the transport rates should reflect the interaction between MTP and the lipid molecules in the membrane once MTP is bound to the membrane surface.
For our analysis, it was assumed that the radiolabeled lipid molecules were equally distributed between the monolayers of the bilayer membrane of the donor vesicles and that only the phospholipid molecules on the surface of the monolayer were available to MTP, whereas all of the nonpolar lipid molecules were available to MTP. This was confirmed for both broad classes of lipid investigated. The maximum transfer observed in the presence of excess MTP was 54-68% for all phospholipid species investigated, comparable with the calculated maximum transfer of 57%. In contrast to the polar lipids, all of the neutral lipids were accessible to MTP as the maximum transfer (range 77-95%) was similar to the maximum calculated transfer (86%), assuming all the lipid was available. This is consistent with the previous findings that uncharged lipid molecules rapidly diffuse between the two monolayers of a phospholipid bilayer, whereas charged lipids slowly diffuse (for review see Zachowski(1993)). Thus, to calculate transfer, the available pool size for phospholipid was reduced to 66% of the total for phospholipid and assumed to be 100% for neutral lipid. This may underestimate the transfer rates for neutral lipid in that at any given time, only two-thirds of the neutral lipid would be found on the vesicle surface.
With the exception of MG, MTP catalyzed the transfer of every lipid species investigated. Under the experimental conditions utilized in these studies, MTP had clear preferences for the lipid molecules it transferred. These trends undoubtedly reflect the structure of the site on MTP which binds and transports the lipid molecules, and thus these results provide insights into the nature of the lipid binding pocket on MTP. MTP displayed a distinct preference for transporting neutral lipids when compared with polar lipid. For all classes of lipid molecules studied, transfer was directly related to the hydrophobicity of the molecules. For example, the addition of a fatty acyl chain or chains to MG, cholesterol, or lyso-PC dramatically increased their transfer rates (Fig. 3).
MTP binds and transports both neutral lipids and phospholipids. Although MTP may accommodate a polar head group, it slows the rate of MTP-mediated transport when compared with a more hydrophobic molecule without the charged head group. However, neither the nature nor the charge of the head group of the phospholipid molecules investigated had any effect on MTP mediated lipid transport. Although MTP binds and transports lipid molecules containing polar head groups, there does not appear to be any specific interactions between the head groups and MTP. Thus, the lipid binding pocket on MTP which binds and transports lipid appears to be hydrophobic in nature; however, it is able to accommodate the polar head groups of phospholipid molecules. It is quite flexible in the nature of the molecules it can bind, suggesting there are minimal specific interactions between the lipid molecules and MTP. In addition, the rate of transport of different lipid classes parallels the binding of these lipids to MTP (Fig. 4). This suggests that the binding of lipid to MTP is the rate-determining step in the MTP-mediated lipid transport process.
Phospholipid binding to MTP
was investigated by incubating donor vesicles containing 0.25 mol %
[C]phospholipid substrate and a trace of
[
H]dipalmitoyl PC. If one assumes
[
H]dipalmitoyl PC or
[
C]dioleoyl PC are representative of all the PC
in the donor vesicles, the binding of PC to MTP was 1.4-1.6
mol/mol of MTP, comparable with the values reported for binding
experiments performed at 23 °C (Atzel and Wetterau, 1993b). This
suggests that MTP has at least two phospholipid binding sites.
Numerous other proteins bind lipid molecules. These proteins can be divided into two classes, those that bind multiple lipid molecules without defined binding sites (for example plasma apolipoproteins or vitellogenin) and those that have specific sites that bind individual lipid molecules. The latter category includes fatty acid-binding proteins and serum albumin which bind fatty acid and lipid transfer proteins which bind a variety of lipid molecules. These proteins display varying preferences for the lipid molecules they bind.
Shoulders et al.(1994) proposed that MTP is a member of the vitellogenin family. However, vitellogenin is more hydrophobic in nature than MTP and unlike MTP, it forms a large lipid binding cavity which simultaneously binds multiple lipid molecules. There is no apparent sequence homology between MTP and other known proteins, including triglyceride lipase, cholesteryl esterase, and cholesterol ester transfer protein (CETP), whose functions involve binding nonpolar lipids. CETP is functionally related to MTP in its ability to catalyze the transport of neutral lipids between lipoproteins (Swenson et al., 1988). However, similar to vitellogenin, CETP is more hydrophobic in nature than MTP and binds multiple PC molecules (up to 11 mol of PC/mol of CETP) (Swenson et al., 1988). Thus, it appears that CETP does not have defined binding sites for PC.
The lipid specificity of MTP suggests it may have properties in common with lipases. Among these, fungal lipase (reviewed in Derewenda and Sharp(1993)) and pancreatic lipase (Verger, 1984) are 1,3-sn-specific triacylglyceride hydrolases. Lipoprotein lipase, although relatively specific for TG, is capable of hydrolyzing phospholipid in TG-rich lipoproteins (Deckelbaum et al., 1992; Shirai et al., 1983), whereas mammalian hepatic lipase shows a very broad substrate spectrum which includes phospholipids and neutral lipids (reviewed in Derewenda and Sharp(1993)). Thus, the character of the lipid binding site on MTP may resemble more the catalytic site of a lipase which has a broad specificity of lipid binding than the more selective lipid transfer proteins.
The lipases characterized to date share common structural features. Brzonzowski et al.(1991) have demonstrated using crystallographic data that the neutral lipid binding site of fungal Rhizomucor miehei triacylglycerol lipase is buried within the protein. A helical flap structure (lid) which covers the binding site is displaced during the conformation change associated with lipid binding. The conformational change exposes the hydrophobic side of the lid and expands the nonpolar surface around the catalytic site. The opening of a lid-like structure is a common feature of fungal lipases, pancreatic lipases, hepatic lipases, and lipoprotein lipases (reviewed in Hide et al.(1992)). Whether MTP contains a buried site covered by a lid-like structure remains to be determined.
MTP is required for the assembly of plasma VLDL and chylomicrons (Sharp et al., 1993; Shoulders et al., 1994)). Presumably it transports newly synthesized lipid from the ER membrane to apolipoprotein B containing particles in the lumen of the ER. MTP transports TG, CE, and phospholipid, all of which are components of VLDL and chylomicrons. Whether all lipid in the mature lipoprotein particle, or only a portion, is complexed to apoB through MTP-mediated transport is not known. If MTP-mediated transport plays a major role in coupling phospholipid to apoB, then the phospholipid composition of newly synthesized VLDL and chylomicrons should reflect the phospholipid composition of the lumenal surface of the ER membrane, because MTP does not discriminate between the various phospholipid species it transports. Of the total phospholipid content of the newly synthesized VLDL isolated from rat liver, 75.5% is PC and 2.6% is PE (Vance and Vance, 1985). In contrast, PE in the rough or smooth microsomes is approximately 25% of the total phospholipid (Bollen and Higgins, 1980), and two-thirds of that is located on the lumenal surface of the membrane (Hutson and Higgins, 1982). Characterization of rat MTP indicated that like bovine MTP, it transports PC and PE at similar rates (data not shown). Thus, the phospholipid composition of newly synthesized VLDL does not appear to be controlled by the ER membrane composition and the transport properties of MTP, suggesting that other factors must play a role in controlling the phospholipid composition of newly synthesized VLDL.
In conclusion, MTP appears to be a unique lipid binding protein. MTP has distinct sites for the molecules it binds in that it has a finite number of lipid molecule binding sites (about two per MTP); however, it binds a very wide variety of molecules and binding does not appear to be controlled by specific protein-lipid interactions, but rather the general hydrophobic nature of the lipid molecule. Our results strongly suggest that the hydrophobic core of the lipid molecule plays the dominant role in promoting its interaction with the transport pocket on MTP. Thus, the lipid binding site on MTP is rather unusual in its properties when compared with previously characterized lipid-binding proteins in that there is a finite number of distinct binding sites, but these binding sites display no strict structural requirements for the lipid molecule they bind and transport.