©1995 by The American Society for Biochemistry and Molecular Biology, Inc.
Microsomal Triglyceride Transfer Protein
SPECIFICITY OF LIPID BINDING AND TRANSPORT (*)

(Received for publication, September 9, 1994; and in revised form, January 4, 1995)

Haris Jamil (§) John K. Dickson Jr. Ching-Hsuen Chu Michael W. Lago J. Kent Rinehart Scott A. Biller Richard E. Gregg John R. Wetterau

From the Bristol-Myers Squibb Pharmaceutical Research Institute, Princeton, New Jersey 08543-4000

ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
FOOTNOTES
ACKNOWLEDGEMENTS
REFERENCES

ABSTRACT

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.


INTRODUCTION

Microsomal triglyceride transfer protein (MTP) (^1)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.


MATERIALS AND METHODS

1,2-Dioleoyl-L-3-phosphatidyl-L-[3-^14C]serine, 1,2-di[1-^14C]oleoyl-L-3-phosphatidylcholine, 1,2-dipalmitoyl-L-3-phosphatidyl[N-methyl-^3H] choline, 1,2-dioleoyl-L-3-phosphatidyl[2-^14C]ethanolamine, [4-^14C]cholesterol, cholesteryl [1-^14C]oleate, and L-3-phosphatidyl[U-^14C]inositol were obtained from Amersham Corp., and [^3H]squalene was obtained from DuPont NEN. All lipids and lipolytic enzymes were obtained from Sigma. Lipids were stored under N(2) 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).

Preparation of Labeled Substrates

Radiolabeled phosphatidic acid (PA), diglyceride (DG), and lysophosphatidylcholine (lyso-PC) were synthesized enzymatically from 1,2-di[1-^14C]oleoyl-L-3-phosphatidylcholine by the action of 25 units of phospholipase D (from Streptomyces chromofuscus, type VI), 2 units of phospholipase C (from Clostridium perfringens, type IX) (Wood and Snyder, 1969), or 2 units of phospholipase Afrom Naja mocambique mocambique), respectively. 1,2-Di(1-[^14C]oleoyl)PC (107 mCi/mmol) was dried under a stream of nitrogen gas and dissolved in 1 ml of diethyl ether, 4.3 ml of 17.5 mM Tris-HCl, pH 7.4, and 1 ml of 8 mM CaCl(2). All enzymatic reactions were at 30 °C for 3 h with vigorous shaking. The lipids were extracted according to the method of Bligh and Dyer(1959). The products were fractionated by thin-layer chromatography on silica gel plates using a petroleum ether/diethyl ether/acetic acid (140:40:8) solvent (solvent A) for DG, or chloroform/methanol/acetic acid/formic acid/water (140:60:24:8:4) solvent (solvent B) for PA or lyso-PC. Radioactivity was scanned on a Betascope 603 blot analyzer (Betagen Corp., Waltham, MA), and bands were identified using unlabeled standards run on the same plate. Radioactive lipid was extracted from the silica gel plate as described by Arvidson(1968).

To prepare monoglyceride (MG), ^14C-labeled PC was incubated with both phospholipase A(2) 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.

Preparation of Radiolabeled R-(Z,Z)-2-[[[2,3-Bis[1-oxo-9-octadecenyl1-oxy]propoxy]ethoxyphosphinyl]oxy]-N,N,N-trimethylethanaminium trifluoromethanesulfonate (ethyl-PC)

1,2-Di[1-^14C]oleoyl-L-3-phosphatidylcholine (100 µCi, 105 mCi/mmol, 0.75 mg, 0.95 µmol) was dissolved in anhydrous chloroform (50 µl). Powdered potassium carbonate (1.31 mg, 9.5 µmol) and ethyl trifluoromethanesulfonate (0.85 mg, 0.62 µl, 4.75 µmol) were added, and the reaction mixture was stirred at room temperature under argon. Progress of the reaction was followed by radioactive thin-layer chromatography using a chloroform/methanol/acetic acid/water (75:17.5:5:2.5) solvent system. Radioactivity was scanned on a Berthold Tracemaster 20 automatic TLC linear analyzer (Berthold, Wildbad, Germany). After 2 h, thin-layer chromatography showed there was still a small amount of starting PC remaining, so additional ethyl trifluoromethanesulfonate (0.85 mg, 4.75 µmol) was added. The reaction was complete after stirring for an additional 2 h. The reaction mixture was purified by silica gel column chromatography (J. T. Baker, 60-200 mesh, 0.6 times 30 mm). The column was eluted with 15 ml of 10% methanol in dichloromethane and 15 ml of 20% methanol in dichloromethane. The product fractions were combined and concentrated to give 46 µCi of ethyl [^14C]PC (46% radiochemical yield). The radiochemical purity of the product was 97.5% by thin-layer chromatography, and the identity was confirmed by fast atom bombardment-mass spectrometry (thioglycerol/glycerol (1:1)) m/z 818 (M).

Lipid Transport Assay

Lipid transfer from donor to acceptor membranes was measured in an assay similar to that previously described by Wetterau et al.(1992). Donor and acceptor SUV were prepared by bath sonication in 15 mM Tris-HCl, pH 7.5, 1 mM (assay buffer). Following sonication, SUV were isolated from larger vesicles by ultracentrifugation at 159,000 times g for 2 h in a Beckman TL-100 ultracentrifuge using a modification of the procedure described by Barenholz et al.(1977).

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](0)e (Wetterau and Zilversmit, 1985), where [S](0) 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.

Lipid Binding Analysis

Four different concentrations of MTP (between 65 and 320 pmol) were incubated with donor vesicles (40 nmol of egg PC, 7.5 mol % cardiolipin, 0.25 mol % [^14C]labeled substrate, and a trace of [^3H]dipalmitoyl PC) for 2 h at 37 °C in assay buffer. At the end of the incubation, the reaction mixture was layered on the top of 3.0 ml of 35% sucrose (prepared in assay buffer) and centrifuged at 412,000 times g for 24 h in a Beckman TL-100 ultracentrifuge. Donor membranes floated on the top of the sucrose solution, and MTP sedimented to the bottom of the tube. The bottom 0.5-ml fraction was collected utilizing a tube slicer, and the amount of lipid substrate bound to MTP was quantitated by measuring radioactivity and protein concentrations according to Bradford(1976). Counts in the bottom 0.5-ml fraction in the absence of MTP (background) were 5-15% of the total radioactivity found in the presence of MTP. In the presence of MTP, the counts ranged from 100 to 2000 for the different concentrations of MTP and various lipids investigated. Protein was not detectable in the top fraction.

To validate the lipid binding assay, MTP (320 pmol) was incubated with donor vesicles containing 0.25 mol % [^14C]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).


RESULTS

Pool Size of Substrates in Donor Vesicles

Donor membranes in the transfer reactions contained small quantities of radioactive substrate (0.25 mol %) in egg PC vesicles. To determine the size of the lipid pool available for transfer, the transfer of [^14C]TG or [^14C]PC (0.25 mol %) in egg PC vesicles was measured at various concentrations of MTP for 1 h at 37 °C (Fig. 1). Assuming that all the neutral lipid is accessible to MTP, the value calculated for the maximum transfer of TG is 86%, since donor vesicles were one-seventh of the total lipid in the assay. In the case of phospholipid substrates, if it is assumed that only the outer monolayer of the bilayer is accessible to MTP and that two-thirds of the phospholipid of an SUV is on the outside monolayer (Huang and Mason, 1978), the estimated value for maximum transfer would be 57% of the total. The values obtained for the maximum transfer of PC and TG were 68 and 95%, respectively, which are comparable with that of the calculated values. This indicates that all the TG and approximately two-thirds of the PC were available for MTP-mediated transfer.


Figure 1: Transport of triglyceride and phosphatidylcholine by MTP. The lipid transfer from donor vesicles containing 0.25 mol % [^14C]TG or [^14C]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.



MTP Has a Distinct Preference for Nonpolar Lipid Transfer

The ability of MTP to transfer different types of lipids was investigated to determine how changes in the lipid structure affect their transfer by MTP. Donor membranes contained a small amount of substrate (0.25 mol %) so the physical properties of the membranes were minimally affected by different lipid substrates. The initial rates of TG transfer were highest among all neutral and phospholipids examined, suggesting TG is the preferred substrate for MTP. The initial rates of transfer of other lipid substrates were measured and compared with the transfer rate of TG (Fig. 2). The transfer of CE was 66% as fast as TG transfer. The transfer activity of other neutral lipid substrates, DG, cholesterol, or squalene were 10.2 ± 2.0%, 8.5 ± 6.4%, or 6.9 ± 0.2% that of TG, respectively. The transfer activity of PC was lowest and had a rate only 4.1 ± 1.1% that of TG transfer. The relative rates of TG, CE, and PC were consistent with what has been reported previously for a different SUV based assay (Wetterau and Zilversmit, 1985).


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.



MTP Does Not Discriminate between Different Polar Head Groups

The effect of adding a polar head group to DG on MTP-mediated lipid transfer was examined (Table 2). As discussed above, the addition of a phosphocholine group to DG (resulting in PC) decreased the transfer rate 60%. This demonstrates that MTP discriminates between neutral lipids and phospholipids. Additional modifications to the head group resulting in zwitterionic PE, or negatively charged PS, PA, and PI, did not change the transfer rate significantly from that of PC (Table 2). These results suggest that there are no specific structural or charge-charge interactions between MTP and the polar head group of the transported phospholipid molecule.



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.

The Addition of Acyl Chains to a Lipid Molecule Increases Its Transfer Rate

The ability of MTP to transport lipid molecules containing a different number of hydrocarbon chains was examined. The initial rate of MTP-mediated transfer of TG, DG, and MG (Fig. 3, left panel), PC and lyso-PC (Fig. 3, middle panel), or CE and cholesterol (Fig. 3, right panel) were determined and then expressed as a percent of the transfer rate of the lipid with the highest number of fatty acyl chains in its class. The initial rate of transfer was decreased by 90, 89, or 87% with loss of one acyl chain from TG to DG (Fig. 3A), from PC to lyso-PC (Fig. 3B), or from CE to cholesterol (Fig. 3C), respectively. Lipid transfer by MTP was not detectable when an acyl chain was removed from DG to form MG (Table 1). These results indicate that the number of acyl chains in a lipid molecule plays an important role in the lipid transfer reaction and that an increase in the size of hydrophobic moiety of the lipid substrate by the addition of fatty acyl chains increases its transport by MTP.


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.



Binding of Lipid to MTP

It has been previously shown that an increase in TG concentration in donor membranes increases both TG binding to MTP and TG mass transfer, whereas the fractional TG transfer rate remains unchanged. This suggests that there is a relationship between the ability of MTP to bind and transport TG (Atzel and Wetterau, 1993b). In this study, the binding of different classes of lipid substrates to MTP was investigated using conditions similar to those used for measuring the initial rates of lipid transport. MTP at various concentrations (between 65 and 320 pmol) was incubated with donor membranes containing 0.25 mol % radiolabeled lipid substrate at 37 °C for 2 h. MTP was reisolated by ultracentrifugation, and the amount of radiolabeled lipid bound to MTP was quantitated as described under ``Materials and Methods.''

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 % [^14C]phospholipid substrate and a trace of [^3H]dipalmitoyl PC. [^3H]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.




DISCUSSION

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 % [^14C]phospholipid substrate and a trace of [^3H]dipalmitoyl PC. If one assumes [^3H]dipalmitoyl PC or [^14C]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.


FOOTNOTES

*
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§
To whom correspondence should be addressed: Dept. of Metabolic Diseases, Bristol-Myers Squibb Pharmaceutical Research Institute, P. O. Box 4000, Princeton, NJ 08543-4000. Tel.: 609-252-6288; Fax: 609-252-6964.

(^1)
The abbreviations used are: MTP, microsomal triglyceride transfer protein; CETP, cholesteryl ester transfer protein; TG, triglyceride; CE, cholesteryl ester; DG, diglyceride; MG, monoglyceride; PC, phosphatidylcholine; PA, phosphatidic acid; lyso-PC, lysophosphatidylcholine; PE, phosphatidylethanolamine; PS, phosphatidylserine; PI, phosphatidylinositol; ethyl-PC, R-(Z,Z)-2-[[[2,3-bis[1-oxo-9-octadecenyl-1-oxy] propoxy]ethoxyphosphinyl]oxy]-N,N,N-trimethylethanaminium trifluoromethanesulfonate; VLDL, very low density lipoproteins; ER, endoplasmic reticulum; SUV, small unilamellar vesicles; apoB, apolipoprotein B.


ACKNOWLEDGEMENTS

We thank Dr. Terry Stouch for helpful discussions.


REFERENCES

  1. Arvidson, G. A. E. (1968) Eur. J. Biochem. 4, 478-486 [Medline] [Order article via Infotrieve]
  2. Atzel, A., and Wetterau, J. R. (1993a) Biochemistry 32, 10444-10450 [Medline] [Order article via Infotrieve]
  3. Atzel, A., and Wetterau, J. R. (1993b) FASEB J. 7, A1268
  4. Barenholz, Y., Gibbes, D., Litman, B. J., Goll, J., Thompson, T. E., and Carlson, F. D. (1977) Biochemistry 16, 2806-2810 [Medline] [Order article via Infotrieve]
  5. Bligh, E. G., and Dyer, W. J. (1959) Can. J. Biochem. Physiol. 37, 911-917
  6. Bollen, I. C., and Higgins, J. A. (1980) Biochem. J. 189, 475-480 [Medline] [Order article via Infotrieve]
  7. Borén, J., White, A., Wettesten, M., Scott, J., Graham, L., and Olofsson, S.-O. (1991) Prog. Lipid Res. 30, 205-218 [Medline] [Order article via Infotrieve]
  8. Boström, K., Borén, Wettesten, M., Sjöberg, A., Bondjers, G., Wiklund, O., Carlsson, P., and Olofsson, S.-O. (1988) J. Biol. Chem. 263, 4434-4442 [Abstract/Free Full Text]
  9. Bradford, M. M. (1976) Anal. Biochem. 72, 248-254 [CrossRef][Medline] [Order article via Infotrieve]
  10. Brzozowski, A. M., Derewenda, U., Derewenda, Z. S., Dodson, G. G., Lawson, D. M., Turkenburg, J. P., Bjorkling, F., Huge-Jensen, B., Patkat, S. A., and Thim, L. (1991) Nature 351, 491-494 [CrossRef][Medline] [Order article via Infotrieve]
  11. Davis, R. A. (1993) Subcellular Biochemistry (Borgese, N., and Harris, J. R., eds) Vol. 21, pp. 169-187, Plenum Press, New York
  12. Deckelbaum, R. J., Ramakrishnan, R., Eisenberg, S., Olivecrona, T., and Bengtsson-Olivecrona, G. (1992) Biochemistry 31, 8544-8551 [Medline] [Order article via Infotrieve]
  13. Derewenda, Z. S., and Sharp, A. M. (1993) Trends Biochem. Sci. 18, 20-25 [CrossRef][Medline] [Order article via Infotrieve]
  14. Gibbons, G. F. (1990) Biochem. J. 268, 1-13 [Medline] [Order article via Infotrieve]
  15. Gregg, R. E., and Wetterau, J. R. (1994) Curr. Opin. Lipidol. 5, 81-86 [Medline] [Order article via Infotrieve]
  16. Hide, W. A., Chan, L., and Li, W.-H. (1992) J. Lipid Res. 33, 167-178 [Abstract]
  17. Higgins, J. A., and Hutson, J. L. (1984) J. Lipid Res. 25, 1295-1305 [Abstract]
  18. Huang, C., and Mason, J. T. (1978) Proc. Natl. Acad. Sci. U. S. A. 75, 308-310 [Abstract]
  19. Hutson, J. L., and Higgins, J. A. (1982) Biochim. Biophys. Acta 687, 247-256 [Medline] [Order article via Infotrieve]
  20. Janero, D. R., and Lane, M. D. (1983) J. Biol. Chem. 258, 14496-14504 [Abstract/Free Full Text]
  21. Laemmli, U. K., and Favre, M. (1973) J. Mol. Biol. 80, 575-599 [Medline] [Order article via Infotrieve]
  22. Rouser, G., Siakotom A. N., and Fleischer, S. (1966) Lipids 1, 85-86
  23. Sharp, D., Blinderman, L., Combs, K. A., Kienzle, B., Ricci, B., Wagner-Smith, K., Gil, C. M., Turck, C. W., Bouma, M.-E., Rader, D. J., Aggerbeck, L. P., Gregg, R. E., Gordon, D. A., and Wetterau, J. R. (1993) Nature 365, 65-69 [CrossRef][Medline] [Order article via Infotrieve]
  24. Shirai, K., Fitzharris, J. T., Shinomiya, M., Muntz, G. H., Harmony, J. A. K., Jackson, R. L., and Quinn, D. M. (1983) J. Lipid Res. 24, 721-730 [Abstract/Free Full Text]
  25. Shoulders, C. C., Brett, D. J., Bayliss, J. D., Narcisi, T. M. E., Jarmuz, A., Grantham, T. T., Leoni, P. R. D., Bhattacharya, S., Pease, R. J., Cullen, P. M., Levi, S., Byfield, P. G. H., Purkiss, P., and Scott, J. (1993) Hum. Mol. Genet. 12, 2109-2116
  26. Shoulders, C. C., Narcisi, T. M. E., Read, J., Chester, S. A., Brett, D. J., Scott, J., Anderson, T. A., Levitt, D. G., and Banaszak, L. J. (1994) Structural Biol. 1, 285-286
  27. Swenson, T. L., Brocia, R. W., and Tall, A. R. (1988) J. Biol. Chem. 263, 5150-5157 [Abstract/Free Full Text]
  28. Wetterau, J. R., and Zilversmit, D. B. (1985) Chem. Phys. Lipids 38, 205-222 [CrossRef][Medline] [Order article via Infotrieve]
  29. Wetterau, J. R., Combs, K. A., Spinner, S. N., and Joiner, B. J. (1990) J. Biol. Chem. 265, 9800-9807
  30. Wetterau, J. R., Combs, K. A., McLean, L. R., Spinner, S. N., and Aggerback, L. P. (1991a) Biochemistry 30, 9728-9735 [Medline] [Order article via Infotrieve]
  31. Wetterau, J. R., Aggerbeck, L. P., Laplaud, M., and McLean, L. R. (1991b) Biochemistry 30, 4406-4412 [Medline] [Order article via Infotrieve]
  32. Wetterau, J. R., Aggerback, L. P., Bouma, M.-E., Eisenberg, C., Munck, A., Hermier, M., Schmitz, J., Gay, G., Rader, D. J., and Gregg, R. E. (1992) Science 258, 999-1001 [Medline] [Order article via Infotrieve]
  33. Wirtz, K. W. A. (1991) Annu. Rev. Biochem. 60, 73-78 [CrossRef][Medline] [Order article via Infotrieve]
  34. Wood, R., and Snyder, F. (1969) Arch. Biochem. Biophys. 131, 478-494 [Medline] [Order article via Infotrieve]
  35. Vance, J. E., and Vance, D. E. (1985) Can. J. Biochem. Cell Biol. 63, 870-881 [Medline] [Order article via Infotrieve]
  36. Vance, D. E., and Vance, J. E. (1990) Annu. Rev. Nutr. 10, 337-356 [CrossRef][Medline] [Order article via Infotrieve]
  37. Verger, R. (1984) in Lipases (Borgstrom, B., and Brockman, H. L., eds) Elsevier Science Publishers B. V., Amsterdam
  38. Yao, Z., and McLeod, R. S. (1994) Biochim. Biophys. Acta 1212, 152-166 [Medline] [Order article via Infotrieve]
  39. Zachowski, A. (1993) Biochem. J. 294, 1-14 [Medline] [Order article via Infotrieve]

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