©1995 by The American Society for Biochemistry and Molecular Biology, Inc.
Characterization of Lipid Exchange Proteins Isolated from Small Intestinal Brush Border Membrane (*)

(Received for publication, April 8, 1994; and in revised form, December 15, 1994)

Gert Lipka Georg Schulthess (1) Herbert Thurnhofer (§) Hans Wacker Ernst Wehrli Karin Zeman Franz E. Weber Helmut Hauser (¶)

From the Laboratorium für Biochemie, Eidgenössische Technische Hochschule, ETH-Zentrum, CH-8092 Zürich, Switzerland Departement für Innere Medizin, Medizinische Poliklinik, Universitätsspital, CH-8091 Zürich, Switzerland

ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
conclusions
FOOTNOTES
ACKNOWLEDGEMENTS
REFERENCES

ABSTRACT

Subjecting rabbit small intestinal brush border membrane vesicles (BBMV) to freeze-thaw cycles releases water-soluble lipid exchange (transfer) proteins into the supernatant. They differ widely in apparent molecular weight and catalyze cholesterol, phosphatidylcholine, and phosphatidylinositol exchange between two populations of small unilamellar lipid vesicles. In order to determine their interrelations, the smallest water-soluble lipid exchange protein was purified to homogeneity by gel filtration on Sephadex G-75 and cation exchange chromatography on Mono S. It is a basic protein of apparent molecular mass of 13 ± 0.5 kDa. The purified protein was used to raise polyclonal antibodies. Polyclonal antibodies were also produced against a lipid exchange protein of apparent molecular mass of 100-120 kDa. By comparing lipid exchange, lipid binding, and immunological properties of the water-soluble lipid exchange proteins it can be shown that the 13-kDa (peak 3) protein is related to the 100-120 kDa (peak 1) protein; the properties of these two proteins are different from those of the peak 2 lipid exchange protein of apparent molecular mass of 22 kDa. Based on the immunological cross-reactivity observed between the 13 and 100-120 kDa and the lipid binding properties of these two proteins, a working hypothesis is proposed: both proteins are probably part of an intergral membrane protein of the brush border membrane that facilitates cholesterol and phosphatidylcholine absorption in this membrane. Evidence derived from immunogold labeling of BBMV supports the notion that this protein is located on the external (luminal) side of the brush border membrane. The analogous behavior of rabbit and human small intestinal brush border membrane in terms of lipid absorption and the release of water-soluble lipid exchange proteins is discussed.


INTRODUCTION

We reported that the absorption of cholesterol by brush border membrane vesicles (BBMV) (^1)made from rabbit small intestines is protein-mediated(1, 2) . It was shown to be a second-order reaction regardless of the nature of the donor particle (1, 2, 3) . The reaction mechanism involves collision-induced transfer of cholesterol between donor and acceptor. We also reported cholesterol absorption to be most efficient from mixed bile salt micelles the composition of which resembles the in vivo situation(2) . From these micelles cholesterol absorption was about 10^3-10^4 times faster than from small unilamellar phospholipid vesicles or mixed micelles consisting of lysophosphatidylcholine, egg phosphatidylcholine, and cholesterol (60:38:2, weight ratio)(2, 3) .

After subjecting BBMV to proteolytic treatment, cholesterol absorption was shown to be a passive process characterized by half-times of the order of hours even with bile salt micelles as donor particles(2) . It involves cholesterol desorption from the donor, diffusion of monomeric cholesterol through the aqueous phase, and incorporation of cholesterol into the bilayer of BBM(1) . The passive process is a true first-order reaction indicating that cholesterol desorption is the rate-limiting step(1) .

Proteolysis of BBM proteins can be mediated by adding extrinsic proteinases, but also by activating intrinsic proteinases(2) . Proteolysis by both extrinsic and intrinsic proteinases results in a significant reduction or loss of protein-mediated cholesterol absorption by BBM. At the same time it liberates water-soluble lipid exchange proteins(1, 2, 3, 4) . These proteins catalyze cholesterol (1) and phosphatidylcholine (PtdCho) exchange between two populations of small unilamellar phospholipid vesicles(2, 3, 4) . Furthermore, some of these proteins bind both cholesterol and PtdCho as shown by ESR spin labeling (1, 3) . Assuming that the water-soluble lipid exchange proteins resulting from the proteolytic treatment are part of the original integral membrane protein(s) responsible for cholesterol and PtdCho absorption, the purification and characterization of these proteins is desirable.

Here we describe the release of water-soluble lipid exchange proteins from BBMV by the action of intrinsic proteinases and the purification of one of these lipid exchange proteins. The purified protein was used to produce polyclonal antibodies in guinea pig which were used to study the immunological relationship between various lipid exchange proteins and to shed light on the question whether these proteins are related to the cholesterol receptor present in the BBM.


EXPERIMENTAL PROCEDURES

Materials

Egg PtdCho, egg phosphatidic acid (PtdOH), and egg lyso-PtdCho were purchased from Lipid Products (South Nutfield, Surrey, United Kingdom); cholesterol (purissimum) from E. Merck (Darmstadt, Germany); [1alpha,2alpha-^3H]cholesterol from Amersham International (Amersham, UK); L-alpha-[myo-inositol-2-^3H]phosphatidylinositol from NEN Products (Boston, MA); 3-doxyl-5alpha-cholestane from Aldrich (Steinheim, Germany); sodium taurocholate and deoxycholate from Sigma; phenylmethylsulfonyl fluoride from Fluka (Buchs, Switzerland); Sephadex G-75 SF from Pharmacia (Dübendorf, Switzerland); and CM Affi-Gel Blue and Bio-Gel P-10 from Bio-Rad (Glattbrugg, Switzerland).

1,2-Dipalmitoyl-sn-phosphatidyl[N-methyl-^3H(U)]choline and the spin-labeled PtdCho, 1-palmitoyl-2-(5-doxylpalmitoyl)-sn-phosphatidylcholine (5-doxyl-PtdCho), were synthesized as described in (3) and (5) , respectively. All lipids used in this work were pure by TLC standard, the two spin labels were also pure by carbon, hydrogen, and nitrogen microanalysis.

Methods

Preparation of BBMV

The small intestines of humans were removed, during autopsy, within 2 h of death of the human donor. The small intestines were cut into pieces of about 30 cm length, each piece was thoroughly rinsed with physiological saline, immediately frozen, and stored at -80 °C prior to the preparation of BBMV. The preparation of BBMV from rabbit and human small intestines was carried out according to (6) .

Preparation and Purification of Supernatant Proteins

Lipid exchange proteins were liberated from the BBM by the action of intrinsic proteinases as described previously(2, 3) . These proteins referred to as supernatant proteins were shown to catalyze the exchange of both cholesterol and PtdCho between two populations of SUV(1, 3) . When these lipid exchange proteins were fractionated by gel filtration on Sephadex G-75 SF(2, 4) , the elution pattern consisted of three well separated peaks (cf. Fig. 1A). Proteins present in peaks 1, 2, and 3 were pooled, concentrated, and exhaustively dialyzed against Hepes/Tris buffer, pH 7.3. The 13-kDa lipid exchange protein present in peak 3 was purified to homogeneity by cation exchange chromatography using Mono S HR 5/5 FPLC (from Pharmacia). For amino acid sequencing the purified protein was subjected to SDS-15% PAGE, transferred to a PVDF membrane, and sequenced on an Applied Biosystems 477A protein sequencer.


Figure 1: A, gel filtration pattern on Sephadex G-75 SF of supernatant proteins liberated from rabbit small intestinal BBMV as described previously(2, 3) . About 10 ml of a protein solution (11 mg/ml) in PBS were applied to the Sephadex G-75 SF column (36 times 4.6 cm; void volume V(0) = 170 ml, total volume V = 600 ml) and proteins were eluted with the same buffer at a flow rate of 32 ml h. Fractions of 6 ml were collected and analyzed for lipid exchange activity: PtdCho (-), cholesterol (+++), and phosphatidylinositol exchange (bulletbulletbullet). B, gel filtration pattern on Sephadex G-75 SF of supernatant proteins liberated by intrinsic proteinases from human small intestinal BBMV. About 1 ml of a protein solution (20 mg/ml) in PBS was applied to a Sephadex G-75 SF column (53.5 times 1.0 cm; V(0) = 16 ml, V = 44 ml) and proteins were eluted with the same buffer at a flow rate of 3.3 ml h. Fractions of 0.44 ml were collected and analyzed for protein(- - -) and PtdCho exchange (-). C, gel filtration on Bio-Gel P-10 of peak 3 proteins. Supernatant proteins released from rabbit small intestinal BBMV were first fractionated on Sephadex G-75 SF and peak 3 proteins were pooled and concentrated. About 50 µl of protein solution (6 mg/ml) in PBS were applied to the Bio-Gel P-10 column (18.5 times 0.9 cm; V(0) = 4.8 ml, V = 11.7 ml) and proteins were eluted with the same buffer at a flow rate of 2 ml h. Fractions of about 0.15 ml were collected and analyzed for protein(- - -) and PtdCho exchange (-). In all three panels lipid exchange is expressed as % of the total amount of lipid present in the donor prior to the start of the exchange reaction.



Preparation of Antisera, IgG, and Affinity-purified Antisera

The 13-kDa lipid exchange protein purified either by affinity high pressure liquid chromatography as described previously (2) or by cation exchange chromatography on Mono S HR 5/5 described here was used to raise polyclonal antibodies in guinea pig. Guinea pig preimmune sera and antisera were filtered through sterile filters (0.22 µm, Millex-GS, Millipore, Molsheim, France) and after addition of 0.1 mM phenylmethylsulfonyl fluoride and 0.04% NaN(3) they were stored at 4 °C prior to use. The IgG fraction of the antisera was prepared by chromatography on CM Affi-Gel Blue followed by ammonium sulfate precipitation(7) . The precipitated IgG was dissolved in 0.01 M potassium phosphate buffer, pH 7.2, containing 0.15 M NaCl, 0.1 mM phenylmethylsulfonyl fluoride, and 0.04% NaN(3) to protein concentrations of 5-10 mg/ml, stored at 4 °C and used within 7 days. Affinity purification of antisera was carried out on PVDF membranes (Immobilon-P from Millipore, Volketswil, Switzerland) according to (37) . Briefly, peak 3 proteins (0.2 mg) obtained by gel filtration on Sephadex G-75 were separated by SDS-15% PAGE. The resolved proteins were transferred electrophoretically to PVDF membranes and visualized by Ponceau S staining. After destaining, the 13-kDa protein band was cut out and incubated with guinea pig antisera. The antibody bound to the 13-kDa protein was eluted from PVDF with 0.1 M glycine/HCl, pH 2.5 (5 min at 23 °C), and the solution neutralized to pH 8.

Polyclonal antibodies against peak 1 lipid exchange protein were raised in guinea pig. Good use was made of the observation (see below) that this protein binds spin-labeled cholestane (Fig. 6G). Upon incubation of peak 1 proteins with mixed taurocholate micelles (sodium taurocholate/oleic acid/monooleoylglycerol/3-doxyl-5alpha-cholestane, 88:6:3.5:2.5, weight %; total lipid/spin label mole ratio, 30:1) in PBS (0.14 M NaCl, 0.01 M sodium phosphate, pH 7.3, containing 2.5 mM EDTA and 0.02% NaN(3)), at room temperature overnight, tiny needles crystallized out consisting of spin label and protein. The crystalline pellet was washed twice by centrifugation at 14,000 times g for 10 min and resuspension in Hepes/Tris buffer, pH 7.3, followed by repeated washing cycles using 2% deoxycholate and finally 10% SDS. Proteins extracted with these detergents were separated by SDS-10% PAGE, protein bands at about 100-120, 90, and 60 kDa were cut out and the proteins were electroeluted from the gel following the Bio-Rad instruction manual. The proteins thus obtained were injected into guinea pigs to produce polyclonal antibodies. Only polyclonal antibodies raised against the 100-120-kDa protein were active and inhibited cholesterol and PtdCho exchange between two populations of SUV induced in the presence of peak 1 proteins.


Figure 6: ESR spectra of 5-doxyl-PtdCho and 3-doxyl-5alpha-cholestane present in different environments were recorded at room temperature on a Varian X-band spectrometer (model E-104A). A, 5-doxyl-PtdCho suspended in PBS at 1 mg of lipid/ml. B, a film of 5-doxyl-PtdCho deposited on the glass wall of a round-bottom flask was exposed to a solution of peak 1 proteins (38 mg/ml) in PBS at 4 °C overnight. Multilamellar liposomes were spun down by centrifugation at 12,000 times g for 10 min and the ESR spectrum of the supernatant was recorded. C, egg PtdCho (4 mg/ml = 5.33 mM) containing 3-doxyl-5alpha-cholestane at a lipid/spin label mole ratio of 127 was dispersed in PBS. D, egg lyso-PtdCho (20 mg/ml = 0.040 M) containing 5-doxyl-PtdCho at a mole ratio of 200:1 dispersed in PBS. E, sodium deoxycholate micelles containing spin-labeled PtdCho (mole ratio 200) dispersed in PBS at 20 mg/ml (=0.048 M). F, sodium deoxycholate micelles spin-labeled with 3-doxyl-5alpha-cholestane (mole ratio 150) and dispersed in PBS at 20 mg/ml (=0.048 M). G, sodium deoxycholate micelles spin-labeled with 3-doxyl-5alpha-cholestane (mole ratio = 85) and dispersed in PBS were mixed with peak 1 protein(s) to final concentrations of bile salt (4.6 mg/ml = 0.011 M) and peak 1 protein of 30 mg/ml, and the ESR spectrum was immediately recorded.



Miscellaneous

Published methods were used for the preparation of lipid SUV and micelles(1, 2, 3) , the measurement of lipid exchange between two different populations of SUV(2, 3) , the measurement of cholesterol and PtdCho absorption by BBMV(1, 2, 3) , the proteolytic treatment of BBMV(1, 3) , immunogold staining of BBMV(8, 9) , SDS-PAGE(2) , and isoelectric focusing(10) . Phospholipid concentrations were determined according to (12) and protein concentrations by the BCA assay(13) . ESR spectra were recorded on a Varian X-band spectrometer at 9.2 GHz as described previously(5) .


RESULTS

Water-soluble Lipid Exchange Proteins Liberated from BBMV

Subjecting BBMV to freeze-thaw cycles and storage of BBMV at temperatures >0 °C was shown to release lipid exchange proteins into the supernatant (supernatant proteins, (2) and (3) ). The gel filtration pattern of supernatant proteins obtained from rabbit BBMV on a large, preparative Sephadex G-75 column consisted of three well separated peaks (Fig. 1A) exhibiting PtdCho and phosphatidylinositol exchange activity. Cholesterol exchange activity was present in peaks 1 and 3, but not in peak 2 (Fig. 1A). The elution volumes of the three peaks were converted to apparent molecular masses by means of a calibration curve (2, 4) and the values thus obtained for peak 1, 2, and 3 proteins were molecular mass >70, 22 ± 2, and 12 ± 1 kDa, respectively. The gel filtration pattern of supernatant proteins obtained from human BBMV on a smaller, analytical Sephadex G-75 column consisted also of three peaks although less well resolved (Fig. 1B). The elution volumes of the three peaks yielded M(r) values in close agreement with those of the supernatant proteins derived from rabbit small intestines. However, the two gel filtration patterns in Fig. 1, A and B, differ markedly in peak intensities. As noted before for supernatant proteins isolated from rabbit small intestinal BBMV(2) , the peak intensities can vary to the extent shown in Fig. 1, A and B.

Proteins present in peak 3 of Fig. 1A were pooled, concentrated, and applied to a Bio-Gel P-10 column in order to test the resolution of these proteins obtained on Sephadex G-75. The elution pattern of peak 3 proteins on Bio-Gel P-10 consisted of a main, fairly symmetric protein peak and two minor ones at larger elution volumes (Fig. 1C). The PtdCho exchange activity was associated with the main protein peak, the shift observed in the peak position reflects the inhomogeneity of the proteins present in the main peak (cf. PAGE pattern in Fig. 2). The peak position of the PtdCho exchange activity was very close to that of ribonuclease A corresponding to an apparent molecular mass of 13.7 kDa.


Figure 2: Cation exchange chromatography (FPLC) of peak 3 proteins on Mono S HR 5/5. Proteins of peak 3 (Fig. 1A) were pooled, exhaustively dialyzed against 0.01 Hepes/Tris buffer, pH 7.3, containing 0.3 MD-mannitol, 5 mM EDTA, and 0.02% NaN(3) and concentrated to 2 mg of protein/ml. The resulting protein solution was centrifuged at 100,000 times g for 20 min, filtered through a sterile Millipore filter, and 2.3 mg of protein were applied to the Mono S column. The column (5 times 0.5 cm) was run with buffer A (5 mM sodium phosphate, pH 7.0, 0.3 MD-mannitol) and after the pass-through peak, proteins bound to the column were eluted with a Na gradient at 0.5 ml/min using buffer A plus 1 M NaCl. All buffers were degassed and filtered through sterile Millipore filters prior to use. Protein (circle); PtdCho exchange (bullet); NaCl concentration (- - -). Panel on the right: SDS-15% PAGE patterns of the proteins present in peak 3 prior to cation exchange chromatography (lane A) and protein eluted from the Mono S column at V = 27 ml (lane B). SDS-PAGE was carried out using the Mini-Protean II dual slab cell from Bio-Rad. Proteins were visualized by silver staining. Bio-Rad low range protein standards were used and the position of apparent molecular masses in kDa of these standards are marked on the right.



Purification of the Smallest Water-soluble Lipid Exchange Protein Liberated from Rabbit Small Intestinal BBMV

The smallest water-soluble lipid exchange protein present in peak 3 (Fig. 1A) was further purified by affinity chromatography as described previously(2) . The purified lipid exchange protein gave a single band on SDS-15% PAGE (2) corresponding to an apparent molecular mass of 13 kDa and two bands on isoelectric focusing with isoelectric points of 9.1 and 9.4 (data not shown). This result indicated that we are dealing with a basic lipid exchange protein and prompted an alternative method of purification. Peak 3 proteins were subjected to cation exchange chromatography on Mono S HR 5/5 and a representative elution pattern which was very well reproducible is shown in Fig. 2. About 70% of the applied protein was eluted in the pass-through peak with practically no lipid exchange activity. About 70% of the lipid exchange activity (25% of the applied protein) was eluted with a NaCl gradient at [NaCl] > 0.18 M. The protein eluted at V(e) = 27 ml ([NaCl] 0.25 M) gave a single band on SDS-15% PAGE using silver staining (shown on the right of Fig. 2) and was used to raise polyclonal antibodies in guinea pig. The specific activity of the purified protein was increased by a factor of 140 referring to PtdCho exchange activity and by a factor of 100 referring to cholesterol exchange activity. The average apparent molecular mass of this protein determined by gel filtration on Bio-Gel P-10 and SDS-PAGE was 13.0 ± 0.5 kDa. The sequence of the first 28 NH(2)-terminal amino acids of this protein was NH(2)-S-S-A-G-D-G-F-K-A-N-L-V-F-K-E-I-E-K-K-L-E-E-E-G-E-Q-F-V- . . . which is highly homologous (86-96%) to liver sterol sterol carrier protein 2 from different sources(14, 15, 16, 17) .

Immunological Experiments

The following series of experiments were carried out with the IgG fraction of guinea pig antisera or affinity-purified guinea pig antisera raised against the 13-kDa protein. Neither the purified 13-kDa protein nor total proteins of peak 3 eluted from the Sephadex G-75 (Fig. 1A) gave a positive precipitin test or precipitation lines with the IgG fraction of the guinea pig antiserum in the gel double diffusion test according to Ouchterlony (18) indicating that the antigen-antibody complex remains soluble.

Immunoblot Analysis of BBMV and Supernatant Proteins

Immunoblot analyses were performed with rabbit small intestinal BBMV, total supernatant proteins prepared from these vesicles, and various fractions of supernatant proteins (peak 1, 2, and 3 proteins) separated by gel filtration on Sephadex G-75 (Fig. 3, A and B). The 13-kDa protein was purified by either affinity chromatography (2) or cation exchange chromatography described here. Polyclonal antibodies raised against the proteins purified by either method gave identical results. The IgG fraction of the guinea pig antisera not only detected the antigen present in total supernatant and peak 3 proteins, but also reacted strongly with a protein of similar size in BBMV. In addition to this protein, the antibody cross-reacted with BBM proteins of higher apparent molecular mass: clearly detectable bands were at 35 (cf. Fig. 3A), 95, and 120 kDa (cf. Fig. 3B). The band pattern of supernatant proteins resembled that of BBMV (Fig. 3A). In addition to the strong 14-kDa band total supernatant proteins gave bands at 35 kDa, a doublet at 100 kDa, and a weak band at 60 kDa. Peak 1 proteins exhibited major bands at 95 and 120 kDa and weak bands at 60 and 130 kDa (cf. Fig. 3, A and B). Proteins of peak 2 gave two bands, a strong one at 35 kDa and a minor one at 38 kDa. As expected, after fractionation of the supernatant proteins on Sephadex G-75, no bands in the 13-kDa range were detected with peak 1 and peak 2 proteins. Inspection of Fig. 3A shows that the immunoblot of supernatant proteins is reasonably well represented by the sum of the band patterns of peaks 1, 2, and 3. The affinity purified antibody gave essentially the same result as shown in Fig. 3in which the IgG fraction of guinea pig antisera was used.


Figure 3: Immunoblotting of BBMV, supernatant proteins, and proteins present in peaks 1, 2, and 3 using IgG of guinea pig antisera raised against the 13-kDa protein. Supernatant proteins from rabbit small intestinal BBMV were prepared as described in Refs. 2 and 3. BBMV (30 µg of protein), total supernatant proteins (30 µg), proteins of peak 1 (30 µg), peak 2 (30 µg), and peak 3 (7 µg) were electrophoresed through SDS-15% PAGE (A) and SDS-10% PAGE (B) and transferred in a Bio-Rad blotting apparatus to PVDF (Immobilon-P from Millipore) using 0.025 M Tris buffer, pH 8.3, containing 0.19 M glycine, 0.01% SDS, and 15% CH(3)OH as the blotting buffer and 30 V overnight at room temperature or 300 V for 2 h at 4 °C. Immunostaining of the transferred protein bands was carried out following the protocol of van Heusden et al.(11) . Su, total supernatant proteins; pk1, pk2, and pk3 refer to peak 1, 2, and 3 proteins, respectively. The numbers on either side of the immunoblots represent the apparent molecular masses in kDa of the marker proteins used.



The Effect of Polyclonal Antibodies against the Purified 13-kDa Protein and the 120-kDa Protein of Peak 1 on PtdCho and Cholesterol Exchange between Two Populations of SUV

The effect of the IgG fraction of guinea pig antisera against the 13-kDa protein on PtdCho exchange between two populations of SUV is shown in Fig. 4. Again antibodies raised against the 13-kDa protein purified by the two different procedures behaved similarly. In this series of experiments PtdCho exchange was mediated by lipid exchange proteins of peaks 1, 2, and 3 and the results are shown in Fig. 4, A-C, respectively. Increasing concentrations of IgG did progressively inhibit PtdCho exchange between two populations of SUV mediated by protein(s) present in peak 1. IgG at 4 mg of protein/ml reduced the PtdCho exchange to 30% (Fig. 4A). In contrast, IgG up to 4 mg of protein/ml had no effect on PtdCho exchange between two populations of SUV mediated by peak 2 protein(s) (Fig. 4B). Increasing concentrations of IgG progressively inhibited the PtdCho exchange mediated by peak 3 protein(s) consistent with a previous report(32) . In this case the PtdCho exchange between two populations of SUV was totally blocked at an IgG concentration of 6 mg of protein/ml (Fig. 4C). In all three cases discussed (Fig. 4, A-C) the IgG fraction of guinea pig preimmune serum had either no or even a stimulating effect of 10-20% compared to the PtdCho exchange measured in the absence of IgG (cf. Fig. 4B). Included in this figure is the effect of the IgG fraction of guinea pig antisera raised against the 100-120-kDa protein of peak 1. This antibody effectively inhibited the exchange of [^3H]cholesterol between two populations of SUV mediated by peak 1 protein (open circles, Fig. 4A). At IgG concentrations of 1 mg of protein/ml the cholesterol exchange was reduced to 30%. This antibody also inhibited the [^3H]cholesterol exchange between two populations of SUV mediated by peak 3 protein (open symbols, Fig. 4C).


Figure 4: Effect of polyclonal antibodies on PtdCho and cholesterol exchange between two populations of SUV mediated by different soluble lipid exchange proteins that were liberated from rabbit small intestinal BBMV(2, 3) . Donor vesicles consisting of egg PtdCho, egg PtdOH, (85:15, mole ratio), and a trace amount of [^3H]dipalmitoyl-PtdCho or [^3H]cholesterol and acceptor vesicles of pure egg PtdCho, both dispersed in Hepes/Tris buffer, pH 7.3, were mixed to give final lipid concentrations of 0.1 and 1 mg/ml, respectively. PtdCho exchange was mediated by peak 1 proteins (27 µg of protein/ml) in A, by peak 2 proteins (37 µg of protein/ml) in B, and peak 3 proteins (32 µg of protein/ml) in C. Closed symbols refer to PtdCho exchange in the presence of IgG of antisera raised against the purified 13-kDa protein. Open symbols in panels A and C refer to cholesterol exchange in the presence of IgG of antisera raised against the 120-kDa protein of peak 1. Different closed symbols refer to different experimental series.



The Effect of Polyclonal Antibodies against the 13-kDa Protein on PtdCho Uptake by BBMV

The radioactivity present in SUV of egg PtdCho decreased exponentially when these SUV were incubated with rabbit small intestinal BBMV approaching the equilibrium distribution x after about 3-4 h. The exponential decay (solid symbols, Fig. 5A) was linearized following standard procedures (19, 20, 21) and the pseudo first-order rate constant derived from the straight line (solid symbols, Fig. 5B) according to was k(1) = 0.97 h.


Figure 5: A, time course of PtdCho absorption by rabbit small intestinal BBMV. SUV of egg PtdCho labeled with [^3H]dipalmitoyl-PtdCho as donor vesicles and BBMV as the acceptor, both dispersed in Hepes/Tris buffer, pH 7.3, were mixed to final concentrations of 0.71 mg of lipid/ml and 8.4 mg of protein/ml (4 mg of total lipid/ml), respectively. The time courses of the PtdCho absorption by BBMV in the absence and presence of IgG (4 mg of protein/ml) of guinea pig anti-13 kDa antisera (closed symbols) are compared. The equilibrium concentration x of the donor was calculated from the lipid pools of donor (b) and acceptor (a): x = 100 times b/(a + b) = 22%. The lipid concentration [a] of the acceptor was experimentally determined to be [a] = [total lipid] times 0.6, where [total lipid] is the total lipid concentration of BBMV(21) . The lipid concentration [b] of the donor is equal to the analytical concentration of egg PtdCho. B, the kinetic data of panel A were linearized according to ln[(x- x)/(x(o) - x)] = -k(1)[(a + b)/a]t, where x = 100% at t = 0 and x and x are the radioactivities (in %) in the donor at time t and at equilibrium (t ), respectively. Closed symbols, in the absence of antibody; open circles, in the presence of IgG at 4 mg of protein/ml. Different closed symbols represent different experiments. The solid lines are least-squares fits to the experimental data points (r^2 = 0.997).



Where x(o), x, and x are the fractions or percentages of exchangeable lipid in the donor at time 0, t, and at equilibrium, respectively, a and b are the effective lipid pools of acceptor and donor, respectively.

The effect of the IgG fraction of the guinea pig antisera raised against the 13-kDa protein on PtdCho uptake by rabbit small intestinal BBMV is included in Fig. 5. IgG at 4 mg of protein/ml produced a 30% inhibition of the PtdCho uptake by BBMV (Fig. 5A) while preimmune serum had a slightly stimulating effect on PtdCho uptake (data not shown). The inhibitory effect was linear with IgG concentrations (data not shown) and more importantly, it exhibited a time lag. The onset of inhibition occurred reproducibly after a 30-min reaction of BBMV with IgG (cf. open circles,Fig. 5A). Linearization of the kinetic data obtained in the presence of IgG yielded two straight lines (open symbols of Fig. 5B). Up to 30 min the experimental points lay on the straight line fitted to the solid symbols representing PtdCho uptake in the absence of IgG. The slope of the straight line fitted to the data points after 30 min (Fig. 5B) yielded a pseudo first-order rate constant k(1) = 0.27 h indicating that the rate of PtdCho uptake was reduced by a factor of about 4. Preincubation of BBMV with IgG for 2.5 h produced slightly but significantly smaller values than k(1) = 0.27 h. Due to the relatively large scatter of the initial data points it is uncertain whether or not preincubation eliminated the time lag.

Lipid Binding Properties of Supernatant Proteins: Evidence from ESR Spectroscopy

We reported before that supernatant proteins produced from rabbit small intestinal BBMV bind both spin-labeled cholestane (1) and PtdCho (3) . Bilayers of 5-doxyl-PtdCho deposited on the glass wall of the ESR capillary or suspended in PBS gave a typical Heissenberg spin-exchange spectrum (Fig. 6A). In contrast, if the dry film of spin-labeled PtdCho was incubated with a solution of peak 1 proteins (38 mg/ml) in PBS at 4 °C overnight and excess multilamellar liposomes were removed by centrifugation at 12,000 times g for 10 min, the supernatant gave the ESR spectrum shown in Fig. 6B. The maximum hyperfine splitting of this spectrum of 2 A = 60 ± 1 G (Table 1) indicated that the spin label becomes highly immobilized in the presence of peak 1 proteins. The 2 A value clearly exceeded the hyperfine splitting values observed when 5-doxyl-PtdCho was present in either egg PtdCho bilayers (2 A = 53 G, (22) ) or egg lyso-PtdCho micelles (2 A = 52 G, Fig. 6D and Table 1). In both these situations the spin probe is known to undergo rapid but highly anisotropic motion. Based on ESR experiments summarized in Fig. 6and Table 1, the immobilized component of the spectrum in Fig. 6B was interpreted to arise from binding of spin-labeled PtdCho to peak 1 protein(s). Addition of egg lyso-PtdCho (34 mM) or deoxycholate micelles (16 mM) to the spin label-protein complex (Fig. 6B) had no effect on the line shape of the ESR spectrum. This result indicated that the spin label remained bound to protein and did not distribute back into the lipid micelles.



When peak 1 proteins (23 mg/ml) were incubated with sodium deoxycholate micelles containing 5-doxyl-PtdCho, the ESR spectrum changed instantaneously from one characteristic of the spin label in the bile salt micelle with 2 A = 36 G (Fig. 6E) to the immobilized spectrum of Fig. 6B with 2 A = 60 G. Similarly, when peak 1 proteins (30 mg/ml) were incubated with sodium deoxycholate micelles containing 3-doxyl-5alpha-cholestane, there was also an instantaneous change from the three-line spectrum characteristic of the cholestane spin label in deoxycholate micelles with 2 A = 38 ± 1 G (Fig. 6F) to a near rigid-limit powder spectrum with 2 A = 66 ± 1 G (Fig. 6G, Table 1). This 2 A value indicated very tight binding of the cholestane spin label to peak 1 proteins. The kinetics of the binding of the two spin labels from deoxycholate micelles was characterized by half-times of the order of seconds. Inspection of the spectra in Fig. 6, F and G, shows that the spectrum in Fig. 6G is a composite one: in addition to the near rigid-limit powder spectrum there is a second minor component of a three-line spectrum arising from some cholestane spin label molecules still being present in bile salt micelles. By progressively increasing the concentration of peak 1 proteins the intensity of the near rigid-limit powder spectrum (Fig. 6G) increased at the expense of the three-line spectrum (Fig. 6F) indicating that the equilibrium is pushed toward the lipid-protein complex (data not shown).

The 13-kDa protein behaved qualitatively similar to peak 1 proteins. Incubation of sodium cholate micelles containing 3-doxyl-5alpha-cholestane with the 13-kDa protein (1.7 mg/ml) in PBS yielded a composite ESR spectrum similar to that shown in Fig. 6G with a maximum hyperfine splitting of 2 A = 63 ± 2 G (Table 1). However, in this case the intensity of the three-line spectral component arising from the spin label in deoxycholate micelles clearly exceeded that of the near rigid-limit powder spectrum (data not shown). The behavior of peak 1 and the 13-kDa protein is contrasted by that of peak 2 proteins. When peak 2 proteins (up to 20 mg/ml) were incubated with sodium cholate micelles containing 3-doxyl-5alpha-cholestane, no binding of spin-labeled cholestane to peak 2 protein(s) was detected.

Electron Microscopy of Immunogold-labeled Rabbit Small Intestinal BBMV

BBMV incubated with the IgG fraction of guinea pig antisera against the 13-kDa protein for 90 min interacted with protein A/10-nm gold as shown in Fig. 7a. No immunogold labeling was observed when BBMV were incubated with guinea pig preimmune sera (Fig. 7b) or when BBMV were treated sequentially with IgG, gold-free protein A, and protein A/10 nm gold (data not shown). Furthermore, no immunogold labeling was detected when BBMV were incubated with IgG for less than 30 min. This finding is consistent with the data in Fig. 4where the inhibition by the antibody was observed only after incubation of the BBMV for more than 30 min.


Figure 7: Electron micrographs of immunogold-stained BBMV. In a, BBMV from rabbit small intestines were subjected to immunogold staining using the IgG fraction of the guinea pig antisera against the rabbit 13-kDa protein. In b the BBMV were treated with guinea pig preimmune serum as a control. BBMV suspended in Hepes/Tris buffer, pH 7.3, were treated as described in (8) and (9) . The IgG fraction of antisera against the 13-kDa protein was diluted to 1 mg of protein/ml with blocking buffer (PBS containing 50 mM glycine and 0.2% gelatine)(9) . The colloidal protein A-10 nm gold suspension obtained from Aurion (Wageningen, The Netherlands) was diluted 1:20 with the same buffer. BBMV were consecutively incubated with dilute IgG for 90 min and protein A-10 nm gold for 60 min, then washed twice each with blocking buffer, PBS, and distilled water and negatively stained with 1% uranyl acetate. The bar represents 0.1 µm.




DISCUSSION

Fig. 1illustrates that various lipid exchange proteins are liberated from BBMV in the absence of externally added proteinases. Control experiments show that one or several steps in the preparation of BBMV, but also subjecting BBMV to freeze-thaw cycles can be responsible for the observed release of membrane proteins. In light of these findings two questions arise, 1) are some or all of these water-soluble lipid exchange proteins interrelated and/or related to the integral membrane protein responsible for cholesterol and/or PtdCho absorption in BBM(^2); 2) in case they are not related to this protein what are their origin and physiological role? The first question is pertinent to the isolation and purification of the cholesterol receptor of the BBM. The major problem of isolating integral proteins from BBMV is that detergent solubilization of BBM is accompanied by the activation of intrinsic proteinases resulting in massive degradation of membrane proteins and total loss of enzymatic activities(24) . It was shown that the activity of the intrinsic proteinases is difficult to control with proteinase inhibitors and various mixtures thereof(24) . If the water-soluble lipid exchange proteins described here were indeed related to the cholesterol receptor of the BBM, the purification of these proteins would be desirable. The usefulness of antibodies raised against these proteins is obvious: these antibodies would be employed in immunoaffinity chromatography for the isolation and purification of the cholesterol receptor of the BBM.

In an attempt to shed light on the first question, the 13-kDa protein was purified to homogeneity and polyclonal antibodies were raised against this protein. It is the smallest water-soluble lipid exchange protein least contaminated as judged by SDS-PAGE(2) . The purification by cation exchange chromatography on Mono S HR 5/5 described here is easier, faster, and significantly cheaper than affinity high pressure liquid chromatography on Nucleosil-phosphatidylcholine described in a previous publication (2) and therefore better suitable as a routine method for purifying this protein.

The lipid exchange protein present in peak 1, which has an apparent molecular mass of 100 kDa(2) , and the 13-kDa protein both catalyze the exchange of PtdCho, phosphatidylinositol, and cholesterol (Fig. 1A) exhibiting features of nonspecific lipid transfer proteins(25) . In contrast, the lipid exchange protein present in peak 2 with an apparent molecular mass of 22 kDa mediates PtdCho and phosphatidylinositol exchange but not cholesterol exchange. The 13-kDa protein is not only functionally related to the 100-kDa lipid exchange protein, but also immunologically: the polyclonal antibody against the 13-kDa protein cross-reacts with the 100-kDa protein, but not with the 22-kDa protein of peak 2 and the polyclonal antibody against the 100-120-kDa protein of peak 1 cross-reacts with the 13-kDa protein (cf. Fig. 4, A-C). The peak 2 protein appears to be functionally and immunologically different from the 13- and 100-kDa lipid exchange proteins.

The relations between the three water-soluble lipid exchange proteins are further corroborated by their lipid binding properties as studied by ESR spin labeling. The 100- and 13-kDa protein bind both spin-labeled cholestane and PtdCho indicating that their lipid binding site have properties reminiscent of nonspecific lipid transfer proteins (25) . Formation of a complex between these proteins and either 3-doxyl-5alpha-cholestane or 5-doxyl-PtdCho is indicated by a strong immobilization of the spin label as evident from the large hyperfine splittings observed (cf. Fig. 6and Table 1). The splittings are significantly larger than those observed when the spin labels are present in lipid bilayers or micelles (Table 1). This behavior is contrasted by that of the 22-kDa lipid exchange protein which under comparable experimental conditions shows no binding of 3-doxyl-5alpha-cholestane. From the comparison of the functional, immunological, and lipid binding properties of the three water-soluble lipid exchange proteins we conclude that the 13-kDa protein is related to the 100-kDa protein and the properties of these two proteins are different from those of the 22-kDa lipid exchange protein.

The origin of the strong 13-kDa band in the immunoblot analysis of BBMV (Fig. 3A) is unclear although a similar observation was reported previously(26) . The 13-kDa protein of the BBM could be either a membrane-bound (membrane-associated) isoform of the 13-kDa protein or it could originate from a larger integral membrane protein by proteolytic cleavage. A third possibility would be that it is identical to cytosolic sterol carrier protein 2 which is absorbed to the BBM and subsequently slowly released. The close resemblance in terms of physicochemical properties between the 13-kDa protein and sterol carrier protein 2, particularly the high homology of the NH(2)-terminal amino acid sequence of these two proteins support this possibility.

The results in Fig. 3would be explicable if the 13-kDa antigen were contaminated with proteins of higher molecular mass giving rise to a heterogeneous immune response. However, SDS-15% PAGE of the purified antigen clearly shows (Fig. 2) that this is not the case although, sometimes, due to the tendency of this protein to aggregate, dimers were detected. Hence the immunoblot results in Fig. 3indicate that the antibody against the 13-kDa protein cross-reacts with BBM proteins of apparent molecular mass greater than 13 kDa. Epitopes present on the 13-kDa protein are presumably also present on brush border membrane proteins of higher molecular mass. This notion is corroborated by using affinity-purified antibodies in the immunoblot analysis in Fig. 3. The results of Fig. 3may be rationalized in terms of membrane-bound isoforms of the 13-kDa protein. In this context results reported recently by Baum et al.(27) are relevant. These authors showed also by immunoblot analysis that polyclonal antibodies raised against rat liver sterol carrier protein 2 detect several high-molecular weight isoforms of this protein in tissue homogenates of rat liver and rat small intestines. Cross-reactions were observed with hepatic proteins of 14, 25, 27, 30, 45, 58, and 120 kDa and with small intestinal proteins of 14 and 30 kDa and a weak one with a 58-kDa protein. Several papers have appeared over the last few years providing convincing evidence for the presence of high-molecular weight isoforms of nonspecific lipid transfer proteins in different cell and cell organelles (see for instance, (11) and (28) -31). Alternatively, the immunoblot analysis of Fig. 3could be rationalized by postulating that the proteins that cross-react with the polyclonal antibody against the 13-kDa protein are derived proteolytically from one large integral BBM protein by the action of intrinsic proteinases. This notion is supported by the observation of similar band patterns in the immunoblot analysis of supernatant proteins and BBMV (cf. Fig. 3). In this case the water-soluble lipid exchange proteins including the 13-kDa protein could be part of a large integral BBM protein from which they are cleaved off proteolytically. We know that intrinsic proteinases are activated during the preparation of BBMV and it is likely that these proteinases are also activated by dissolving BBMV in SDS for PAGE.

The partial inhibition shown in Fig. 5is consistent with a previous report showing that the same antibody partially inhibits cholesterol absorption from mixed taurocholate micelles(32) . Unless the observed inhibition is an unspecific effect, it suggests that the 13-kDa protein is related to the integral membrane protein responsible for cholesterol/PtdCho absorption, i.e. to the cholesterol receptor of the BBM. The time lag observed may be due to the antigenic site being initially inaccessible to the antibody. The integral membrane protein may have to undergo a conformational change prior to the binding of the antibody. Immunogold staining (Fig. 7) lends support to this interpretation. BBMV as used here are oriented right side out (33, 38) and are impermeable to proteins of the size of trypsin(24, 34) . For positive immunogold labeling to be observed as shown in Fig. 7a, BBMV have to be incubated with the antibody for more than 30 min. The immunogold labeling clearly shows that the antibody interacts with a protein exposed on the external or luminal side of the BBM. In this context the results of the papain treatment of rabbit small intestinal BBMV are pertinent(35) . By using actin as a marker for proteins located on the cytosolic side of the BBM, papain was shown to cleave only proteins exposed on the external or luminal side of the BBM and to have no access to the cytosolic side(36) . Papain treatment of BBMV under standard conditions (35) liberates about 65% of the total membrane protein. The loss in protein is accompanied by a dramatic reduction in activity of enzymes known to be exposed on the external (luminal) side of BBM. For instance, the activities of aminopeptidase and sucrase isomaltase were shown to drop to levels of 25 and 18% of their original activity, respectively(35) . Concomitantly the protein-mediated absorption of cholesterol from mixed bile salt micelles is significantly reduced and the absorption of PtdCho from egg PtdCho SUV is practically abolished(1, 2, 3) . After papain treatment of BBMV, immunogold labeling was also greatly reduced. Immunogold labeling and papain digestion of BBM taken together provide therefore good evidence that the cholesterol receptor is exposed on the external or luminal side of the BBM. These experiments also support the notion that the 13-kDa protein is related to the cholesterol receptor.

The behavior of BBMV prepared from human small intestines and supernatant proteins produced from these vesicles merits discussion. The human BBMV closely resembled rabbit small intestinal BBMV in their lipid absorption properties. Both cholesterol and PC absorption by human BBMV are protein-mediated analogous to rabbit small intestinal BBMV reported before(1, 2, 3) . The rate constants are also comparable to those measured with rabbit BBMV. What was said for rabbit small intestinal BBMV concerning the reaction order and mechanism of cholesterol absorption is also valid for human BBMV. After proteolytic treatment of human BBMV with papain or proteinase K, cholesterol absorption became a passive diffusion process with half-times on the order of 10 h (data not shown).

The analogous behavior of the two kinds of BBMV is extended to the production of supernatant proteins. As evident from Fig. 1B, water-soluble lipid exchange proteins of very similar molecular mass and lipid exchange properties are liberated from human BBM by freeze-thaw cycles. Human peak 3 proteins mediated cholesterol and PC exchange between two populations of SUV and this lipid exchange was totally inhibited by the anti-rabbit 13-kDa antibody (data not shown). The inhibition was identical to that shown in Fig. 4C in which rabbit peak 3 protein was used as catalyst(32) . The strong immunological cross-reactivity between the anti-rabbit antibody and the human 13-kDa protein points to a high homology between the rabbit and the human 13-kDa protein, indicating that this class of proteins is highly conserved. The high homology observed in this class of lipid exchange proteins may account for the generally pour quality of the antibodies raised against these lipid exchange proteins. This manifests itself in weak or lacking immune responses (cf. Fig. 4, 5, and 7). For instance, with some antibodies raised against the 13-kDa protein no effect on cholesterol and PC absorption by BBMV was observed.


conclusions

It is clear that the work presented does not provide an answer to the two questions raised at the outset of the discussion. At the best it may provide a working hypothesis. Taken all experimental evidence together we propose as a working hypothesis that the 13- and 100-kDa proteins are proteolytic products of the cholesterol receptor of the BBM. Evidence in favor of this proposal comes from 1) the similarity in functional, imunological, and lipid binding properties of the 13- and 100-kDa lipid exchange proteins; 2) the partial inhibition of lipid absorption by BBM in the presence of the anti-13-kDa antibody; 3) the positive immunogold labeling of BBMV using this antibody together with the papain treatment of BBMV; and 4) the immunoblot analysis of BBMV and supernatant proteins yielding similar band patterns.

The 22-kDa lipid exchange protein is both functionally and immunologically different from the other two water-soluble lipid exchange proteins and very likely of a different origin. Clearly more work is required to better characterize it and to clarify its relationship to other lipid exchange proteins.

In order to test our working hypothesis we are currently purifying the 100-kDa lipid exchange protein. By raising antibodies against this protein we should stand a good chance of elucidating the interrelationship between the water-soluble lipid exchange proteins liberated from the BBM and also answering the important question of whether or not the 100-kDa protein is related to the cholesterol receptor.


FOOTNOTES

*
This work was supported by Swiss National Science Foundation Grants 31-32441.91 and 32-36577.92. The molecular mass determination of the 13-kDa protein and the effect of the anti-13 kDa antibody on PC exchange between two populations of SUV mediated by the antigen were presented at the British Biochemical Society meeting in London, 1991 (32) . The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore by hereby marked ``advertisement'' in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

§
Present address: Vienna Biocenter, Dept. of Cell Biology and Microbiology, Institute for Microbiology and Genetics, Dr. Bohr-Gasse 9, A-1030 Vienna, Austria.

To whom correspondence should be addressed.

(^1)
The abbreviations used are: BBMV, brush border membrane vesicle(s); BBM, brush border membrane; FPLC, fast protein liquid chromatography; PBS, phosphate-buffered saline; PtdCho, phosphatidylcholine; PtdOH, phosphatidic acid; PVDF, polyvinylidene difluoride; SUV, small unilamellar vesicle(s); TLC, thin-layer chromatography; PC phosphatidylcholine.

(^2)
We propose to designate this protein as the cholesterol receptor of the BBM.


ACKNOWLEDGEMENTS

We are indebted to Dr. R. Falchetto for determining the amino acid sequence.


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