©1995 by The American Society for Biochemistry and Molecular Biology, Inc.
A Sphingomyelin-transferring Protein from Chicken Liver
USE OF PYRENE-LABELED PHOSPHOLIPID (*)

Jan Westerman , Klaas-Jan de Vries , Pentti Somerharju (1), Johanna L. P. M. Timmermans-Hereijgers , Gerry T. Snoek , Karel W. A. Wirtz (§)

From the (1)Centre for Biomembranes and Lipid Enzymology, Utrecht University, 3508 TB Utrecht, The Netherlands and Department of Basic Chemistry, University of Helsinki, SF-00014 Helsinki, Finland

ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS AND DISCUSSION
FOOTNOTES
ACKNOWLEDGEMENTS
REFERENCES

ABSTRACT

A phospholipid transfer protein was purified from chicken liver which, in addition to phosphatidylinositol (PI) and phosphatidylcholine (PC), carries sphingomyelin (SM) between membranes. For comparison, the PI-transfer protein from chicken liver only carries PI and PC. Specificity was established by use of phospholipids that carry a pyrene-labeled acyl chain. Based on the N-terminal sequence and Western blot analysis we conclude that this protein is an isoform of the PI-transfer protein. At increasing length of the pyrene-labeled acyl chain, the isoform expresses a high activity toward SM, a low activity toward PI, and virtually no activity toward PC.


INTRODUCTION

Phospholipid transfer proteins have been purified from mammals, plants, fungi, and bacteria (Wirtz, 1991). The best characterized ones are the phosphatidylcholine (PC)()-transfer protein from bovine liver (Kamp et al., 1973), the phosphatidylinositol (PI)-transfer protein (PI-TP) from bovine and rat brain (Helmkamp et al., 1974; Venuti and Helmkamp, 1988) and yeast (Aitken et al., 1990), and the nonspecific lipid transfer protein (nsL-TP) from bovine, rat, and human liver (Crain and Zilversmit, 1980; Bloj and Zilversmit, 1977; Van Amerongen et al., 1987) and plants (Kader, 1975). Recently, PI-TP has raised considerable interest because of its proposed roles in the phosphoinositide cycle (Thomas et al., 1993) and the ATP-dependent, Ca-activated secretory process (Hay and Martin, 1993). Furthermore, PI-TP from yeast is identical to the protein SEC14p, which is an essential factor in the secretory vesicle flow from the trans-Golgi network to the plasma membrane (Bankaitis et al., 1989, 1990). Extensive studies have been carried out to establish that the lipid transfer activity of SEC14p is related to its function in protein secretion (Cleves et al., 1991; Skinner et al., 1993; McGee et al., 1994).

Recently, the agonist-controlled metabolism of sphingomyelin (SM) has raised considerable interest because of its crucial role in regulating cellular function (Hannun, 1994). Given this novel role of SM, we thought it of interest to investigate whether mammalian tissues have a SM-transferring protein. A nsL-TP with a pronounced activity toward SM was purified from rat hepatoma (Dyatlovitskaya et al., 1978). Distinct SM-transfer activity was identified in chicken liver (Koumanov et al., 1982). Here we report the purification to homogeneity of the SM-transferring protein from this latter tissue. While this study was in progress, a cDNA encoding an isoform of PI-TP from rat brain was identified (Tanaka and Hosaka, 1994). Evidence will be provided that the SM-transferring protein is very similar, if not identical, to this isoform.


EXPERIMENTAL PROCEDURES

Materials

Q-Sepharose, Sephacryl S-100, heparin-Sepharose CL-6B, Mono P, and Polybuffer 74 were obtained from Pharmacia (Uppsala, Sweden). Hydroxylapatite (Bio-Gel, HTP) was from Bio-Rad. Egg yolk PC, PA, and TNP-PE were from Sigma. Pyr(x)SM, Pyr(x)PI, and Pyr(x)PC were synthesized as described (Kasurinen and Somerharju, 1992). High bonding microtiter plates were from Greiner (Alphen a/d Rijn, The Netherlands). Goat anti-rabbit IgG conjugated with peroxidase (GAR-PO) was obtained from Nordic Immunological Laboratories (Tilburg, The Netherlands) and goat anti-rabbit IgG conjugated with alkaline phosphatase (GAR-AP) from Sigma. Nitrocellulose membranes were from Schleicher and Schuell (Dassel, Germany). The anti-PI-TP antibody was raised in rabbits against predicted epitopes of rat brain PI-TP (Snoek et al., 1992).

Purification of Phospholipid Transfer Proteins

All steps were performed at 4 °C.

Step 1

A 33% (w/v) homogenate of fresh chicken liver (2000 g) was prepared in 0.9% NaCl, 20 mM Tris-HCl, pH 7.4, 1 mM EDTA, 10 mM NaF, 1 mM sodium pyrophosphate, 0.17 mg of trypsin inhibitor/liter, and 1 mM phenylmethylsulfonyl fluoride. After centrifugation at 14,000 g for 1 h, the supernatant was collected.

Step 2

A 40-90% ammonium sulfate precipitate of the supernatant was prepared and sedimented by centrifugation at 14,000 g for 60 min. The pellet was dissolved in 20 mM Tris-HCl, pH 7.4, 1 mM EDTA, 10 mM -mercaptoethanol (TEM buffer). After dialysis overnight against TEM buffer, the solution was centrifuged for 1 h at 100,000 g to remove insoluble material.

Step 3

The dialysate was applied to a column (11.5 21 cm) of Q-Sepharose equilibrated with TEM buffer. After rinsing with TEM buffer (2 liters), the protein was eluted with a linear gradient of TEM buffer and 500 mM NaCl/TEM buffer (2 2.5 liters) (flow rate of 400 ml/h; 20-ml fractions). SM-transfer activity eluted between 350 and 400 mM NaCl.

Step 4

The active fractions were applied to a Sephacryl S-100 column (5 94 cm) equilibrated with 10 mM potassium phosphate, pH 6.8, 10 mM -mercaptoethanol. The column was rinsed with the same buffer (flow rate of 180 ml/h; 18-ml fractions). SM-transfer activity eluted at about half the column volume.

Step 5

The active fractions were applied to a hydroxylapatite column (1.4 40 cm) equilibrated with 10 mM potassium phosphate, pH 6.8, 10 mM -mercaptoethanol. The column was rinsed with a linear gradient of 10-50 mM potassium phosphate, pH 6.8, 10 mM -mercaptoethanol (2 300 ml) (flow rate of 25 ml/h; 5-ml fractions). SM-transfer activity eluted at 20 mM phosphate. This activity co-eluted with PI-transfer activity. A second peak of PI-transfer activity devoid of SM-transfer activity eluted at 35 mM phosphate (see Fig. 1).


Figure 1: Separation of PI/SM-TP and PI-TP by chromatography on hydroxyl-apatite. Column fractions were assayed for PI-transfer activity () and SM-transfer activity ().



Step 6

The PI/SM- and PI-transfer activity peaks were pooled separately, and each active pool was dialyzed against 20 mM Tris-HCl, pH 7.2, 50 mM NaCl, 5 mM -mercaptoethanol and then applied to a heparin-Sepharose CL-6B column (2.3 24 cm). By isocratic elution SM/PI-transfer activity was recovered between 120 and 200 ml and PI-transfer activity between 200 and 360 ml of elution buffer. The active fractions were pooled and dialyzed against 25 mM methylpiperazine pH 5.7, 10 mM -mercaptoethanol.

Step 7

Each active pool was applied to a Mono P column (0.5 20 cm). The column was eluted with 25 ml of 10% Polybuffer 74-HCl, pH 4, 10 mM -mercaptoethanol (flow rate of 0.8 ml/min, 1-ml fractions). Both PI/SM-TP and PI-TP activity eluted from the column in two peaks between pH 4 and 5.

Phospholipid Transfer Assay

The assay is based on the increase of the pyrene monomer fluorescence intensity as a result of the transfer of pyrenylacyl(Pyr(x))-labeled phospholipid from quenched donor vesicles (2 nmol of total phospholipid) to a 25-fold excess of unquenched acceptor vesicles (Van Paridon et al., 1988). The donor vesicles consisted of Pyr(x)SM or Pyr(x)PC, TNP-PE, PA, and egg PC (10:10:10:70 mol %) and Pyr(x)PI, TNP-PE, and egg PC (10:10:80 mol %) and the acceptor vesicles of PA and egg PC (5:95 mol %). The reaction was carried out in 2 ml of 20 mM Tris-HCl, 5 mM EDTA, 200 mM NaCl (pH 7.4) containing 0.1 mg of BSA at 37 °C. The reaction was initiated by the addition of fractions (10-50 µl) containing phospholipid transfer activity. The initial slope of the progress curve was taken as an arbitrary unit of transfer activity. Measurements were performed on a SLM-Aminco SPF-500C fluorimeter equipped with a thermostated cuvette holder and a stirring device.

ELISA Procedure

The ELISA for the qualitative detection of PI-TP was performed using high bonding microtiter plates. Aliquots of the column fractions were added to the wells and incubated for 1 h at 37 °C. After washing the wells six times with PBS-0 (phosphate-buffered saline: 137 mM NaCl, 2.7 mM KCl, 6.5 mM NaHPO, and 1.5 mM KHPO), the wells were incubated for 1 h at 37 °C with PBS-0 containing 1% (w/v) BSA as blocking agent. Further manipulations were carried out at room temperature. After washing the wells with PBS/Tween (PBS-0 containing 0.05% (v/v) Tween 20), the wells were incubated for l h with anti-PI-TP antibody in PBS/Tween/BSA (1%, w/v). The wells were washed with PBS/Tween and incubated with GAR-PO (0.10 ml; 1:1000 diluted in PBS/Tween/BSA) for 1 h. After washing the wells with PBS/Tween, the substrate solution was added and the absorbance determined at 492 nm.

Gel Electrophoresis and Blotting

Samples were analyzed by electrophoresis on a 15% SDS-polyacrylamide gel (Laemmli, 1970). Proteins were electrophoretically transferred from the gel to nitrocellulose membranes at 1 mA/cm for 75 min. 3% gelatin (w/v) in Tris-buffered saline (20 mM Tris-HCl, 0.5 M NaCl, pH 7.4) was used as a blocking agent. PI-TP was identified by incubating the nitrocellulose membranes with antibody in Tris-buffered saline containing 1% (w/v) gelatin followed by an incubation with GAR-AP and color development.

Amino Acid Sequence Determination

The N-terminal amino acid sequence of the purified phospholipid transfer proteins was determined by automated Edman degradation using the 476A protein sequencer (Applied Biosystems).


RESULTS AND DISCUSSION

Purification

Based on the evidence that the membrane-free supernatant of chicken liver contains SM-transfer activity (Koumanov et al., 1982), we have selected this tissue for the purification of the active protein (see ``Experimental Procedures''). Fractionation on both Q-Sepharose (see ``Step 2'') and Sephacryl S-100 (see ``Step 3'') yielded a single SM-transfer activity peak. Initially, this activity was detected by measuring the transfer of bovine [N-methyl-C]SM from unilamellar vesicles to bovine heart mitochondria in an assay similar to that described for the transfer of [C]phosphatidylethanolamine (Poorthuis and Wirtz, 1983). Subsequently, transfer activities were routinely measured by making use of pyrene-labeled phospholipid analogs (see ``Experimental Procedures''). Since in a previous study PI-TP purified from bovine brain was shown to have a limited capacity to transfer SM between membranes (DiCorletto et al., 1979), the column fractions were tested also for PI-TP by use of a specific ELISA. With both Q-Sepharose and Sephacryl S-100 one immunoreactive PI-TP peak was detected that coincided with the SM-transfer activity peak. Hence, in the subsequent purification steps the column fractions were tested for both SM- and PI-transfer activity. Fractionation on hydroxylapatite (see ``Step 4'') yielded two peaks with PI-transfer activity, the first peak of which only contained the SM-transfer activity (Fig. 1). Further purification on a heparin-affinity column (see ``Step 5'') and Mono P (see ``Step 6'') demonstrated convincingly that, in addition to PI-TP, a protein exists with both PI- and SM-transfer activity (denoted as PI/SM-TP). Fractionation on the isoelectrofocusing column (Mono P) showed that PI-TP consists of two forms (PI-TP I and II), which differ slightly in isoelectric point. In agreement with previous observations on bovine brain PI-TP, this charge difference is probably due to one form containing an endogenously bound PI molecule and the other form a PC molecule (Van Paridon et al., 1987). Possibly for similar reasons, two forms of PI/SM-TP (I and II) were collected from the Mono P column. Analysis by SDS-polyacrylamide gel electrophoresis (Fig. 2A) indicated that by fractionation on Mono P both PI-TP I and II (lanes3 and 4) and PI/SM-TP I (lane5) were purified to homogeneity. PI/SM-TP II (lane6) was contaminated with one major protein and was not further purified. Starting from 2000 g of liver the yields were 0.2 mg of PI-TP I, 0.15 mg of PI-TP II, and 0.4 mg of PI/SM-TP I.


Figure 2: SDS-polyacrylamide gel electrophoresis (A) and Western blot analysis (B) of phospholipid-transfer proteins purified from chicken liver. Lane1, bovine brain PI-TP (control); lane2, marker proteins; lane3, PI-TP I, lane4, PI-TP II; lane5, PI/SM-TP I; lane6, PI/SM-TP II.



Homology

Analysis by Western blotting (Fig. 2B) using the antibody elicited against epitope segments of rat brain PI-TP showed that the antibody was cross-reactive with both forms of PI-TP (lanes3 and 4) and PI/SM-TP (lanes5 and 6). This strongly suggests an extensive similarity between chicken liver and rat brain PI-TP, as well as between PI-TP and PI/SM-TP. On the other hand, one may infer from this analysis that the molecular mass of PI-TP (35 kDa) is different from that of PI/SM-TP (39 kDa). To further corroborate the similarity between these proteins, the N-terminal amino acid sequence was analyzed by automated Edman degradation. As shown in Fig. 3, the first 21 amino acid residues of PI/SM-TP I were identical to that of the isoform of rat brain PI-TP (designated -PI-TP), the sequence of which was deduced from the cDNA nucleotide sequence (Tanaka and Hosaka, 1994). The N-terminal sequence (first 21 amino acid residues) of PI-TP I was identical to that of PI-TP II and nearly homologous with that of rat brain PI-TP with conservative replacements at position 3 (Ile for Leu) and at position 15 (Glu for Asp). PI/SM-TP was highly homologous with PI-TP I and II with conservative replacements at position 6 (Phe for Tyr) and position 9 (Val for Ile). The residue at position 12, which is presumed to be Cys, was replaced for Val and Gln at position 15 for Glu. A similar high homology was observed for the N-terminal sequence of -PI-TP and PI-TP from rat brain (Fig. 3) (Tanaka and Hosaka, 1994). In the latter study it was reported that the total amino acid sequence of -PI-TP showed 77% identity to that of PI-TP and about 94% similarity.


Figure 3: N-terminal amino acid sequences of chicken liver PI/SM-TP I and PI-TP I and II. For comparison, the sequence of the isoform of rat brain PI-TP (-PI-TP) deduced from the cDNA nucleotide sequence (Tanaka and Hosaka, 1994) and rat brain PI-TP sequence (Dickeson et al., 1989) are shown. The sequence method used is not suitable to identify Cys (see PI/SM-TP I, position 12).



The amino acid sequences of mouse, rat, and human PI-TP share about 99% identity, which makes this protein exceedingly conserved (Dickeson et al., 1989, 1994; Geijtenbeek et al., 1994). Recently, we established that, in addition to chicken liver PI/SM-TP, the N-terminal amino acid sequence of bovine brain PI/SM-TP is also identical to that of rat brain -PI-TP.()This strongly suggests that, similar to PI-TP, PI/SM-TP is highly conserved among species.

Specificity

During the purification of PI/SM-TP and PI-TP, the transfer activities were determined in a continuous fluorescent transfer assay using as substrates 1-palmitoyl-2-pyrenyldecanoyl-PI (Pyr(10)PI) and N-pyrenylmyristoyl-SM (Pyr(14)SM). Previously it was found that by varying the acyl chain length at the sn-1- and sn-2-position, PI-TP from bovine brain had a preference for certain molecular species of PI (Van Paridon et al., 1988). In order to establish whether this was also the case for PI/SM-TP, the pyrenylacyl chain length was varied at the sn-2-position of 1-palmitoyl-2-Pyr(x)PI and at the amine moiety of Pyr(x)SM. As shown in Fig. 4A, the protein expressed the highest transfer activity with Pyr(10)PI and, relative to this species, a 5-fold lower activity with Pyr(14)PI. Very characteristically, PI/SM-TP expressed a very low activity with Pyr(10)SM and, relative to this species, a 9-10-fold higher activity with Pyr(12)SM and Pyr(14)SM. With the Pyr(14) species the protein expressed a more than 3-fold higher activity with SM than with PI. Under conditions where PI/SM-TP is incubated with the donor vesicles only, this protein binds Pyr(x)PI and Pyr(x)SM species with a preference similar to that observed for transfer (data not shown). Given the occurrence of long chain fatty acids (>18 carbon atoms) in natural PI and SM, we predict that in situ PI/SM-TP has a distinct preference for SM.


Figure 4: Effect of acyl chain length on the transfer of pyrenyl acyl-labeled phospholipid species by PI/SM-TP (A) and PI-TP (B). , PI; , SM; , PC. x indicates the number of carbon atoms of the pyrenyl-labeled acyl chain.



As was previously observed for bovine brain PI-TP (Van Paridon et al., 1988; Kasurinen et al., 1990) PI/SM-TP may also transfer PC, albeit at a relatively low rate (Fig. 4A). It is quite remarkable that with both PC and SM having the same polar headgroup, the protein expressed virtually no activity with Pyr(12)PC and Pyr(14)PC. This strongly suggests that, provided the correct chain length is present, PI/SM-TP recognizes molecular features of SM that are alien to PC like the sphingosine moiety. On the other hand, the polar headgroup is required for activity as the protein failed to transfer the analogous pyrene-labeled ceramide species (data not shown).

Under identical conditions of transfer, the preference of PI-TP for molecular species was determined (Fig. 4B). The activity profiles were quite similar to what was observed for bovine brain PI-TP (Van Paridon et al., 1988). For the species measured, the protein transferred PI 5-7 times faster than PC. Transfer of Pyr(x)SM species by PI-TP was negligible. Another striking point is that PI-TP has a 4-5-fold lower activity toward both PI and PC, compared with PI/SM-TP (Fig. 4A).

Conclusion

A novel phospholipid transfer protein has been identified with a distinct affinity for SM. This protein is an isoform of PI-TP with the remarkable feature that it expresses a high activity toward long chain SM, concomitant with a low activity toward PI and PC. This protein also occurs in mammalian tissues.

It remains to be established whether in analogy with the involvement of PI-TP in the phosphoinositide cycle, the isoform of PI-TP plays a role in the sphingomyelin cycle.


FOOTNOTES

*
This research was carried out under auspices of the Netherlands Foundation for Chemical Research (SON) and with financial aid from the Netherlands Organization for Scientific Research (NWO). 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.

§
To whom correspondence should be addressed: Centre for Biomembranes and Lipid Enzymology, Utrecht University, P. O. Box 80.054, 3508 TB Utrecht, The Netherlands. Tel.: 31-30-533443; Fax: 31-30-522478; E-mail: k.w.a.wirtz@chem.ruu.nl.

The abbreviations used are: PC, phosphatidylcholine; PI, phosphatidylinositol; SM, sphingomyelin; PA, phosphatidic acid; TNP-PE, 2,4,6-trinitrophenylphosphatidylethanolamine; Pyr(x), pyrene fatty acid, x equals the number of carbon atoms in the acyl chain; Pyr(x)SM, N-(1-pyrenyl)acyl-labeled sphingomyelin; Pyr(x)PC, 1-palmitoyl-2-(1-pyrenyl)acyl-sn-3-glycerophosphocholine; Pyr(x)PI, 1-palmitoyl-2-(1-pyrenyl)acyl-sn-3-glycerophosphoinositol; PI-TP, phosphatidylinositol-transfer protein; PI/SM-TP, phosphatidylinositol/sphingomyelin-transfer protein; nsL-TP, nonspecific lipid transfer protein; BSA, bovine serum albumin; GAR-PO, goat anti-rabbit IgG conjugated with peroxidase; GAR-AP, goat anti-rabbit IgG conjugated with alkaline phosphatase; ELISA, enzyme-linked immunosorbent assay; PBS, phosphate-buffered saline.

K. J. de Vries, manuscript in preparation.


ACKNOWLEDGEMENTS

We thank F. van der Lecq and Dr. A. J. Aarsman of the Sequence Centre Utrecht for performing the sequence analyses.


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©1995 by The American Society for Biochemistry and Molecular Biology, Inc.