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GRP94 is a putative high density lipoprotein-binding protein in liver
Biochimica et Biophysica Acta 1437 (1999) 378^392
Gp96/GRP94 is a putative high density lipoprotein-binding protein in liver Rini de Crom *, Rien van Haperen, Rick Janssens, Pim Visser, Rob Willemsen, Frank Grosveld, Arthur van der Kamp The Medical Genetics Center, Department of Cell Biology and Genetics, Erasmus University Rotterdam, Dr. Molenwaterplein 50, P.O. Box 1738, 3000 DR Rotterdam, The Netherlands Received 9 November 1998; received in revised form 18 January 1999; accepted 22 January 1999
Abstract We have previously shown that three high density lipoproteins (HDL)-binding proteins in liver, of 90, 110 and 180 kDa, are structurally related. In this study, these proteins are identified as gp96/GRP94. This protein is known to occur as a homodimer and has a dual subcellular localization : it is both an endoplasmic reticulum resident protein, where it is supposed to act as a chaperonin, and a plasma membrane protein, whose significance is unknown. In ultrastructural studies the plasma membrane localization of the homodimeric form was verified. The 90-kDa protein was abundantly present at the membranes of the endosomal/lysosomal vesicles as well as at the apical hepatocyte membranes, comprising the bile canaliculi. The monomeric protein is scarcely present at the basolateral membrane of the hepatocytes, but could be demonstrated in coated pits, suggesting involvement in receptor-mediated endocytosis. Labeling of the endoplasmic reticulum was virtually absent. Gp96/GRP94 was transiently expressed in COS-1 cells. However, the expressed protein was exclusively localized in the endoplasmic reticulum. Transfection with constructs in which the C-terminal KDEL sequence had been deleted, resulted in plasma membrane localized expression of protein, but only in an extremely low percentage of cells. In order to evaluate the HDL-binding capacities of this protein, stably transfected cells were generated, using several cell types. It appeared to be difficult to obtain a prolonged high level expression of gp96. In these cases, however, a marked increase of HDL-binding activity compared with the control cells could be observed. ß 1999 Elsevier Science B.V. All rights reserved. Keywords: High density lipoprotein; HDL-binding protein ; Ultrastructural localization; Bile canaliculus; KDEL; Murine erythroleukemia cell
1. Introduction
Abbreviations: apo, apolipoprotein; CHO, Chinese hamster ovary; HDL, high density lipoprotein; LDL, low density lipoprotein ; LRP, LDL receptor-related protein; PAGE, polyacrylamide gel electrophoresis ; SDS, sodium dodecylsulfate; SR-BI, scavenger receptor class B, type I * Corresponding author. Fax: +31 (10) 408-9468; E-mail: [email protected]
Plasma levels of HDL are among the best indicators of the risk of coronary artery disease [1,2]. Although this notion has led to intensive research on HDL metabolism, many aspects are still under debate [1,3]. One of these aspects is the role of HDL-binding proteins in the liver [4]. We identi¢ed HDL-binding proteins of 90, 110 and 180 kDa in porcine liver by ligand blotting (mo-
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lecular masses estimated by sodium dodecylsulfatepolyacrylamide gel electrophoresis (SDS-PAGE) under non-reducing conditions) [5]. In a later study, we showed that these three proteins are structurally related [6]. The principal arguments for this conclusion are: a polyclonal antibody raised against the puri¢ed 90-kDa HDL-binding protein shows crossreactivity with the 110- and 180-kDa proteins on an immunoblot; under reducing conditions, puri¢ed protein preparations of 90, 110 and 180 kDa all migrate at the 90-kDa position in SDS-PAGE, and still show immunoreactivity with the antiserum as well as HDL-binding activity in a ligand blot assay; CNBrfragment maps of the three proteins are highly similar. We concluded that the 180-kDa HDL-binding protein is a homodimer of the 90-kDa HDL-binding protein [6]. In this paper, we show that the 90-, 110- and 180kDa proteins have the same N-terminal amino acid sequence. Amino acid sequences of several CNBr fragments of the 90-kDa protein show an almost 100% identity with a protein known under various names, like mouse endoplasmin or ERp99, and human tra1, gp96 or GRP94 [7]. This protein is known to occur both in a monomeric and in a dimeric conformation [8]. Gp96 has the endoplasmic reticulum (ER) retention signal KDEL [9]. Therefore, we performed both light and electron-microscopical localization studies. They show that at least part of the protein is localized at the plasma membrane of liver cells. cDNA clones, encoding both the porcine and human homolog of gp96 were isolated and tested in expression studies using various transfected cell lines. 2. Materials and methods
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applied to a 473A Applied Biosystems protein sequencer for N-terminal amino acid sequence determination. 2.2. Synthesis of a cDNA probe Oligonucleotides, based on the published sequence of mouse ERp99 [7], were synthesized using an Applied Biosystems oligonucleotide synthesizer. From RNA isolated from murine liver, ¢rst-strand cDNA was synthesized with reverse transcriptase (RT). This was used as a template for polymerase chain reaction (PCR), performed in a Perkin Elmer Cetus PCR apparatus. Mouse cDNA fragments were cloned into the EcoRI site of pTZ19 via the restriction sites incorporated in the synthetic oligonucleotide primers of the PCR reaction. 2.3. Screening of cDNA libraries Vgt11 cDNA libraries from porcine liver (CloneTech PL1001b) and human liver (CloneTech HL1115b) were screened with cDNA probes labeled with [K-32 P]ATP. V clones with the largest inserts were subcloned into pTZ19. Partial cDNA clones were digested with restriction enzymes and ligated in order to obtain full length cDNA inserts by standard techniques [11]. 2.4. DNA sequencing The DNA sequence was determined on both strands by the dideoxy-nucleotide chain-termination method (T7 Sequencing kit, Pharmacia), using a series of synthetic oligonucleotides. 2.5. Computer analyses
2.1. N-Terminal sequencing Puri¢cation of HDL-binding proteins and CNBr cleavage of puri¢ed protein preparations were performed as described before [6]. The protein preparations or CNBr fragments were subjected to SDSPAGE and electrophoretically transferred to ProBlott membranes (Applied Biosystems) [10]. These membranes were stained with Coomassie brilliant blue and protein bands of interest were carefully cut out. These pieces of membrane were directly
Comparisons for homology were performed by FASTA using the data bases of GenBank/EMBL, and Swiss-Prot. 2.6. Electron-microscopy studies Porcine liver specimens were immediately ¢xed in 3% paraformaldehyde and prepared for ultracryotomy as described previously [12]. Ultrathin cryosections (60 nm) were immunolabeled with either rabbit
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polyclonal antibodies against the 90-kDa protein or against the 180-kDa protein [5]. Antibodies directed against cathepsin D were kindly provided by Dr. H. Aerts, University of Amsterdam. Antigen^antibody complexes were visualized with goat anti-(rabbit Ig) conjugated with 10 nm colloidal gold particles. To localize two antigens simultaneously, the ¢rst antibody was labeled with a 6-nm protein A-gold probe and the second with a 10-nm probe, according to Geuze et al. [13]. Sections were stained with uranyl salts and embedded in 1.5% methylcellulose and examined with a Philips CM100. The speci¢city of the labeling procedure was checked by incubation of the sections with normal rabbit serum as substitution for the primary antibody, or with the di¡erent gold probes alone. Background labeling was negligible.
porcine and human cDNA (PvuII site at positions 2395 and 2497, respectively; BamHI site at positions 2434 and 2536, respectively). This results in a K801 CSTOP mutation, which completely eliminates the KDEL sequence. COS-1 cells were grown in 98mm-diameter dishes and transfected with 10 Wg of plasmid DNA by the DEAE-dextran method [11]. The transfected cells were analyzed at 48^72 h post-transfection. For immunocytochemistry experiments, cells were grown and transfected in 24-well plates on 15-mm-diameter coverslips. Alternative transfection protocols that were tested in order to achieve a higher percentage of expressing cells used Lipofectin (Gibco BRL), Lipofectamin (Gibco BRL), or Transfectam (Promega), all applied according to the manufacturers instructions.
2.7. Protein expression in Escherichia coli and generation of antisera
2.9. Transfection experiments
cDNA sequences were subcloned into the vector pGEX (Pharmacia) and transfected to BL21 E. coli cells by standard techniques [11]. Induction of protein expression and isolation of bacterially expressed protein was performed according to Smith and Corcoran [14]. Bacterially expressed protein was puri¢ed by preparative SDS-PAGE using a Bio-Rad Prep Cell. Polyclonal antisera were raised in rabbits as described previously [6]. All animals were cared for in compliance with the Guidelines of the Ethical Committee on the Use of Laboratory Animals of the Erasmus University Rotterdam (prot. no. 132.94.01) and with the European Committee Standards on the care and use of laboratory animals (Ministry of Welfare, Health and Cultural A¡airs, The Netherlands). 2.8. Transient protein expression in COS-1 cells Porcine or human cDNA, either full length (gp96) or carrying a mutation that gives rise to a protein that lacks the C-terminal KDEL tetrapeptide (gp96vKDEL ), was inserted into the eukaryotic expression vector pCD-X [15]. vKDEL cDNA was constructed by inserting a linker oligonucleotide (5PCTGAATAAGATGAACTGTAAATTATACTCTCACCATTTG-3P) by ligation between the PvuII and BamHI restriction sites that are present both in
COS-1 cells, Hep G2 cells, CHO-9 cells, L cells and murine erythroleukemia (MEL) cells were cultured in DMEM/F10 (1:1) supplemented with 2 mM L-glutamine, 100 IU penicillin/ml, 100 mg streptomycin/ml and 10% FCS at 37³C in a 5% CO2 atmosphere. All cell types were transfected with Lipofectin (Promega) according to the manufacturer's instructions, except for MEL cells, that were transfected by electroporation using a Bio-Rad Gene Pulser (settings: 117 V, 1200 mF, 10 ms). The next day following transfection, cells were selected on culture medium containing 800 mg/ml G418. MEL cells were induced by adding 2% dimethylsulfoxide to the medium 4 days after transfection and were harvested for analysis after 24 h [16]. For cotransfection to COS, CHO and HepG2 cells, 1 mg of pCD-X-neo was used, containing a neomycin resistance gene into a derivative of the mammalian expression vector pCD-X [15] and 10 mg of either pCD-X-p-gp96 or pCD-X-h-gp96 containing the porcine or human gp96 cDNA, respectively. L cells were transfected with 10 mg of a mammalian expression vector pJG1 containing the CMV promoter and the TK-neo gene (gift of Jacky Guy, Rotterdam), and the porcine or human gp96 cDNA cloned into the polylinker (pJG1-p-gp96 and pJG1h-gp96, respectively). MEL cells were transfected with a vector named
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pEV3, which is a derivative of pEC3 [17], containing the L-globin locus control region and parts of the Lglobin gene for mRNA stability, into which porcine or human gp96 cDNA was cloned (pEV3-p-gp96 and pEV3-h-gp96, respectively). 2.10. DNA and RNA analysis DNA and RNA isolation from cells, as well as Southern and Northern blotting were performed using standard techniques [11]. Blots were hybridized with radioactively labeled probes as indicated in the ¢gure legends. Hybridizing bands were visualized by autoradiography or using a Phosphor Imager (Molecular Dynamics). 2.11. HDL-binding to MEL cells Isolation and radiolabeling of human HDL with I was performed exactly as described before [5]. Two hundred microliters of MEL cell suspension in medium containing 10% lipoprotein-de¢cient serum (LPDS) was incubated with the indicated amounts of 125 I-HDL at 37³C for 2 h. Cells were washed four times in PBS containing 5% LPDS-FCS, two times in PBS and resuspended in 0.5 ml PBS. Associated radioactivity was determined in a gamma counter. Then cell protein was dissolved by adding NaOH to a ¢nal concentration of 0.1 M and determined according to Lowry [18]. Aspeci¢c cell association was measured in the presence of a 25-fold excess of unlabeled HDL. Speci¢c cell association was calculated by subtracting the aspeci¢c cell association from the total cell association. All measurements were performed in triplicate. 125
2.12. Immunoblotting, immunocytochemistry and immunohistochemistry Immunoblotting was performed exactly as described before [6]. Immunocytochemistry was performed on cells grown on coverslips as described by Van Dongen et al. [19]. For immunohistochemistry, porcine liver was ¢xed and prepared for ultracryotomy as described before [20]. Semithin cryosections (0.5^1.0 Wm) were incubated with a Protein A Sepharose CL-4B (Pharmacia) puri¢ed IgG fraction of polyclonal antibodies
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against HDL-binding proteins for 1 h at room temperature in a humid incubator. Antigen^antibody complexes were visualized with £uorescein isothiocyanate-conjugated goat anti-rabbit immunoglobulins (Nordic, Tilburg, The Netherlands) (diluted 1:80; incubated at room temperature for 1 h in a humid incubator) and studied by £uorescence microscopy. 3. Results The N-terminal amino acid sequence of puri¢ed 90-, 110- and 180-kDa HDL-binding proteins and of four di¡erent CNBr fragments from the 90-kDa protein were determined. As shown in Fig. 1, the 90-, 110- and 180-kDa HDL-binding proteins have identical N-termini. The variation at the 9th amino acid is due to occasional di¤culties in the interpretation of the output of the sequencer at that particular position. The 32-kDa CNBr fragment has the same Nterminus, indicating that this is the N-terminal fragment of the protein. Data-base searches revealed that all the determined amino acid sequences have a very high homology with three known proteins from different species: mouse ERp99, human gp96 and chicken hsp108. A cDNA probe was obtained by RT-PCR and used to screen a porcine liver Vgt11 cDNA library. In several screenings, only clones with partial cDNA inserts were found. Therefore, a cDNA clone containing the full-length cDNA was assembled from two individual clones. From this clone, the complete cDNA sequence of porcine gp96 (p-gp96) was determined (Fig. 2). The deduced protein sequence contains both an N-terminal signal sequence, which is no longer present in the mature protein (Fig. 1), and a C-terminal KDEL sequence, which in many proteins serves as an endoplasmic reticulum (ER) retention signal. The 21^23 amino acids beginning at Leu-191 that possibly comprise a membrane spanning region [7] are completely conserved in the porcine sequence (Fig. 2). The identi¢cation of an HDL-binding protein as a KDEL-containing protein prompted us to perform electron-microscopical localization studies. The ultrastructural localization of the 90-kDa protein was studied by indirect immunogold labeling of
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Fig. 1. N-Terminal amino acid sequences. N-Terminal amino acid sequences of puri¢ed porcine protein preparations of the 90-, 110and 180-kDa HDL-binding proteins and of 9-, 10-, 25- and 32-kDa CNBr fragments of the porcine 90-kDa protein are presented in bold. Uncertainties and variations within the porcine sequences are due to occasional di¤culties in the interpretations of the output of the sequencer. Comparisons with data-base sequences of mouse, human and chicken homologs are presented (accession numbers J02735, X15187, M14772, respectively).
electron-microscopic frozen sections of porcine liver using an antibody speci¢cally recognizing the 90-kDa protein [5]. Most label was found along the membranes of intracellular vesicles belonging to the endosomal/lysosomal compartment and along the membranes of the microvilli of the bile canaliculi (Fig. 3a). Using an antibody directed against cathepsin D, a lysosomal enzyme, it was con¢rmed in a double labeling procedure that the 90-kDa protein is co-localized with cathepsin D (Fig. 3b). Cathepsin D label is mainly found in the lumen of the vesicles, while the 90-kDa protein label is found preferentially localized at the limiting membranes of the vesicles, suggesting it is a membrane-associated protein. High amounts of label were found associated with the apical membrane of the hepatocytes, comprising the bile canaliculi.
Apart from these two sites, no intracellular label was observed in the hepatocytes. Special attention was paid to the ER, since gp96 is reported to be an ER protein. However, only very few gold particles could be detected in the ER (Fig. 3c). At the basolateral membrane of the hepatocytes, facing the space of Disse, the 90-kDa protein is present in very low quantities. However, labeling in coated pits is observed (Fig. 3d). The distribution of the 180-kDa protein was studied in a similar way as the localization of the 90-kDa protein, using an antiserum speci¢cally recognizing the 180-kDa protein [5]. The 180-kDa protein appeared to be localized primarily at the plasma membrane of both endothelial cells (Fig. 4a) and Kup¡er cells (Fig. 4b). Gold particles can be found at the sinusoidal side and at the side facing the space C
Fig. 2. Nucleotide and deduced amino acid sequences of porcine gp96 cDNA. The putative signal sequence is underlined. The putative transmembrane region, homologous to the putative transmembrane region in mouse Erp99 [7], is underlined as well. The cysteine residue that is involved in dimerization [42] is encircled. The sequence has been deposited at the EMBL database with the accession number Y09136.
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of Disse. Labeling is also present at the microvilli of the hepatocytes protruding in the space of Disse. Intracellular labeling of the hepatocytes was found only occasionally. No ER labeling was found (not shown). Using an antiserum recognizing both the 90- and 180-kDa proteins [6], a combination of both localization patterns was found (bile canalicular membranes, membranes of vesicles of the endosomal/lysosomal system, basolateral membranes of the hepatocytes and plasma membranes of endothelial cells and Kup¡er cells), while labeling of other intracellular compartments, including the ER, was virtually absent (not shown). We wanted to extend our analyses to the human protein (h-gp96). Since none of the antibodies we raised against the porcine proteins cross-react with sections of human liver in immunohistochemistry studies, we raised antibodies recognizing h-gp96. Therefore, we used a protein fragment expressed in E. coli transfected with human cDNA. With the porcine cDNA as a probe, we screened a human liver Vgt11 cDNA library in order to isolate a cDNA clone encoding the human cDNA. A clone containing the full-length cDNA was obtained by ligation of the inserts of two partial overlapping clones. The cDNA was completely sequenced; it perfectly matched the published sequence of human gp96 (GenBank accession number X15187; not shown). A cDNA fragment, comprising the C-terminal part of the protein (amino acids 677^801) was cloned into pGEX for expression in E. coli. Produced protein was puri¢ed by preparative SDS-PAGE. Rabbits were injected with this protein preparation and an antiserum was obtained. In immunoblotting studies, this anti-(h-gp96) recognizes protein bands of 90- and 180-kDa in human liver (Fig. 5a). A protein band of 110 kDa was not
found. However, in porcine liver, this is a minor band that is usually hardly visible in immunoblots of crude material [6]. Therefore, it is probably below detection level in the human liver. It might also be degraded, since the liver used in this experiment came from autopsy. In immunohistochemistry studies, using human liver biopsies, the newly developed antiserum showed a localization pattern along the lining of the sinusoids as found before in porcine liver [5,6]; the localization within the hepatocellular plates was less obvious due to a rather high background (Fig. 5b). Occasionally, cells were found with a prominent intracellular £uorescence pattern, that might represent abundant ER localization (Fig. 5b, insert). To determine if the isolated cDNA clones encode an HDL-binding protein, both the human and porcine cDNAs were cloned in a vector for transient expression in COS-1 cells. Cells were analyzed at 2 or 3 days following transfection. In immunoblotting experiments, it was found that non-transfected COS-1 cells express gp96 while in COS-1 cells transfected with either porcine or human cDNA there is no signi¢cant increase in gp96-expression (not shown). Immunocytochemistry studies, however, that permit the inspection of individual cells, revealed that a very low number of cells did express the protein, and that this protein is located intracellularly, showing a distribution highly suggestive for ER localization (Fig. 6a). In order to circumvent this ER localization in transfected COS-1 cells, vectors were generated containing porcine gp96 cDNA with a deletion of the C-terminal KDEL sequence (p-gp96vKDEL ). COS-1 cells were transfected and analyzed after 2 or 3 days. Immunocytochemistry revealed, that only a very small minority ( 6 1%) of the cells expressed gp96. The protein was localized at the periphery of C
Fig. 3. Subcellular localization of the 90-kDa HDL-binding protein in porcine liver. Immunohistochemistry was performed as described in Section 2. (a) The 90-kDa protein is present in high amounts at the membrane of the bile canaliculus (BC) and at the membranes of the vesicles of the endosomal/lysosomal system (V). G, Golgi complex; M, mitochondrion. Magni¢cation: U36 120. Scale bar: 0.25 Wm. (b) Colocalization of the 90-kDa HDL-binding protein (6-nm gold particles; white arrowheads) and cathepsin D (10nm gold particles; black arrowheads) in an endosomal/lysosomal vesicle. Magni¢cation: U65 520. Scale bar: 0.1 Wm. (c) Only a few gold particles are present in the rough endoplasmic reticulum (R), indicating that the protein is expressed there in minor quantities (arrows). M, mitochondrion. Magni¢cation: U50 400. Scale bar: 0.25 Wm. (d) The 90-kDa HDL-binding protein is detected in a coated pit (arrow) in the basolateral plasma membrane of the hepatocyte (H), facing the space of Disse (SD). EC, endothelial cell. Magni¢cation: U70 560. Scale bar: 0.1 Wm.
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the cell, indicating plasma membrane localization (Fig. 6b). Thus the plasma membrane localization found in liver, can be simulated in cultured cells. Several attempts to increase the percentage of expressing cells by using other or modi¢ed transfection protocols proved to be unsuccessful.
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Because of the di¤culties in obtaining high level expression of transiently transfected COS cells, we decided to generate stably transfected cell lines. We tested COS-1, HepG2, CHO-9 and L-cells using different constructs (see Section 2). Although fairly large numbers of G418-resistant clones were isolated,
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Fig. 4. Subcellular localization of the 180-kDa HDL-binding protein in porcine liver. Immunohistochemistry was performed as described in Section 2. The 180-kDa protein is found at the plasma membrane of endothelial cells (EC), Kup¡er cells (K) and hepatocytes (H). SD, space of Disse. Magni¢cation: a, U43 680; b, U37 800. Scale bar: 0.25 Wm.
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the level of protein expression appeared to be highly variable and unstable. Immunohistochemistry studies revealed that each of the clones initially expressed the protein, but the percentage of expressing cells varied considerably, from þ 5% to þ 50%. However, when later passages of the cells in culture were rescreened, percentages of expressing cells had dropped to 6 5% in all cases, indicating that there is no stable expression. Probably, the non-expressing cells quickly overgrow the expressing cells. Expression of gp96 possibly interferes with cellular proliferation. This would result in a selection of cells that do not express, or somehow switch o¡ the expression of the protein. Therefore, we decided to use MEL cells. These cells can be transfected with a plasmid containing the neomycin resistance gene and the DNA of interest under the control of a minimal promoter and the L-globin locus control region (LCR). This DNA will not be expressed in undi¡erentiated cells, but will be expressed when the MEL cells are induced by agents like dimethylsulfoxide to undergo erythroid di¡erentiation, that will activate the L-globin gene [17]. This allows the selection of cells that have taken up the transfected DNA under conditions in which the protein of interest is not expressed. In this way, an eventual selective e¡ect of the disadvantageous in£uence of gp96 expression is avoided. We cloned either porcine or human gp96 cDNA in this plasmid (pEV3-[h/p]-gp96; Fig. 7a), that contains L-globin sequences as well [17]. MEL cells were transfected with pEV3 (four independent populations, C1^C4), pEV3-p-gp96 (eight independent populations, P1^P8), pEV3-h-gp96 (eight independent populations, H1^H8) by electroporation and grown in the presence of G418. Cells were induced by dimethylsulfoxide and analyzed. Southern blotting showed that each of the cell populations P1^ P8 and H1^H8 had incorporated gp96 cDNA (Fig. 7b). Each of the cell populations expressed gp96 protein, as demonstrated by immunoblotting (Fig. 7c). HDL-binding was studied to C4, P3 and H5 (Fig. 8). Binding to the h-gp96 expressing cells appeared to be considerably higher than to control cells, while p-gp96 expressing cells bind equal amounts of HDL as control cells. In subsequent experiments, it became clear, that also in MEL cells, prolonged expression of gp96 was only observed in exceptional
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Fig. 5. Immunoblot and immunohistochemistry of human liver. (a) Immunoblot of anti-(h-gp96) with a protein homogenate of human liver. Protein bands of 90 and 180 kDa are indicated by arrows. (b) Immunohistochemistry of human liver. Semi-thin cryosections of human liver were incubated with anti-(h-gp96) and £uorescein isothiocyanate-conjugated goat anti-(rabbit IgG) as a secondary antibody as described in Section 2. Expression of gp96 was detected at the borders of the hepatocellular plates (H) and sinusoids (S) (arrows) and within the hepatocellular plates (arrowheads). Next to the (green) £uorescence of the label that was used, (yellow) auto£uorescence of fat droplets is visible (small arrows). Inset: Occasionally, hepatocytes were found with a strong intracellular £uorescence, probably representing localization in the ER.
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Fig. 6. Immunocytochemistry of transiently transfected COS cells. COS cells were transiently transfected with p-gp96 (a) or p-gp96vKDEL (b). Transfection of COS cells and immunocytochemistry were performed as described in Section 2. The observed localization pattern in a is indicative for expression in the ER. The results in b were examined by the use of confocal laser microscopy, and clearly demonstrate localization at the cell border, presumably at the plasma membrane.
cases. Only these allow the detection of increased binding of 125 I-HDL. 4. Discussion In this paper, we identify the HDL-binding proteins detected in porcine liver [5,6] as the porcine homolog of gp96. This protein is known from several species and has been given various names: mouse ERp99 [7,21] and endoplasmin [22], human GRP94 [23], tra1 and gp96 [24,25], and chicken hsp108 [26]. Gp96 has 50% identity with the cytosolic hsp90 proteins [24] and is therefore included in the stress-90 protein family [27]. It is quite an exceptional member of this family, though, since it contains an N-terminal signal sequence for ER translocation and the Cterminal KDEL sequence for ER retrieval [9]. The protein is induced by glucose starvation and by dis-
ruption of the intracellular calcium stores, but not by heat [28,29]. Its function is unknown [27,30], although several groups suggested that the protein is a chaperonin like GRP78 (BiP). This hypothesis is based on data showing that both GRP78 and gp96 expression is induced by the presence of malfolded proteins in the ER [31] and that both proteins associate with unassembled immunoglobulin chains [29,32]. Besides, the promoters of both GRP78 and gp96 have several regulatory elements in common and the genes have been shown to be coordinately expressed [28,33,34]. Others have implicated gp96 in antigen presentation [30,35,36]. Dechert et al. [37] isolated porcine gp96 from brain microvessels and showed that the protein has protein kinase activity. Its function is unknown. The identi¢cation of an HDL-binding protein as an ER localized protein is unexpected. However, several lines of evidence indicate that expression of gp96
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Fig. 7. Transfection of MEL-cells. (a) MEL-cells were transfected with the vector pEV3 in which human or porcine gp96 cDNA was cloned. The L-globin sequence represents exon II and III from human L-globin and intronic sequences, and is derived from the vector pEC3 [17]. (b) DNA was isolated from G418-resistant MEL-cell clones, digested with EcoRI and subjected to Southern blotting. N, non-transfected MEL cells; C1^C4, populations transfected with pEV3; P1^P8, populations transfected with pEV3-p-gp96; H1^H8, populations transfected with pEV3-h-gp96. Blots were hybridized with full-length h-gp96 cDNA. A band representing endogenous gp96 in MEL cells (m-gp96) is visible in every lane. (c) Protein was isolated from the same MEL cell clones and immunoblotting was performed. An antiserum directed against human p96 was used, which cross-reacts with porcine gp96.
is not restricted to the ER, but is also present in the plasma membrane, where it might exert a di¡erent function. Gp96 could be immunoprecipitated from cell surface radiolabeled cells and puri¢ed from isolated plasma membranes [24,38]. In rat pancreas, gp96 was shown to be present both in ER and plasma membranes by immunoblotting and electron-microscopical immunohistochemistry [39]. Hayes et al. [40] showed that gp96 puri¢ed from rat liver or chicken liver or oviduct does not react with an anti-KDEL antibody, suggesting that the KDEL sequence has been removed or modi¢ed. Several KDEL-containing proteins have been described to escape ER retention [41], including rat growth hormone, the K-subunit of chorionic gonadotropin [42]
and protein disul¢de-isomerase [39]. In the case of at least one protein, the plant cysteine endopeptidase SH-EP, the KDEL sequence is removed during processing of the mature protein [43]. It has been suggested that gp96 is transported to the cell surface as part of a larger molecular complex [44]. Our data show that gp96 is indeed localized at the cell surface in porcine liver [5,6] and in human liver (Fig. 5b). In electron-microscopical localization studies, we describe the ultrastructural localization of gp96 in porcine liver, which is con¢ned to the plasma membrane and the endosomal/lysosomal compartment, while the protein was hardly detectable in the ER (Figs. 3 and 4). In light microscopic immunohistochemistry studies in human liver sections,
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Fig. 8. Cell association of 125 I-HDL to transfected MEL cell populations. MEL cells were incubated in medium containing 10% lipoprotein de¢cient serum (LPDS) at 37³C for 2 h. Cells were washed with PBS and speci¢c cell association was determined as described in Section 2. Values are means of triplicate measurements. W, MEL cells transfected with pEV3 (C4); a, MEL cells transfected with pEV3-p-gp96 (P3); v, MEL cells transfected with pEV3-h-gp96 (H3).
patterns that might represent ER localization were observed in some cells (Fig. 5b, insert). The distinct localization of monomeric versus dimeric gp96 in porcine liver is intriguing. Although it is known that gp96 occurs both as a monomer and as a dimer [8], nothing is known about functional di¡erences between the two forms. The dual and possibly tissue-dependent localization pattern of gp96 suggest multi-functionality. Recently, a number of proteins have been assumed to be multi-functional, including proteins involved in lipoprotein metabolism, like the low density lipoprotein (LDL) receptor related protein (LRP) [45]. Ligand blotting studies suggest a role of gp96 in HDL metabolism by the liver [5]. The presence of the dimeric protein at the plasma membrane of the hepatocytes in the space of Disse and at the plasma membrane of endothelial cells and Kup¡er cells possibly facilitate the interaction of hepatic lipase with HDL, uptake of HDL particles, or selective uptake of HDL-cholesterol esters, via an anchoring or docking mechanism, as has been suggested for another putative hepatic HDL receptor, SR-BI [4]. It is also possible that the dimeric protein represents excreted
gp96 since Takemoto et al. [39] showed that gp96 in pancreas cells is both plasma membrane bound and secreted. The protein found associated with endothelial cells and Kup¡er cells could be hepatocyte-derived and, in analogy with the situation of hepatic lipase [46], bound to glycosaminoglycans, since we found that gp96 strongly associates with heparin (unpublished results). On the other hand, the ¢nding of 90-kDa protein in coated pits in hepatocytes strongly suggest the involvement of receptor-mediated endocytosis, a process that has been described for a number of receptors, including the low density lipoprotein receptor [47]. Conformation of these speculations awaits further research. The monomeric protein found in the endosomal/ lysosomal system and bile canaliculi could be a mere degenerative of the (functional) dimeric protein, without any function in HDL metabolism. On the other hand, it is possible that the monomeric protein guides HDL along its intracellular route. The localization in the bile canaliculi is in agreement with this, since HDL/apolipoprotein (apo) A-I can be found in bile canaliculi and apoA-I has been suggested to inhibit cholesterol crystal formation [48]. Future experiments, including analysis of HDL-binding and uptake by hepatocytes in culture, are needed to evaluate the intracellular fate of HDL and apoA-I and the involvement of HDL-binding proteins in these processes. Expression of a gp96vKDEL construct in COS-1 cells gave rise to exclusively plasma membrane localized expression, albeit in a very low percentage of cells. This result shows that the plasma membrane localization of gp96 that was found in liver cells, can be simulated in this system. This indicates that cells have the ability to express the gp96 protein at the plasma membrane. Probably, plasma membrane localization can only be found in cells with the necessary regulatory processes. The nature of the plasma membrane association is unknown. This membrane association is a strong one, however, since the protein has been puri¢ed by detergent treatments of an isolated plasma membrane enriched cellular fraction [5]. Considerable e¡ort was put into the isolation of cells that stably express transfected gp96, in order to evaluate their 125 I-HDL-binding activity. However, it proved to be practically impossible to obtain stably
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expressing cells. This probably indicates that constitutive and/or elevated expression of gp96 is detrimental for cell growth. In rare cases, however, gp96 expression could be induced in MEL cells for a longer period, that allowed the comparison of HDL-binding activity with non-expressing cells. This binding appeared to be considerably higher, strongly supporting the suggested function of gp96 as an HDL-binding protein. Acton et al. [4,49] identi¢ed SR-BI as an HDLbinding protein in the liver. SR-BI is probably also a multifunctional protein, since it can bind a number of other ligands, including native or chemically modi¢ed LDL and anionic phospholipids [50,51]. Mice de¢cient in SR-BI have been generated by gene targeting [52,53]. They have elevated levels of HDL in plasma, demonstrating an important role of SR-BI in HDL metabolism. The selective uptake of HDL-cholesterol by the liver is decreased in these mice, but is not completely absent. Uptake of HDL-protein is unchanged. Therefore, these ¢ndings do not exclude the presence of other HDL-binding proteins in the liver. There could be a redundancy of HDL-binding proteins in the liver. Redundancy has been shown to exist for several proteins, including proteins involved in lipid metabolism: apolipoprotein (apo) E mediated uptake of lipoproteins by the liver can occur via the apoB/E receptor (LDL receptor) or by a speci¢c apoE receptor, which is possibly LRP [45]. It has been suggested that for vital functions, such as cholesterol metabolism, redundancy of functional proteins is predictable [54]. Other HDL-binding proteins in the liver that have been cloned include HBP [55] and HB2 [56]. Both proteins are unrelated to gp96. HBP has been shown to be identical to vigilin, which is believed to be cytoplasmic protein [57,58]. Rat HB2 is very homologous to the human protein ALCAM, a member of the immunoglobulin superfamily [56]. The protein is probably involved in cell adhesion. HB2 might therefore be a multifunctional protein. The quantitative contribution to various aspects of HDL-metabolism by HDL-binding proteins in liver that have been reported has to be determined. Genetically modi¢ed mice will be a very helpful tool in this process.
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Acknowledgements The authors would like to thank Mirko Kuit for photographic artwork, Marian Kroos and Dr. Arnold Reuser for protein sequencing, Dr. Adriaan Houtsmuller for confocal laser microscopy and Ton Verkerk for assistance with computer analyses. R.J. was supported by the Netherlands Heart Foundation (Grant 92.131).
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[42] P. Zagouras, J.K. Rose, J. Cell Biol. 109 (1989) 2633^ 2640. [43] T. Okamoto, H. Nakayama, K. Seta, T. Isobe, T. Minamikawa, FEBS Lett. 351 (1994) 31^34. [44] A. Altmeyer, R.G. Maki, A.M. Feldweg, M. Heike, V.P. Protopopov, S.K. Masur, P.K. Srivastava, Int. J. Cancer (Pred. Oncol.) 69 (1996) 340^349. [45] M. Krieger, J. Herz, Annu. Rev. Biochem. 63 (1994) 601^ 637. [46] A. Bensadoun, D.E. Berryman, Curr. Opin. Lipidol. 7 (1996) 77^81. [47] M.S. Brown, J.L. Goldstein, Science 232 (1986) 34^47. [48] A. Kibe, R.T. Holzbach, N.F. LaRusso, S.J. Mao, Science 225 (1984) 514^516. [49] S. Acton, A. Rigotti, K.T. Landschulz, S. Xu, H.H. Hobbs, M. Krieger, Science 271 (1996) 518^520. [50] S. Acton, P.E. Scherer, H.F. Lodish, M. Krieger, J. Biol. Chem. 269 (1994) 21003^21009. [51] A. Rigotti, S. Acton, M. Krieger, J. Biol. Chem. 270 (1995) 16221^16224. [52] A. Rigotti, B.L. Trigatti, M. Penman, H. Rayburn, J. Herz, M. Krieger, Proc. Natl. Acad. Sci. USA 94 (1997) 12610^ 12615. [53] M.L. Varban, F. Rinninger, N. Wang, V. Fairchild-Huntress, J.H. Dunmore, Q. Fang, M.L. Gosselin, K.L. Dixon, J.D. Deeds, S.L. Acton, A.R. Tall, D. Huszar, Proc. Natl. Acad. Sci. USA 95 (1998) 4619^4624. [54] P.J. Barter, K.A. Rye, Curr. Opin. Lipidol. 7 (1996) 82^ 87. [55] G.L. McKnight, J. Reasoner, T. Gilbert, K.O. Sundquist, B. Hokland, P.A. McKernan, J. Champagne, C.J. Johnson, M.C. Bailey, R. Holly, P.J. O'Hara, J.F. Oram, J. Biol. Chem. 267 (1992) 12131^12141. [56] A. Matsumoto, A. Mitchell, H. Kurata, L. Pyle, K. Kondo, H. Itakura, N. Fidge, J. Biol. Chem. 272 (1997) 16778^ 16782. [57] G. Neu-Yilik, H. Zorbas, T.R. Gloe, H.M. Raabe, T.A. Hopp-Christensen, P.K. Muller, Eur. J. Biochem. 213 (1993) 727^736. [58] G. Plenz, S. Kugler, S. Schnittger, H. Rieder, C. Fonatsch, P.K. Muller, Hum. Genet. 93 (1994) 575^582.
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Gp96/GRP94 is a putative high density lipoprotein-binding protein in liver Rini de Crom *, Rien van Haperen, Rick Janssens, Pim Visser, Rob Willemsen, Frank Grosveld, Arthur van der Kamp The Medical Genetics Center, Department of Cell Biology and Genetics, Erasmus University Rotterdam, Dr. Molenwaterplein 50, P.O. Box 1738, 3000 DR Rotterdam, The Netherlands Received 9 November 1998; received in revised form 18 January 1999; accepted 22 January 1999
Abstract We have previously shown that three high density lipoproteins (HDL)-binding proteins in liver, of 90, 110 and 180 kDa, are structurally related. In this study, these proteins are identified as gp96/GRP94. This protein is known to occur as a homodimer and has a dual subcellular localization : it is both an endoplasmic reticulum resident protein, where it is supposed to act as a chaperonin, and a plasma membrane protein, whose significance is unknown. In ultrastructural studies the plasma membrane localization of the homodimeric form was verified. The 90-kDa protein was abundantly present at the membranes of the endosomal/lysosomal vesicles as well as at the apical hepatocyte membranes, comprising the bile canaliculi. The monomeric protein is scarcely present at the basolateral membrane of the hepatocytes, but could be demonstrated in coated pits, suggesting involvement in receptor-mediated endocytosis. Labeling of the endoplasmic reticulum was virtually absent. Gp96/GRP94 was transiently expressed in COS-1 cells. However, the expressed protein was exclusively localized in the endoplasmic reticulum. Transfection with constructs in which the C-terminal KDEL sequence had been deleted, resulted in plasma membrane localized expression of protein, but only in an extremely low percentage of cells. In order to evaluate the HDL-binding capacities of this protein, stably transfected cells were generated, using several cell types. It appeared to be difficult to obtain a prolonged high level expression of gp96. In these cases, however, a marked increase of HDL-binding activity compared with the control cells could be observed. ß 1999 Elsevier Science B.V. All rights reserved. Keywords: High density lipoprotein; HDL-binding protein ; Ultrastructural localization; Bile canaliculus; KDEL; Murine erythroleukemia cell
1. Introduction
Abbreviations: apo, apolipoprotein; CHO, Chinese hamster ovary; HDL, high density lipoprotein; LDL, low density lipoprotein ; LRP, LDL receptor-related protein; PAGE, polyacrylamide gel electrophoresis ; SDS, sodium dodecylsulfate; SR-BI, scavenger receptor class B, type I * Corresponding author. Fax: +31 (10) 408-9468; E-mail: [email protected]
Plasma levels of HDL are among the best indicators of the risk of coronary artery disease [1,2]. Although this notion has led to intensive research on HDL metabolism, many aspects are still under debate [1,3]. One of these aspects is the role of HDL-binding proteins in the liver [4]. We identi¢ed HDL-binding proteins of 90, 110 and 180 kDa in porcine liver by ligand blotting (mo-
1388-1981 / 99 / $ ^ see front matter ß 1999 Elsevier Science B.V. All rights reserved. PII: S 1 3 8 8 - 1 9 8 1 ( 9 9 ) 0 0 0 1 7 - 7
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lecular masses estimated by sodium dodecylsulfatepolyacrylamide gel electrophoresis (SDS-PAGE) under non-reducing conditions) [5]. In a later study, we showed that these three proteins are structurally related [6]. The principal arguments for this conclusion are: a polyclonal antibody raised against the puri¢ed 90-kDa HDL-binding protein shows crossreactivity with the 110- and 180-kDa proteins on an immunoblot; under reducing conditions, puri¢ed protein preparations of 90, 110 and 180 kDa all migrate at the 90-kDa position in SDS-PAGE, and still show immunoreactivity with the antiserum as well as HDL-binding activity in a ligand blot assay; CNBrfragment maps of the three proteins are highly similar. We concluded that the 180-kDa HDL-binding protein is a homodimer of the 90-kDa HDL-binding protein [6]. In this paper, we show that the 90-, 110- and 180kDa proteins have the same N-terminal amino acid sequence. Amino acid sequences of several CNBr fragments of the 90-kDa protein show an almost 100% identity with a protein known under various names, like mouse endoplasmin or ERp99, and human tra1, gp96 or GRP94 [7]. This protein is known to occur both in a monomeric and in a dimeric conformation [8]. Gp96 has the endoplasmic reticulum (ER) retention signal KDEL [9]. Therefore, we performed both light and electron-microscopical localization studies. They show that at least part of the protein is localized at the plasma membrane of liver cells. cDNA clones, encoding both the porcine and human homolog of gp96 were isolated and tested in expression studies using various transfected cell lines. 2. Materials and methods
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applied to a 473A Applied Biosystems protein sequencer for N-terminal amino acid sequence determination. 2.2. Synthesis of a cDNA probe Oligonucleotides, based on the published sequence of mouse ERp99 [7], were synthesized using an Applied Biosystems oligonucleotide synthesizer. From RNA isolated from murine liver, ¢rst-strand cDNA was synthesized with reverse transcriptase (RT). This was used as a template for polymerase chain reaction (PCR), performed in a Perkin Elmer Cetus PCR apparatus. Mouse cDNA fragments were cloned into the EcoRI site of pTZ19 via the restriction sites incorporated in the synthetic oligonucleotide primers of the PCR reaction. 2.3. Screening of cDNA libraries Vgt11 cDNA libraries from porcine liver (CloneTech PL1001b) and human liver (CloneTech HL1115b) were screened with cDNA probes labeled with [K-32 P]ATP. V clones with the largest inserts were subcloned into pTZ19. Partial cDNA clones were digested with restriction enzymes and ligated in order to obtain full length cDNA inserts by standard techniques [11]. 2.4. DNA sequencing The DNA sequence was determined on both strands by the dideoxy-nucleotide chain-termination method (T7 Sequencing kit, Pharmacia), using a series of synthetic oligonucleotides. 2.5. Computer analyses
2.1. N-Terminal sequencing Puri¢cation of HDL-binding proteins and CNBr cleavage of puri¢ed protein preparations were performed as described before [6]. The protein preparations or CNBr fragments were subjected to SDSPAGE and electrophoretically transferred to ProBlott membranes (Applied Biosystems) [10]. These membranes were stained with Coomassie brilliant blue and protein bands of interest were carefully cut out. These pieces of membrane were directly
Comparisons for homology were performed by FASTA using the data bases of GenBank/EMBL, and Swiss-Prot. 2.6. Electron-microscopy studies Porcine liver specimens were immediately ¢xed in 3% paraformaldehyde and prepared for ultracryotomy as described previously [12]. Ultrathin cryosections (60 nm) were immunolabeled with either rabbit
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polyclonal antibodies against the 90-kDa protein or against the 180-kDa protein [5]. Antibodies directed against cathepsin D were kindly provided by Dr. H. Aerts, University of Amsterdam. Antigen^antibody complexes were visualized with goat anti-(rabbit Ig) conjugated with 10 nm colloidal gold particles. To localize two antigens simultaneously, the ¢rst antibody was labeled with a 6-nm protein A-gold probe and the second with a 10-nm probe, according to Geuze et al. [13]. Sections were stained with uranyl salts and embedded in 1.5% methylcellulose and examined with a Philips CM100. The speci¢city of the labeling procedure was checked by incubation of the sections with normal rabbit serum as substitution for the primary antibody, or with the di¡erent gold probes alone. Background labeling was negligible.
porcine and human cDNA (PvuII site at positions 2395 and 2497, respectively; BamHI site at positions 2434 and 2536, respectively). This results in a K801 CSTOP mutation, which completely eliminates the KDEL sequence. COS-1 cells were grown in 98mm-diameter dishes and transfected with 10 Wg of plasmid DNA by the DEAE-dextran method [11]. The transfected cells were analyzed at 48^72 h post-transfection. For immunocytochemistry experiments, cells were grown and transfected in 24-well plates on 15-mm-diameter coverslips. Alternative transfection protocols that were tested in order to achieve a higher percentage of expressing cells used Lipofectin (Gibco BRL), Lipofectamin (Gibco BRL), or Transfectam (Promega), all applied according to the manufacturers instructions.
2.7. Protein expression in Escherichia coli and generation of antisera
2.9. Transfection experiments
cDNA sequences were subcloned into the vector pGEX (Pharmacia) and transfected to BL21 E. coli cells by standard techniques [11]. Induction of protein expression and isolation of bacterially expressed protein was performed according to Smith and Corcoran [14]. Bacterially expressed protein was puri¢ed by preparative SDS-PAGE using a Bio-Rad Prep Cell. Polyclonal antisera were raised in rabbits as described previously [6]. All animals were cared for in compliance with the Guidelines of the Ethical Committee on the Use of Laboratory Animals of the Erasmus University Rotterdam (prot. no. 132.94.01) and with the European Committee Standards on the care and use of laboratory animals (Ministry of Welfare, Health and Cultural A¡airs, The Netherlands). 2.8. Transient protein expression in COS-1 cells Porcine or human cDNA, either full length (gp96) or carrying a mutation that gives rise to a protein that lacks the C-terminal KDEL tetrapeptide (gp96vKDEL ), was inserted into the eukaryotic expression vector pCD-X [15]. vKDEL cDNA was constructed by inserting a linker oligonucleotide (5PCTGAATAAGATGAACTGTAAATTATACTCTCACCATTTG-3P) by ligation between the PvuII and BamHI restriction sites that are present both in
COS-1 cells, Hep G2 cells, CHO-9 cells, L cells and murine erythroleukemia (MEL) cells were cultured in DMEM/F10 (1:1) supplemented with 2 mM L-glutamine, 100 IU penicillin/ml, 100 mg streptomycin/ml and 10% FCS at 37³C in a 5% CO2 atmosphere. All cell types were transfected with Lipofectin (Promega) according to the manufacturer's instructions, except for MEL cells, that were transfected by electroporation using a Bio-Rad Gene Pulser (settings: 117 V, 1200 mF, 10 ms). The next day following transfection, cells were selected on culture medium containing 800 mg/ml G418. MEL cells were induced by adding 2% dimethylsulfoxide to the medium 4 days after transfection and were harvested for analysis after 24 h [16]. For cotransfection to COS, CHO and HepG2 cells, 1 mg of pCD-X-neo was used, containing a neomycin resistance gene into a derivative of the mammalian expression vector pCD-X [15] and 10 mg of either pCD-X-p-gp96 or pCD-X-h-gp96 containing the porcine or human gp96 cDNA, respectively. L cells were transfected with 10 mg of a mammalian expression vector pJG1 containing the CMV promoter and the TK-neo gene (gift of Jacky Guy, Rotterdam), and the porcine or human gp96 cDNA cloned into the polylinker (pJG1-p-gp96 and pJG1h-gp96, respectively). MEL cells were transfected with a vector named
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pEV3, which is a derivative of pEC3 [17], containing the L-globin locus control region and parts of the Lglobin gene for mRNA stability, into which porcine or human gp96 cDNA was cloned (pEV3-p-gp96 and pEV3-h-gp96, respectively). 2.10. DNA and RNA analysis DNA and RNA isolation from cells, as well as Southern and Northern blotting were performed using standard techniques [11]. Blots were hybridized with radioactively labeled probes as indicated in the ¢gure legends. Hybridizing bands were visualized by autoradiography or using a Phosphor Imager (Molecular Dynamics). 2.11. HDL-binding to MEL cells Isolation and radiolabeling of human HDL with I was performed exactly as described before [5]. Two hundred microliters of MEL cell suspension in medium containing 10% lipoprotein-de¢cient serum (LPDS) was incubated with the indicated amounts of 125 I-HDL at 37³C for 2 h. Cells were washed four times in PBS containing 5% LPDS-FCS, two times in PBS and resuspended in 0.5 ml PBS. Associated radioactivity was determined in a gamma counter. Then cell protein was dissolved by adding NaOH to a ¢nal concentration of 0.1 M and determined according to Lowry [18]. Aspeci¢c cell association was measured in the presence of a 25-fold excess of unlabeled HDL. Speci¢c cell association was calculated by subtracting the aspeci¢c cell association from the total cell association. All measurements were performed in triplicate. 125
2.12. Immunoblotting, immunocytochemistry and immunohistochemistry Immunoblotting was performed exactly as described before [6]. Immunocytochemistry was performed on cells grown on coverslips as described by Van Dongen et al. [19]. For immunohistochemistry, porcine liver was ¢xed and prepared for ultracryotomy as described before [20]. Semithin cryosections (0.5^1.0 Wm) were incubated with a Protein A Sepharose CL-4B (Pharmacia) puri¢ed IgG fraction of polyclonal antibodies
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against HDL-binding proteins for 1 h at room temperature in a humid incubator. Antigen^antibody complexes were visualized with £uorescein isothiocyanate-conjugated goat anti-rabbit immunoglobulins (Nordic, Tilburg, The Netherlands) (diluted 1:80; incubated at room temperature for 1 h in a humid incubator) and studied by £uorescence microscopy. 3. Results The N-terminal amino acid sequence of puri¢ed 90-, 110- and 180-kDa HDL-binding proteins and of four di¡erent CNBr fragments from the 90-kDa protein were determined. As shown in Fig. 1, the 90-, 110- and 180-kDa HDL-binding proteins have identical N-termini. The variation at the 9th amino acid is due to occasional di¤culties in the interpretation of the output of the sequencer at that particular position. The 32-kDa CNBr fragment has the same Nterminus, indicating that this is the N-terminal fragment of the protein. Data-base searches revealed that all the determined amino acid sequences have a very high homology with three known proteins from different species: mouse ERp99, human gp96 and chicken hsp108. A cDNA probe was obtained by RT-PCR and used to screen a porcine liver Vgt11 cDNA library. In several screenings, only clones with partial cDNA inserts were found. Therefore, a cDNA clone containing the full-length cDNA was assembled from two individual clones. From this clone, the complete cDNA sequence of porcine gp96 (p-gp96) was determined (Fig. 2). The deduced protein sequence contains both an N-terminal signal sequence, which is no longer present in the mature protein (Fig. 1), and a C-terminal KDEL sequence, which in many proteins serves as an endoplasmic reticulum (ER) retention signal. The 21^23 amino acids beginning at Leu-191 that possibly comprise a membrane spanning region [7] are completely conserved in the porcine sequence (Fig. 2). The identi¢cation of an HDL-binding protein as a KDEL-containing protein prompted us to perform electron-microscopical localization studies. The ultrastructural localization of the 90-kDa protein was studied by indirect immunogold labeling of
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Fig. 1. N-Terminal amino acid sequences. N-Terminal amino acid sequences of puri¢ed porcine protein preparations of the 90-, 110and 180-kDa HDL-binding proteins and of 9-, 10-, 25- and 32-kDa CNBr fragments of the porcine 90-kDa protein are presented in bold. Uncertainties and variations within the porcine sequences are due to occasional di¤culties in the interpretations of the output of the sequencer. Comparisons with data-base sequences of mouse, human and chicken homologs are presented (accession numbers J02735, X15187, M14772, respectively).
electron-microscopic frozen sections of porcine liver using an antibody speci¢cally recognizing the 90-kDa protein [5]. Most label was found along the membranes of intracellular vesicles belonging to the endosomal/lysosomal compartment and along the membranes of the microvilli of the bile canaliculi (Fig. 3a). Using an antibody directed against cathepsin D, a lysosomal enzyme, it was con¢rmed in a double labeling procedure that the 90-kDa protein is co-localized with cathepsin D (Fig. 3b). Cathepsin D label is mainly found in the lumen of the vesicles, while the 90-kDa protein label is found preferentially localized at the limiting membranes of the vesicles, suggesting it is a membrane-associated protein. High amounts of label were found associated with the apical membrane of the hepatocytes, comprising the bile canaliculi.
Apart from these two sites, no intracellular label was observed in the hepatocytes. Special attention was paid to the ER, since gp96 is reported to be an ER protein. However, only very few gold particles could be detected in the ER (Fig. 3c). At the basolateral membrane of the hepatocytes, facing the space of Disse, the 90-kDa protein is present in very low quantities. However, labeling in coated pits is observed (Fig. 3d). The distribution of the 180-kDa protein was studied in a similar way as the localization of the 90-kDa protein, using an antiserum speci¢cally recognizing the 180-kDa protein [5]. The 180-kDa protein appeared to be localized primarily at the plasma membrane of both endothelial cells (Fig. 4a) and Kup¡er cells (Fig. 4b). Gold particles can be found at the sinusoidal side and at the side facing the space C
Fig. 2. Nucleotide and deduced amino acid sequences of porcine gp96 cDNA. The putative signal sequence is underlined. The putative transmembrane region, homologous to the putative transmembrane region in mouse Erp99 [7], is underlined as well. The cysteine residue that is involved in dimerization [42] is encircled. The sequence has been deposited at the EMBL database with the accession number Y09136.
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of Disse. Labeling is also present at the microvilli of the hepatocytes protruding in the space of Disse. Intracellular labeling of the hepatocytes was found only occasionally. No ER labeling was found (not shown). Using an antiserum recognizing both the 90- and 180-kDa proteins [6], a combination of both localization patterns was found (bile canalicular membranes, membranes of vesicles of the endosomal/lysosomal system, basolateral membranes of the hepatocytes and plasma membranes of endothelial cells and Kup¡er cells), while labeling of other intracellular compartments, including the ER, was virtually absent (not shown). We wanted to extend our analyses to the human protein (h-gp96). Since none of the antibodies we raised against the porcine proteins cross-react with sections of human liver in immunohistochemistry studies, we raised antibodies recognizing h-gp96. Therefore, we used a protein fragment expressed in E. coli transfected with human cDNA. With the porcine cDNA as a probe, we screened a human liver Vgt11 cDNA library in order to isolate a cDNA clone encoding the human cDNA. A clone containing the full-length cDNA was obtained by ligation of the inserts of two partial overlapping clones. The cDNA was completely sequenced; it perfectly matched the published sequence of human gp96 (GenBank accession number X15187; not shown). A cDNA fragment, comprising the C-terminal part of the protein (amino acids 677^801) was cloned into pGEX for expression in E. coli. Produced protein was puri¢ed by preparative SDS-PAGE. Rabbits were injected with this protein preparation and an antiserum was obtained. In immunoblotting studies, this anti-(h-gp96) recognizes protein bands of 90- and 180-kDa in human liver (Fig. 5a). A protein band of 110 kDa was not
found. However, in porcine liver, this is a minor band that is usually hardly visible in immunoblots of crude material [6]. Therefore, it is probably below detection level in the human liver. It might also be degraded, since the liver used in this experiment came from autopsy. In immunohistochemistry studies, using human liver biopsies, the newly developed antiserum showed a localization pattern along the lining of the sinusoids as found before in porcine liver [5,6]; the localization within the hepatocellular plates was less obvious due to a rather high background (Fig. 5b). Occasionally, cells were found with a prominent intracellular £uorescence pattern, that might represent abundant ER localization (Fig. 5b, insert). To determine if the isolated cDNA clones encode an HDL-binding protein, both the human and porcine cDNAs were cloned in a vector for transient expression in COS-1 cells. Cells were analyzed at 2 or 3 days following transfection. In immunoblotting experiments, it was found that non-transfected COS-1 cells express gp96 while in COS-1 cells transfected with either porcine or human cDNA there is no signi¢cant increase in gp96-expression (not shown). Immunocytochemistry studies, however, that permit the inspection of individual cells, revealed that a very low number of cells did express the protein, and that this protein is located intracellularly, showing a distribution highly suggestive for ER localization (Fig. 6a). In order to circumvent this ER localization in transfected COS-1 cells, vectors were generated containing porcine gp96 cDNA with a deletion of the C-terminal KDEL sequence (p-gp96vKDEL ). COS-1 cells were transfected and analyzed after 2 or 3 days. Immunocytochemistry revealed, that only a very small minority ( 6 1%) of the cells expressed gp96. The protein was localized at the periphery of C
Fig. 3. Subcellular localization of the 90-kDa HDL-binding protein in porcine liver. Immunohistochemistry was performed as described in Section 2. (a) The 90-kDa protein is present in high amounts at the membrane of the bile canaliculus (BC) and at the membranes of the vesicles of the endosomal/lysosomal system (V). G, Golgi complex; M, mitochondrion. Magni¢cation: U36 120. Scale bar: 0.25 Wm. (b) Colocalization of the 90-kDa HDL-binding protein (6-nm gold particles; white arrowheads) and cathepsin D (10nm gold particles; black arrowheads) in an endosomal/lysosomal vesicle. Magni¢cation: U65 520. Scale bar: 0.1 Wm. (c) Only a few gold particles are present in the rough endoplasmic reticulum (R), indicating that the protein is expressed there in minor quantities (arrows). M, mitochondrion. Magni¢cation: U50 400. Scale bar: 0.25 Wm. (d) The 90-kDa HDL-binding protein is detected in a coated pit (arrow) in the basolateral plasma membrane of the hepatocyte (H), facing the space of Disse (SD). EC, endothelial cell. Magni¢cation: U70 560. Scale bar: 0.1 Wm.
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the cell, indicating plasma membrane localization (Fig. 6b). Thus the plasma membrane localization found in liver, can be simulated in cultured cells. Several attempts to increase the percentage of expressing cells by using other or modi¢ed transfection protocols proved to be unsuccessful.
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Because of the di¤culties in obtaining high level expression of transiently transfected COS cells, we decided to generate stably transfected cell lines. We tested COS-1, HepG2, CHO-9 and L-cells using different constructs (see Section 2). Although fairly large numbers of G418-resistant clones were isolated,
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Fig. 4. Subcellular localization of the 180-kDa HDL-binding protein in porcine liver. Immunohistochemistry was performed as described in Section 2. The 180-kDa protein is found at the plasma membrane of endothelial cells (EC), Kup¡er cells (K) and hepatocytes (H). SD, space of Disse. Magni¢cation: a, U43 680; b, U37 800. Scale bar: 0.25 Wm.
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the level of protein expression appeared to be highly variable and unstable. Immunohistochemistry studies revealed that each of the clones initially expressed the protein, but the percentage of expressing cells varied considerably, from þ 5% to þ 50%. However, when later passages of the cells in culture were rescreened, percentages of expressing cells had dropped to 6 5% in all cases, indicating that there is no stable expression. Probably, the non-expressing cells quickly overgrow the expressing cells. Expression of gp96 possibly interferes with cellular proliferation. This would result in a selection of cells that do not express, or somehow switch o¡ the expression of the protein. Therefore, we decided to use MEL cells. These cells can be transfected with a plasmid containing the neomycin resistance gene and the DNA of interest under the control of a minimal promoter and the L-globin locus control region (LCR). This DNA will not be expressed in undi¡erentiated cells, but will be expressed when the MEL cells are induced by agents like dimethylsulfoxide to undergo erythroid di¡erentiation, that will activate the L-globin gene [17]. This allows the selection of cells that have taken up the transfected DNA under conditions in which the protein of interest is not expressed. In this way, an eventual selective e¡ect of the disadvantageous in£uence of gp96 expression is avoided. We cloned either porcine or human gp96 cDNA in this plasmid (pEV3-[h/p]-gp96; Fig. 7a), that contains L-globin sequences as well [17]. MEL cells were transfected with pEV3 (four independent populations, C1^C4), pEV3-p-gp96 (eight independent populations, P1^P8), pEV3-h-gp96 (eight independent populations, H1^H8) by electroporation and grown in the presence of G418. Cells were induced by dimethylsulfoxide and analyzed. Southern blotting showed that each of the cell populations P1^ P8 and H1^H8 had incorporated gp96 cDNA (Fig. 7b). Each of the cell populations expressed gp96 protein, as demonstrated by immunoblotting (Fig. 7c). HDL-binding was studied to C4, P3 and H5 (Fig. 8). Binding to the h-gp96 expressing cells appeared to be considerably higher than to control cells, while p-gp96 expressing cells bind equal amounts of HDL as control cells. In subsequent experiments, it became clear, that also in MEL cells, prolonged expression of gp96 was only observed in exceptional
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Fig. 5. Immunoblot and immunohistochemistry of human liver. (a) Immunoblot of anti-(h-gp96) with a protein homogenate of human liver. Protein bands of 90 and 180 kDa are indicated by arrows. (b) Immunohistochemistry of human liver. Semi-thin cryosections of human liver were incubated with anti-(h-gp96) and £uorescein isothiocyanate-conjugated goat anti-(rabbit IgG) as a secondary antibody as described in Section 2. Expression of gp96 was detected at the borders of the hepatocellular plates (H) and sinusoids (S) (arrows) and within the hepatocellular plates (arrowheads). Next to the (green) £uorescence of the label that was used, (yellow) auto£uorescence of fat droplets is visible (small arrows). Inset: Occasionally, hepatocytes were found with a strong intracellular £uorescence, probably representing localization in the ER.
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Fig. 6. Immunocytochemistry of transiently transfected COS cells. COS cells were transiently transfected with p-gp96 (a) or p-gp96vKDEL (b). Transfection of COS cells and immunocytochemistry were performed as described in Section 2. The observed localization pattern in a is indicative for expression in the ER. The results in b were examined by the use of confocal laser microscopy, and clearly demonstrate localization at the cell border, presumably at the plasma membrane.
cases. Only these allow the detection of increased binding of 125 I-HDL. 4. Discussion In this paper, we identify the HDL-binding proteins detected in porcine liver [5,6] as the porcine homolog of gp96. This protein is known from several species and has been given various names: mouse ERp99 [7,21] and endoplasmin [22], human GRP94 [23], tra1 and gp96 [24,25], and chicken hsp108 [26]. Gp96 has 50% identity with the cytosolic hsp90 proteins [24] and is therefore included in the stress-90 protein family [27]. It is quite an exceptional member of this family, though, since it contains an N-terminal signal sequence for ER translocation and the Cterminal KDEL sequence for ER retrieval [9]. The protein is induced by glucose starvation and by dis-
ruption of the intracellular calcium stores, but not by heat [28,29]. Its function is unknown [27,30], although several groups suggested that the protein is a chaperonin like GRP78 (BiP). This hypothesis is based on data showing that both GRP78 and gp96 expression is induced by the presence of malfolded proteins in the ER [31] and that both proteins associate with unassembled immunoglobulin chains [29,32]. Besides, the promoters of both GRP78 and gp96 have several regulatory elements in common and the genes have been shown to be coordinately expressed [28,33,34]. Others have implicated gp96 in antigen presentation [30,35,36]. Dechert et al. [37] isolated porcine gp96 from brain microvessels and showed that the protein has protein kinase activity. Its function is unknown. The identi¢cation of an HDL-binding protein as an ER localized protein is unexpected. However, several lines of evidence indicate that expression of gp96
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Fig. 7. Transfection of MEL-cells. (a) MEL-cells were transfected with the vector pEV3 in which human or porcine gp96 cDNA was cloned. The L-globin sequence represents exon II and III from human L-globin and intronic sequences, and is derived from the vector pEC3 [17]. (b) DNA was isolated from G418-resistant MEL-cell clones, digested with EcoRI and subjected to Southern blotting. N, non-transfected MEL cells; C1^C4, populations transfected with pEV3; P1^P8, populations transfected with pEV3-p-gp96; H1^H8, populations transfected with pEV3-h-gp96. Blots were hybridized with full-length h-gp96 cDNA. A band representing endogenous gp96 in MEL cells (m-gp96) is visible in every lane. (c) Protein was isolated from the same MEL cell clones and immunoblotting was performed. An antiserum directed against human p96 was used, which cross-reacts with porcine gp96.
is not restricted to the ER, but is also present in the plasma membrane, where it might exert a di¡erent function. Gp96 could be immunoprecipitated from cell surface radiolabeled cells and puri¢ed from isolated plasma membranes [24,38]. In rat pancreas, gp96 was shown to be present both in ER and plasma membranes by immunoblotting and electron-microscopical immunohistochemistry [39]. Hayes et al. [40] showed that gp96 puri¢ed from rat liver or chicken liver or oviduct does not react with an anti-KDEL antibody, suggesting that the KDEL sequence has been removed or modi¢ed. Several KDEL-containing proteins have been described to escape ER retention [41], including rat growth hormone, the K-subunit of chorionic gonadotropin [42]
and protein disul¢de-isomerase [39]. In the case of at least one protein, the plant cysteine endopeptidase SH-EP, the KDEL sequence is removed during processing of the mature protein [43]. It has been suggested that gp96 is transported to the cell surface as part of a larger molecular complex [44]. Our data show that gp96 is indeed localized at the cell surface in porcine liver [5,6] and in human liver (Fig. 5b). In electron-microscopical localization studies, we describe the ultrastructural localization of gp96 in porcine liver, which is con¢ned to the plasma membrane and the endosomal/lysosomal compartment, while the protein was hardly detectable in the ER (Figs. 3 and 4). In light microscopic immunohistochemistry studies in human liver sections,
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Fig. 8. Cell association of 125 I-HDL to transfected MEL cell populations. MEL cells were incubated in medium containing 10% lipoprotein de¢cient serum (LPDS) at 37³C for 2 h. Cells were washed with PBS and speci¢c cell association was determined as described in Section 2. Values are means of triplicate measurements. W, MEL cells transfected with pEV3 (C4); a, MEL cells transfected with pEV3-p-gp96 (P3); v, MEL cells transfected with pEV3-h-gp96 (H3).
patterns that might represent ER localization were observed in some cells (Fig. 5b, insert). The distinct localization of monomeric versus dimeric gp96 in porcine liver is intriguing. Although it is known that gp96 occurs both as a monomer and as a dimer [8], nothing is known about functional di¡erences between the two forms. The dual and possibly tissue-dependent localization pattern of gp96 suggest multi-functionality. Recently, a number of proteins have been assumed to be multi-functional, including proteins involved in lipoprotein metabolism, like the low density lipoprotein (LDL) receptor related protein (LRP) [45]. Ligand blotting studies suggest a role of gp96 in HDL metabolism by the liver [5]. The presence of the dimeric protein at the plasma membrane of the hepatocytes in the space of Disse and at the plasma membrane of endothelial cells and Kup¡er cells possibly facilitate the interaction of hepatic lipase with HDL, uptake of HDL particles, or selective uptake of HDL-cholesterol esters, via an anchoring or docking mechanism, as has been suggested for another putative hepatic HDL receptor, SR-BI [4]. It is also possible that the dimeric protein represents excreted
gp96 since Takemoto et al. [39] showed that gp96 in pancreas cells is both plasma membrane bound and secreted. The protein found associated with endothelial cells and Kup¡er cells could be hepatocyte-derived and, in analogy with the situation of hepatic lipase [46], bound to glycosaminoglycans, since we found that gp96 strongly associates with heparin (unpublished results). On the other hand, the ¢nding of 90-kDa protein in coated pits in hepatocytes strongly suggest the involvement of receptor-mediated endocytosis, a process that has been described for a number of receptors, including the low density lipoprotein receptor [47]. Conformation of these speculations awaits further research. The monomeric protein found in the endosomal/ lysosomal system and bile canaliculi could be a mere degenerative of the (functional) dimeric protein, without any function in HDL metabolism. On the other hand, it is possible that the monomeric protein guides HDL along its intracellular route. The localization in the bile canaliculi is in agreement with this, since HDL/apolipoprotein (apo) A-I can be found in bile canaliculi and apoA-I has been suggested to inhibit cholesterol crystal formation [48]. Future experiments, including analysis of HDL-binding and uptake by hepatocytes in culture, are needed to evaluate the intracellular fate of HDL and apoA-I and the involvement of HDL-binding proteins in these processes. Expression of a gp96vKDEL construct in COS-1 cells gave rise to exclusively plasma membrane localized expression, albeit in a very low percentage of cells. This result shows that the plasma membrane localization of gp96 that was found in liver cells, can be simulated in this system. This indicates that cells have the ability to express the gp96 protein at the plasma membrane. Probably, plasma membrane localization can only be found in cells with the necessary regulatory processes. The nature of the plasma membrane association is unknown. This membrane association is a strong one, however, since the protein has been puri¢ed by detergent treatments of an isolated plasma membrane enriched cellular fraction [5]. Considerable e¡ort was put into the isolation of cells that stably express transfected gp96, in order to evaluate their 125 I-HDL-binding activity. However, it proved to be practically impossible to obtain stably
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expressing cells. This probably indicates that constitutive and/or elevated expression of gp96 is detrimental for cell growth. In rare cases, however, gp96 expression could be induced in MEL cells for a longer period, that allowed the comparison of HDL-binding activity with non-expressing cells. This binding appeared to be considerably higher, strongly supporting the suggested function of gp96 as an HDL-binding protein. Acton et al. [4,49] identi¢ed SR-BI as an HDLbinding protein in the liver. SR-BI is probably also a multifunctional protein, since it can bind a number of other ligands, including native or chemically modi¢ed LDL and anionic phospholipids [50,51]. Mice de¢cient in SR-BI have been generated by gene targeting [52,53]. They have elevated levels of HDL in plasma, demonstrating an important role of SR-BI in HDL metabolism. The selective uptake of HDL-cholesterol by the liver is decreased in these mice, but is not completely absent. Uptake of HDL-protein is unchanged. Therefore, these ¢ndings do not exclude the presence of other HDL-binding proteins in the liver. There could be a redundancy of HDL-binding proteins in the liver. Redundancy has been shown to exist for several proteins, including proteins involved in lipid metabolism: apolipoprotein (apo) E mediated uptake of lipoproteins by the liver can occur via the apoB/E receptor (LDL receptor) or by a speci¢c apoE receptor, which is possibly LRP [45]. It has been suggested that for vital functions, such as cholesterol metabolism, redundancy of functional proteins is predictable [54]. Other HDL-binding proteins in the liver that have been cloned include HBP [55] and HB2 [56]. Both proteins are unrelated to gp96. HBP has been shown to be identical to vigilin, which is believed to be cytoplasmic protein [57,58]. Rat HB2 is very homologous to the human protein ALCAM, a member of the immunoglobulin superfamily [56]. The protein is probably involved in cell adhesion. HB2 might therefore be a multifunctional protein. The quantitative contribution to various aspects of HDL-metabolism by HDL-binding proteins in liver that have been reported has to be determined. Genetically modi¢ed mice will be a very helpful tool in this process.
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Acknowledgements The authors would like to thank Mirko Kuit for photographic artwork, Marian Kroos and Dr. Arnold Reuser for protein sequencing, Dr. Adriaan Houtsmuller for confocal laser microscopy and Ton Verkerk for assistance with computer analyses. R.J. was supported by the Netherlands Heart Foundation (Grant 92.131).
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