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Biology of Reproduction 61, 1042-1049 (1999)
©Copyright 1999 Society for the Study of Reproduction, Inc.

Articles

Glycosyl Phosphatidylinositol-Anchored Ceruloplasmin Is Expressed by Rat Sertoli Cells and Is Concentrated in Detergent-Insoluble Membrane Fractions1

Ryan R. Fortna3,a, Hadiya A. Watson4,a, and Sally E. Nyquist2,a

a Department of Biology, Bucknell University, Lewisburg, Pennsylvania 17837


   ABSTRACT
TOP
 ABSTRACT
INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
The copper-binding protein, ceruloplasmin, is both a serum component and a secretory product of Sertoli cells. Studies on serum ceruloplasmin have demonstrated it to be a ferroxidase that is essential for iron transport throughout the body. We report here that a glycosyl phosphatidylinositol (GPI)-anchored form of ceruloplasmin is expressed by Sertoli cells. Sertoli cell GPI-anchored proteins were selectively released by phosphatidylinositol-specific phospholipase C and were analyzed by Western blotting. A 135-kDa band was identified as ceruloplasmin by multiple antibody recognition and by amino acid sequence analysis. The presence of the GPI anchor on ceruloplasmin was confirmed by Triton X-114 phase partitioning experiments and by recognition with an antibody to the GPI anchor. GPI-anchored ceruloplasmin was enriched in detergent-insoluble glycolipid-enriched membrane microdomains (DIGs) of Sertoli cells. This is the first report of GPI-anchored ceruloplasmin in Sertoli cells and the first study of GPI-anchored ceruloplasmin in DIGs. We suggest that GPI-anchored ceruloplasmin may be the dominant form expressed by Sertoli cells and that Sertoli cell DIGs may play a role in iron metabolism within the seminiferous tubule.


   INTRODUCTION
TOP
 ABSTRACT
 INTRODUCTION
MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
Low and Zilversmit [1] reported the first glycosyl phosphatidylinositol (GPI)-anchored protein, alkaline phosphatase, on intestinal epithelial cells. Since then more than 100 examples of GPI-anchored proteins have been reported in a broad range of cell types and organisms [2], but no studies have examined the existence of GPI-anchored proteins on Sertoli cells and their possible roles in Sertoli-germ cell interactions. This led us to investigate the Sertoli cell surface for novel proteins anchored to the cell in this manner.

GPI-anchored proteins are located on the extracellular surface of membranes and are covalently attached to the membrane lipid phosphatidylinositol via a core glycan linkage that attaches to the COOH-terminus of the protein [3]. GPI proteins are apically sorted in polarized cells [2, 4, 5], although exceptions have been reported [6]. After protein synthesis and GPI anchor addition, some GPI-anchored proteins are believed to partition into specialized membrane microdomains known as lipid rafts, or DIGs (detergent-insoluble glycolipid-enriched membrane microdomains), within the trans-Golgi membranes [7, 8]. This sorting into DIGs may be responsible for the apical distribution of many GPI-anchored proteins. DIGs are specialized membrane regions enriched in cholesterol, glycolipids, and certain proteins, including GPI-anchored proteins. They may be associated with small membrane invaginations known as caveolae, although the relationship between DIGs and caveolae is controversial [8–16]. DIGs and caveolae have been implicated in a number of processes, including cell signaling, potocytosis (uptake of low-molecular weight molecules such as folic acid), and localization of molecules with a related function [17, 18]. Although no studies of Sertoli cell DIGs have been reported, membrane pinocytotic invaginations similar to caveolae were reported in studies of prenatal mouse [19] and adult rat [20] Sertoli cells.

Ceruloplasmin (Cp) is a copper-binding protein with ferroxidase activity. Studies of serum Cp suggest that Cp-catalyzed oxidation of Fe2+ to Fe3+ mediates the incorporation of iron into transferrin (Tf), which binds only Fe3+ (reviewed in [21]). Serum Cp is essential for proper iron transport and homeostasis throughout the body. Evidence for this has recently been provided by studies of the hereditary Cp deficiency disease, aceruloplasminemia [22–24]. In aceruloplasminemic individuals, Cp deficiency results in iron accumulation within multiple tissues, whereas disruption of copper homeostasis is apparently minimal. Other studies have shown that Cp mediates iron uptake by cells in a Tf-independent system [25, 26]. The yeast Cp homolog, Fet3p, is a membrane-bound protein required for high-affinity iron uptake [21, 27–31], and it has also been shown to mediate incorporation of iron into apo-Tf [28].

Sertoli cells are known to secrete both Cp and a testis-specific form of Tf, testicular Tf (tTf) [32, 33]. Iron taken up basally by Sertoli cells as diferric serum Tf is released from the serum Tf complex within the cell and reincorporated into tTf for delivery to the spermatogenic cells located in the adluminal compartment [20, 34, 35]. Sertoli cell Cp was originally suggested to function either in copper transport or as a ferroxidase, with the majority of the literature referring to it as a copper transport protein. In consideration of the many recent studies of serum Cp, Sertoli cell Cp may be expected to function primarily as a ferroxidase with an essential role in transepithelial iron transport within the seminiferous tubule, although a possible role in copper transport cannot be disregarded. The present discovery of a membrane-bound GPI-anchored form of Cp on the Sertoli cell and further characterization of this protein may shed light on the function of Sertoli cell Cp.


   MATERIALS AND METHODS
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
RESULTS
 DISCUSSION
 REFERENCES
 
Cell Cultures

Sertoli cell cultures were established from 19- to 21-day-old male Sprague-Dawley rat testes (Hilltop Lab Animals, Inc., Scottsdale, PA) according to established laboratory procedures [36]. Sertoli cells isolated from 15 rat pups were plated into approximately six 75-cm2 polystyrene flasks or 6-well plates containing bicameral chamber inserts (Millicel-PCF polycarbonate membranes, 0.4 µm; Millipore, Bedford, MA) for confluent cultures. The cultures were maintained in Dulbecco's modified Eagle's/Ham's F-12 medium (DME/F-12; Sigma Chemical Co., St. Louis, MO) with penicillin (50 U/ml) and streptomycin (50 µl/ml) at 34°C in 5% CO2. After one day in culture, the medium was supplemented with testosterone (10-8 M), estradiol (10-8 M), hydrocortisone (10-9 M), insulin (2 µg/ml), epidermal growth factor (10 ng/ml), Tf (5 µg/ml), and retinol (50 ng/ml). Sertoli cells were cultured for four days before use. Maden-Darby canine kidney (MDCK) cells (#CCL-34; American Type Culture Collection, Manassas, VA) were maintained in DME/F-12 with 10% fetal bovine serum (FBS; HyClone, Logan, UT) at 37°C and were grown to confluence before use.

Cell-Surface Biotinylation and Phosphatidylinositol-Specific Phospholipase C (PI-PLC) Digestion

Cultures of primary Sertoli cells were washed three times in cold PBS, pH 7.6. For biotin labeling of cell-surface proteins (Figs. 1 and 2), some cultures were incubated in PBS containing the cell-impermeant biotinylating reagent sulfosuccinimidyl-6-(biotinamido) hexanoate (0.5 mg/ml; EZ-link sulfo-NHS-LC-biotin; Pierce, Rockford, IL) for 30 min at room temperature and then washed four times with cold PBS. Cells were collected by scraping and were pelleted at low speed. The pellets were washed twice in DME/F-12 and then resuspended in 1.5 ml of DME/F-12 per original 75-cm2 flask of cells or in 3 ml per 6-well plate. A protease inhibitor cocktail (PIC; Sigma; 20 µl/ml) and PMSF (200 µM) were added to the cell suspensions, which were then incubated with or without PI-PLC (Boehringer-Mannheim, Indianapolis, IN; 0.5 U/ml) for 1 h at 37°C with shaking. After incubation, cells were removed by centrifugation (15 700 x g, 30 min, 4°C), and protein was precipitated from the supernatants by addition of ten volumes of ice-cold acetone/methanol (1:1) and centrifugation (11 300 x g, 20 min, 4°C). Protein pellets were resuspended in PBS to a protein concentration of ~0.75–1.00 µg/µl. Aliquots of resuspended protein from both experimental and negative control samples were saved for protein determination [37], and the remainder of the samples were prepared for electrophoresis.



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  		FIG. 1. Detection of Sertoli cell surface proteins released by PI-PLC digestion. Sertoli cells were grown in either bicameral chambers or control (plastic) flasks. Cell surface proteins were apically biotinylated and then incubated with PI-PLC to release GPI-anchored proteins. PI-PLC digestion was omitted for the negative controls. Proteins from experimental (+) and control (-) PI-PLC digest supernatants were separated by SDS-PAGE and transferred to nitrocellulose. Biotinylated proteins were detected with avidin/peroxidase and visualized by ECL

SDS-PAGE, Western Blotting, and Antibodies

Protein samples were prepared for electrophoresis by addition of one volume of double-strength sample treatment buffer (0.125 M Tris pH 6.8, 4% SDS, 20% glycerol, 10% ß-mercaptoethanol, and 0.002% bromophenol blue) and were heated to 95°C for 3–5 min (heating was omitted for some samples, see Fig. 7). All protein samples were stored at -80°C. Proteins were separated by SDS-PAGE [38] on 7% gels and then electroblotted onto nitrocellulose (Bio-Rad, Richmond, CA) by wet transfer in 25 mM Tris, 190 mM glycine, and 20% methanol. Blots were stained reversibly with Ponceau-S dye (0.2% Ponceau-S, 3% trichloroacetic acid, 3% sulfosalicylic acid) to visualize molecular weight markers and then washed twice with distilled water. All blots were blocked with 1% BSA in Tris-buffered saline (TBS) containing 0.1% Tween-20 (TBS-T) for 1 h. For detection of biotin-labeled proteins (Fig. 1), the blot was incubated in ExtrAvidin/peroxidase (Sigma; 1:2000 in TBS-T containing 1% BSA) for 12 min. For detection of ceruloplasmin (Figs. 2–4A, 5, and 7), blots were incubated in goat anti-human ceruloplasmin polyclonal antibody (anti-Cp pAb) (Sigma; 1:400 in TBS-T containing 1% BSA) for 2 h at room temperature, and then incubated in rabbit anti-goat IgG/peroxidase (Sigma; 1:10 000 in TBS-T containing 1% BSA) for 1 h at room temperature. For detection of the cross-reacting determinant (CRD) (Fig. 4B), the blot was incubated in rabbit anti-CRD polyclonal antibody (pAb; generously provided by Dr. P. Englund, Johns Hopkins Medical School, Baltimore, MD; 1:500 in TBS-T containing 1% BSA) for 2 h at room temperature, and then incubated in anti-rabbit IgG/peroxidase (Kierkegard & Perry Laboratories, Gaithersburg, MD; 1:1600 in TBS-T containing 1% BSA) for 2 h at room temperature. For detection with Ran-2 antibody (Fig. 6), the blot was incubated in anti-Ran-2 monoclonal antibody (mAb) prepared as described below for 2 h at room temperature, and then in anti-mouse IgG/peroxidase (Kierkegard & Perry Laboratories; 1:2000 in TBS-T containing 1% BSA) for 1 h at room temperature. All blots were washed with TBS-T between incubations and were visualized by enhanced chemiluminescence (ECL; Amersham, Buckinghamshire, England). Stripping of antibodies (Fig. 4) was done by incubation of the blot in stripping buffer (100 mM ß-mercaptoethanol, 2% SDS, 62.5 mM Tris, pH 6.7) for 40 min at 50°C, followed by washing in TBS-T.



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  		FIG. 7. Comparison of the migration of Sertoli cell GPI-Cp and serum Cp in heat-denatured and nondenatured samples. Serum Cp and Sertoli cell DIG fractions were prepared for electrophoresis in standard Laemmli buffer containing ß-mercaptoethanol. Samples of each were either heat-denatured at 95°C for 3–5 min (+) or not heated (-). The samples were separated by SDS-PAGE, transferred to nitrocellulose, and detected with anti-Cp pAb



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  		FIG. 2. Recognition of the 135-kDa GPI-anchored protein by anti-ceruloplasmin pAb. Experimental and negative control samples of both biotinylated and nonbiotinylated Sertoli cell surface proteins from PI-PLC digestions were separated by SDS-PAGE, transferred to nitrocellulose, and detected with a pAb to human Cp. Human serum Cp was used as a positive control



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  		FIG. 4. Anti-CRD detection of Sertoli cell GPI-Cp and serum Cp. A) Protein from a PI-PLC digestion of Sertoli cells (+), along with an equal protein load from a negative control sample in which PI-PLC was omitted (-) and purified serum Cp were electrophoresed, transferred to nitrocellulose, and detected with anti-Cp. B) The same blot was stripped and re-probed with anti-CRD pAb



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  		FIG. 6. Anti-Ran-2 mAb recognition of Sertoli cell GPI-Cp. Equal protein loads of experimental (+) and negative control (-) samples from a PI-PLC digestion of Sertoli cells were electrophoresed along with Sertoli cell DIG protein and human serum Cp. Proteins were transferred to nitrocellulose and detected with anti-Ran-2 mAb. Serum Cp was loaded at a concentration approximately 5-fold greater than in previous gels that were probed with anti-Cp pAb

Anti-Ran-2 mAb was produced with a Ran-2 hybridoma cell line (ATCC, #TIB-119). The cells were maintained in RPMI-1640 medium (HyClone) containing 10% FBS. Culture supernatants were precipitated with 52% (NH4)2SO4 and centrifuged (8000 rpm in a Beckman JA10 rotor; Beckman Instruments, Palo Alto, CA) at 4°C. The protein pellet was resuspended in PBS (pH 7.4; ~1/20 of the original supernatant volume) and dialyzed against two changes of PBS at 4°C. The dialyzed solution was adjusted to 0.1% Tween-20/1% BSA, then applied directly to the blot as described above.

Isolation of DIGs

DIG fractions were isolated by a modification of a previously described method [39]. Approximately seven 75-cm2 flasks of confluent cells (primary Sertoli cells or MDCK cells) were used for each isolation. All procedures were carried out at 4°C. Cultures were washed thoroughly with cold PBS. The cells were extracted with ~1 ml/flask of 1% Triton X-100 in MES-buffered saline (MBS; 25 mM 2-[N-morpholino]ethanesulfonic acid [MES] pH 6.5, 0.15 M NaCl, 0.2 mM PMSF), scraped off the flasks, and transferred to a potter-Elvehjem tissue homogenizer packed in ice. The extract was homogenized with 10 passes of the homogenizer and adjusted to 40% sucrose, then divided into six clear 5-ml ultracentrifuge tubes. The extract was overlaid sequentially with 25% sucrose in MBS (~0.8 ml), 15% sucrose in MBS (~0.8 ml), and 5% sucrose in MBS (~0.8 ml). The gradients were centrifuged for 18 h at 46 000 rpm in a Beckman SW55Ti rotor. The insoluble material at the 15%/25% interface was collected, diluted in MBS, and pelleted by centrifugation (46 000 rpm in a Beckman SW55Ti rotor, 45 min). The pellets were resuspended in a total of 100 µl of PBS containing 0.2 mM PMSF. Aliquots were saved for protein determination, and the remainder was prepared for SDS-PAGE as described above.

Preparation of Sertoli Cell Lysates

Sertoli cell cultures were washed twice with cold PBS. One milliliter of lysis buffer (1% SDS, 1.0 mM sodium vanadate, 10 mM Tris pH 7.4, 0.2 mM PMSF) was added to each 75-cm2 flask. Lysed cells were scraped out of the flasks and heated to 95°C for 5 min. PIC (20 µl/ml) and PMSF (0.2 mM) were added, and the lysates were sonicated briefly to decrease viscosity. Insoluble material was removed by brief microcentrifugation. Aliquots of the supernatants were removed for protein determination, and the remainder was prepared for SDS-PAGE as described above.

Triton X-114 Extraction and Phase Partitioning

Triton X-114 (TX114) extraction of whole cells and DIG fractions was performed as described [40]. For whole-cell extraction, primary Sertoli cells were first removed from 75-cm2 flasks by scraping, pelleted by centrifugation, resuspended in 0.17 ml TBS (pH 7.4)/flask, and preincubated with or without PI-PLC (5.4 U/ml) for 80 min at 37°C. One-fifth volume of ice-cold precondensed TX114 was added to the samples, which were then extracted on ice for ~25 min with occasional agitation. Undissolved material was removed by centrifugation (10 000 x g, 10 min, 4°C) and prepared for electrophoresis. The supernatants were heated to 37°C and centrifuged (1000 x g, 10 min) to separate detergent and aqueous phases, and each phase was collected and prepared for electrophoresis. For TX114 extraction of DIG fractions, two 10-µl aliquots of a DIG sample were diluted 5x with PBS containing 0.2 mM PMSF and were preincubated with or without PI-PLC (4.5 U/ml) for 1 h at 37°C. TX114 extraction and phase separation were then performed as described above for whole cells, and the detergent and aqueous phases were prepared for electrophoresis.

NH3-Terminal Sequencing

Protein from a DIG fraction was used for amino acid sequence analysis of the 135-kDa GPI-anchored protein because the protein was highly concentrated in this membrane fraction. DIG protein was separated by SDS-PAGE as described above. The gel was soaked in CAPS buffer (10 mM 3-[cyclohexylamino]1-propane sulfonic acid, pH 11, 10% methanol) for 5 min, and a polyvinylidene difluoride (PVDF; Bio-Rad) membrane was soaked briefly in methanol. The gel and PVDF membrane were assembled for electroblotting in CAPS transfer buffer (50 V, 90 min at room temperature). The PVDF was removed and rinsed with distilled water, then stained briefly with Coomassie Blue (R250; 0.1% Coomassie in 40% methanol/1% acetic acid) and rinsed with 50% methanol. The 135-kDa band was excised and sequenced by Edman degradation at Hershey Medical Center (Macromolecular Core Facility; Hershey, PA). A sequence homology search was performed using the BLAST program (National Center for Biotechnology Information, National Library of Medicine, National Institutes of Health; http://www.ncbi.nlm.nih.gov/).


   RESULTS
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
DISCUSSION
 REFERENCES
 
Most GPI-anchored proteins can be released from cell surfaces by the action of the bacterial enzyme PI-PLC. We employed this method to investigate the possibility that GPI-anchored proteins are present on the apical-lateral domain of the Sertoli cell membrane. After 4 days in culture, primary Sertoli cells from 20-day-old rats were subjected to cell-surface biotinylation with a cell-impermeant biotinylating reagent to label cell surface proteins. After biotinylation, the cells were harvested and subjected to PI-PLC incubation. Protein from the digest supernatants was acetone/methanol-precipitated and analyzed by Western blotting. Biotinylated proteins were detected with avidin/peroxidase and visualized by ECL. A prominent band of approximately 135 kDa was seen in PI-PLC-digested samples but not in negative controls that had been subjected to a mock incubation without PI-PLC (Fig. 1). This indicated that the protein was specifically released by PI-PLC activity. Sertoli cell cultures were also grown on porous bicameral inserts, which allow the cells to assume a more polarized morphology with basal occludens junctions [41, 42]. PI-PLC digestion of apically biotinylated bicameral cultures produced a 135-kDa protein identical to that from the plastic-grown cultures (Fig. 1). PI-PLC digestion of basally biotinylated bicameral cultures produced no identifiable GPI-anchored proteins (data not shown).

Since Sertoli cells have been shown to secrete the ~135-kDa protein Cp [32, 33], we collected PI-PLC digest supernatants from Sertoli cell cultures and probed for Cp on Western blots to determine whether the previously observed 135-kDa protein was a GPI-anchored form of Sertoli cell Cp. Cp was detected at ~135 kDa in PI-PLC digest supernatants but not in negative controls (Fig. 2), indicating that the protein was a GPI-anchored form of Cp (GPI-Cp). Cp antibody binding was clearly inhibited by biotinylation (Fig. 2).

To confirm that the observed Cp was itself covalently modified with GPI rather than associated with other PI-PLC-releasable GPI-anchored proteins, we tested whether the protein would partition into the detergent phase of TX114-extracted cells [43]. This detergent in aqueous solution remains miscible with water at low temperatures and separates into detergent and aqueous phases at higher temperatures. GPI-anchored proteins containing the intact GPI anchor partition into the detergent phase after TX114 extraction because of the presence of hydrophobic lipid chains on the GPI anchor, while PI-PLC digestion of GPI-anchored proteins removes the hydrophobic portions of the GPI anchor and causes these proteins to partition into the aqueous phase. Sertoli cells collected from equal numbers of flasks were preincubated with or without PI-PLC, and TX114 extraction was then done as described in Materials and Methods. Proteins from the aqueous phase, the detergent phase, and the undissolved pellet were analyzed by Western blotting (Fig. 3). Cp from the PI-PLC-digested sample partitioned exclusively into the aqueous phase, while Cp from untreated samples partitioned primarily into the detergent phase. A low level of Cp present in the aqueous phase of undigested samples was probably due to background soluble Cp. Cp was also present in the pellet of undigested samples, presumably because of incomplete extraction of the cells. These observations confirmed that Cp is anchored to the Sertoli cell directly by GPI, rather than through association with some other GPI-anchored protein. Interestingly, the very low level of Cp in the aqueous phase of the undigested sample along with the absence of Cp in the pellet of the PI-PLC-treated sample (Fig. 3) suggests that the majority of Cp present in Sertoli cells is GPI-Cp rather than soluble Cp, which would partition into the aqueous phase regardless of PI-PLC treatment.



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  		FIG. 3. Phase partitioning of GPI-Cp in TX114 Sertoli cell extracts. Sertoli cells were subjected to either a PI-PLC digestion (+) to remove the hydrophobic portion of the GPI anchor or to a mock digestion (-). After extraction with TX114 and partitioning of detergent and aqueous phases, proteins were separated by SDS-PAGE, transferred to nitrocellulose, and detected with anti-Cp pAb as before. Loading amounts were approximately equal for the PI-PLC-treated and untreated samples

The presence of the GPI anchor on Sertoli cell Cp was further confirmed by detection with an antibody to the cross-reacting determinant (CRD), which is the portion of the GPI anchor remaining after PI-PLC cleavage. A Western blot containing serum Cp and protein from a Sertoli cell PI-PLC digest supernatant was first probed for Cp (Fig. 4A) and then stripped and reprobed with anti-CRD (Fig. 4B). After stripping and before reprobing with anti-CRD, ECL reagents were re-applied and the blot was exposed to film for an extended period to verify that the antibodies had been completely removed (data not shown). Probing with anti-CRD detected a 135-kDa band identical to the band detected with anti-Cp in Sertoli cell digests. The heavily loaded serum Cp also showed a weak binding of anti-CRD antibody (Fig. 4B), possibly nonspecific in nature.

Many GPI-anchored proteins have been suggested as constituents of DIGs [8, 11]. To determine whether GPI-Cp was present in Sertoli cell DIGs, we isolated 1% Triton X-100-insoluble membrane fractions from primary Sertoli cells and MDCK cells, which yield well-characterized DIG fractions. These samples were analyzed by Western blotting using anti-Cp detection (Fig. 5A). Cp was present in large quantity in Sertoli cell DIGs but not in MDCK DIGs. An equivalent quantity of protein from Sertoli cell lysates was also analyzed and lacked detectable Cp, suggesting that the Cp is highly enriched in the DIG fraction. A TX114 phase-partitioning experiment similar to the one described above for whole cells was performed on the DIG fraction to confirm that the Cp found in this fraction was GPI-Cp (Fig. 5B). Cp from the DIG fraction partitioned into the detergent phase in undigested samples and into the aqueous phase in PI-PLC-treated samples, indicating that the Cp within the DIG fraction was GPI-anchored.



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  		FIG. 5. Ceruloplasmin in DIG fractions. A) Equal protein loads from Sertoli cell DIGs, Sertoli cell lysate, and MDCK DIGs were separated by SDS-PAGE, transferred to nitrocellulose, and detected with anti-Cp pAb. B) Equal aliquots of Sertoli cell DIG suspensions were incubated with or without PI-PLC, followed by extraction with Triton X-114 and phase partitioning. The entire aqueous (Aq) and detergent (Det) phases from both aliquots were separated by SDS-PAGE, transferred to nitrocellulose, and detected with anti-Cp pAb

To confirm that the anti-Cp-reactive 135-kDa band seen in the above experiments was in fact Cp, we obtained a partial sequence on the 135-kDa protein from a DIG fraction. A DIG sample was separated by SDS-PAGE and transferred to PVDF membrane. The 135-kDa band was excised and sequenced by Edman degradation. A 10-amino acid sequence was obtained (REKHYYIGIT) and was found to be identical to residues 20–29 of rat Cp, thus confirming that the 135-kDa GPI-anchored protein was Cp. As further confirmation, the protein was found to be recognized by a mAb to Ran-2 (Fig. 6), a glial surface antigen that has recently been shown to be a GPI-anchored form of Cp [44]. Anti-Ran-2 mAb recognized the 135-kDa protein from a PI-PLC digestion of whole Sertoli cells and from a Sertoli cell DIG fraction but did not recognize human serum Cp (Fig. 6).

GPI-Cp from rat Sertoli cells migrated slightly faster than human serum Cp (Figs. 2–5). To further characterize the migration pattern of this protein, we analyzed both heat-denatured and nondenatured (nonheated) serum Cp and Sertoli cell GPI-Cp from a DIG fraction by SDS-PAGE and Western blotting (Fig. 7). The slightly faster migration of heat-denatured Sertoli cell DIG Cp was again observed, and the altered migration became even more prominent in the nondenatured sample. The major species in nondenatured serum Cp migrated at ~84 kDa, as noted previously by Sato and Gitlin [45]. Nondenatured Cp from Sertoli cell DIGs migrated at ~66 kDa (Fig. 7). The migration differences seen here between human serum Cp and rat Sertoli cell GPI-Cp may be due to cell-specific or species-specific differences in glycosylation.


   DISCUSSION
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
REFERENCES
 
Ceruloplasmin (Cp) was first identified as a secretory product of Sertoli cells more than fifteen years ago by Skinner and Griswold [33]. In this and other reports, Cp was suggested to function either in transport of copper to spermatogenic cells or possibly as a ferroxidase. It is now clear that serum ceruloplasmin functions primarily as a mediator of iron transport and that its role in copper transport is either secondary or nonexistent. Models of Cp both as a mediator of Tf loading for iron egress from cells [21] and as a mediator of cellular iron uptake [26] have been proposed. The former is supported by studies of iron accumulation within various organs of aceruloplasminemia patients [22–24], while the latter is supported by analogy to the role of the yeast Cp homologue, Fet3p, in high-affinity iron uptake. With respect to testicular iron transport, it has been observed that aceruloplasminemia patients (who arrive at the clinic late in life) show elevated testicular iron levels (J. Gitlin, personal communication). This suggests that Cp may be essential for transepithelial iron transport in the seminiferous tubule.

Transepithelial transport of iron across Sertoli cell epithelia in a basal to an adluminal direction has been clearly established (reviewed in [35]). Basal uptake of diferric serum Tf occurs via binding to basal Tf receptors followed by endocytosis and dissociation of iron from Tf within the endosomal compartment in the ferrous (soluble) state. The mechanisms involved in transport of iron out of this vesicle and to the apical portion of the Sertoli cell, where loading of tTf is thought to occur, remain unclear. Also unclear is exactly how and where tTf is loaded, although some evidence suggests that the majority of tTf is loaded intracellularly [46]. Iron bound to either Tf or ferritin is in the Fe3+ state, while unbound iron is generally in the more soluble Fe2+ state. This suggests that intracellular iron must be oxidized before incorporation into tTf. Newly synthesized apo-tTf is presumably loaded with Fe3+ within trans- or post-Golgi secretory vesicles, so Cp would be expected to be present within these same vesicles to catalyze the oxidation of Fe2+. GPI-Cp would be positioned appropriately to oxidize iron immediately after it crosses the vesicular membrane. Interestingly, the stage-specific patterns of Cp and tTf secretion from Sertoli cells do not coincide [47]. If intravesicular iron oxidation and tTf loading is mediated by soluble Cp, secretion of Cp and tTf would be expected to approximately coincide. Membrane anchorage of the ferroxidase may account for this apparent discrepancy, especially if GPI-Cp is the precursor to secreted Cp. GPI-Cp could carry out the oxidation within the secretory vesicles and could later be released (secreted) from the plasma membrane as soluble Cp. Additionally, GPI-Cp may be expected to function while anchored to the plasma membrane. GPI-Cp may load apo-tTf that has been recycled to the extracellular space after delivering iron to spermatogenic cells, or it may oxidize free Fe2+ at the extracellular surface to reduce free radical generation within the extracellular fluid.

The presence of GPI-Cp in the DIGs raises some interesting possibilities. This membrane microdomain may play a role in organizing proteins involved in iron transport. An evaluation of other components of this membrane region that could be involved in iron transport is currently underway. DIGs could be involved in either egress or uptake of iron. If Sertoli cell DIGs are associated with caveolae, iron uptake could be accomplished by potocytosis in a manner similar to folate uptake [18]. Although Sertoli cells would not be expected to take in iron apically, this possibility cannot be excluded since the cells may need to scavenge free iron from the surrounding fluid to prevent free radical injury. Cp has been implicated in a mammalian Tf-independent iron uptake system [25, 26]. Analogy to the yeast high-affinity iron uptake pathway further supports an iron uptake role for GPI-Cp. The yeast Cp homologue, Fet3p, is anchored to the plasma membrane by a single transmembrane domain [26], and the necessity of Fet3p for iron uptake by yeast has been well documented [21, 29, 30]. Fet3p is an iron oxidase that appears to work in concert with an iron permease, Ftr1p, and two iron reductases, Fre1p and Fre2p [21, 29]. An analysis of whether homologous proteins are present within the DIG would be useful. Our laboratory is currently investigating whether the mammalian divalent cation transporter Nramp2/DCT1 [48, 49] is contained in Sertoli cell DIGs. This gene was found to be expressed in the testis by Northern blot analysis, and in situ hybridization showed expression specifically in Sertoli cells [48].

This study raises the issue of the relationship between GPI-Cp and secreted Cp. GPI-Cp may be a precursor to secreted ceruloplasmin, such that GPI anchoring of the protein would simply be an apical sorting signal, with GPI-Cp being released from the plasma membrane as soluble Cp by an endogenous phospholipase or protease after arriving at the cell surface. The observation that very little soluble Cp is detected in whole-cell TX114 extracts of Sertoli cells (Fig. 3) suggests that GPI-Cp may be the dominant form expressed by Sertoli cells. This may favor the possibility that GPI-Cp is a precursor to secreted Cp. This type of apical secretion mechanism has been demonstrated for alkaline phosphatase, acetylcholine esterase, decay-accelerating factor, and glycoprotein 2 [50, 51]. However, whether GPI-Cp and soluble Cp are alternatively processed forms of the same gene, the products of two separate genes, or the same protein at different stages of the secretory pathway is not yet known.

Of major significance to this study are the recent reports of a GPI-anchored form of Cp expressed by mammalian astrocytes [44, 52] and Schwann cells [44]. Both testis and brain possess a blood/tissue barrier, with Sertoli cells and astrocytes, respectively, acting as nutritional gatekeepers across these barriers. An interesting difference exists between these two tissues, however, in that Tf and Cp are expressed by two different cell types in the brain (oligodendrocytes and astrocytes, respectively), whereas Sertoli cells produce both tTf and Cp. Apo-Tf must be loaded extracellularly in the brain, so GPI-Cp may play an essential role in the loading of Tf immediately after Fe2+ crosses the astrocyte plasma membrane to ensure a minimum of free Fe2+ [52]. As we have suggested, Sertoli cell GPI-Cp could play a similar role in the testis, either within secretory vesicles or on the extracellular surface. Also of note is that immunocytochemical studies by Salzer et al. [44] showed GPI-anchored Cp in a punctate pattern on Schwann cells, suggesting that it may be contained in caveolae or DIGs. This agrees well with our finding that GPI-Cp is contained in Sertoli cell DIG fractions.


   ACKNOWLEDGMENTS
 
 The authors wish to acknowledge the initial work of Zarine Patel, Constance Sedon, and Amy Millen, who began the search for GPI-anchored proteins in Sertoli cells, and Mike Keeley, who first investigated the Sertoli cell DIGs. We also thank Dr. Jonathan Gitlin, Dr. Marie Pizzorno, Dr. Kathleen Page, Dr. Mitch Chernin, and Dr. Charlie Clapp for helpful discussions and critical readings of this manuscript, Dr. Paul Englund for providing the anti-CRD antibody, and Anne Stanley for sequence analysis and helpful suggestions.


   FOOTNOTES
 
1 Financial support for this research was received from Dr. James Kase, M.D., F.A.C.S. Back

2 Correspondence: FAX: 570 577 3537; nyquist{at}bucknell.edu Back

3 Current address: University of Pennsylvania Medical School; fortna{at}mail.med.upenn.edu Back

4 Current address: Johns Hopkins University; haw2{at}jhunix.hcf.jhu.edu Back

Accepted: May 26, 1999.

Received: April 6, 1999.


   REFERENCES
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 ABSTRACT
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 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
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