©1995 by The American Society for Biochemistry and Molecular Biology, Inc.
The Chicken Oocyte Receptor for Lipoprotein Deposition Recognizes -Macroglobulin (*)

(Received for publication, September 23, 1994; and in revised form, January 18, 1995)

Linda Jacobsen (§) Marcela Hermann Päivi M. Vieira (¶) Wolfgang J. Schneider Johannes Nimpf (**)

From the Department of Molecular Genetics, University and Biocenter Vienna, A-1030 Vienna, Austria

ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
FOOTNOTES
ACKNOWLEDGEMENTS
REFERENCES

ABSTRACT

alpha(2)-Macroglobulin (alpha(2)M), a major plasma component in all vertebrates, is proposed to function as a broad spectrum protease inhibitor. The alpha(2)M-proteinase complex (activated alpha(2)M; alpha(2)M) is removed rapidly by receptor-mediated endocytosis in the liver. Here we demonstrate by Western blotting that alpha(2)M is also present in the yolk of chicken oocytes. Plasma levels of alpha(2)M are increased by estrogen, and yolk alpha(2)M is partially proteolyzed, consistent with the action of cathepsin D on endocytosed alpha(2)M. Two known estrogen-induced ligands of the oocyte-specific 95-kDa very low density lipoprotein/vitellogenin receptor (OVR) are also fragmented by yolk cathepsin D (Retzek, H., Steyrer, E., Sanders, E. J., Nimpf, J., and Schneider, W. J.(1992) DNA Cell Biol. 11, 661-672). Since these findings suggested a common uptake mechanism for lipoproteins and alpha(2)M by oocytes, we investigated whether OVR, a member of the low density lipoprotein receptor family, functions in the metabolism of alpha(2)M. Ligand blotting of oocyte membrane extracts with chicken alpha(2)M revealed that it binds to OVR. Surprisingly, the oocyte receptor also recognizes native alpha(2)M, in sharp contrast to the hepatic receptor, which only binds alpha(2)M. Receptor interaction of both forms requires Ca; however, competition experiments suggest that alpha(2)M and alpha(2)M interact with slightly different, or overlapping, sites on the receptor. Colocalization of alpha(2)M and OVR in coated vesicles isolated from growing oocytes, and internalization and degradation of methylamine-activated alpha(2)M by COS-7 cells transfected with OVR, strongly suggest that alpha(2)M is transported into growing oocytes via OVR. We propose that this multifunctional receptor mediates pathways at the metabolic crossroads of lipoproteins and protease inhibitor complexes.


INTRODUCTION

During the last 7 days of the development of a hen's oocyte, the giant cell takes up about 5 g of lipid and protein in the form of very low density lipoprotein (VLDL) (^1)and vitellogenin (VTG)(1, 2) . These major precursors of yolk mass are synthesized in the liver and taken up by the oocytes from the circulation via receptor-mediated endocytosis. Despite the absence of obvious extensive structural similarity, VLDL and VTG are both recognized by the same receptor, termed the oocyte VLDL/VTG receptor (OVR), which is expressed at high levels in the plasma membrane of growing female germ cells(3, 4) . The receptor was purified from chicken follicles, and partial amino acid sequences together with immunological evidence clearly showed that it belongs to the LDL receptor gene family(5, 6) . Recent cloning of OVR (7) revealed that it is the avian representative of the so-called VLDL receptor family branch. The structural hallmark characteristics of this protein(8, 9, 10, 11, 12) were defined based on the structure of the rabbit receptor(13) . The protein contains a cluster of eight cysteine-rich complement-type binding repeats, in contrast to the seven repeats found in all LDL receptors(14) . The term VLDL receptor is based on the mammalian receptor's high affinity for apolipoprotein (apo) E-containing lipoproteins, especially VLDL(13) ; but in contrast to OVR, the true physiological function(s) of mammalian VLDL receptors are not understood.

In this respect it is of particular interest that the chicken OVR exerts a broad ligand specificity, since it binds the major protein constituent of VLDL, apoB(15) , VTG(3) , as well as mammalian apoE (16) , an apo not produced in chicken(17, 18) . To date, an even wider ligand specificity has been found for the LDL receptor-related protein/alpha(2)-macroglobulin receptor (LRP/alpha(2)MR), another member of the LDL receptor gene family(19) . This large membrane protein contains four clusters of 2-11 binding repeats and can bind, at least in vitro, such diverse ligands as alpha(2)M(20) ; apoE (21) and apoE-enriched lipoproteins(22) ; lipoprotein lipase(23) ; plasminogen activators and/or complexes with their respective endogenous inhibitors(24, 25, 26) ; receptor-associated protein, a small 39-kDa intracellular protein that copurifies with the receptor(27) ; lactoferrin(28) ; and rhinoviruses of the minor group(29) . A homologue of this receptor is expressed and has been characterized in somatic cells of the chicken(30) . Besides functioning as a backup receptor system for apoE-containing chylomicron remnants in mammals(31, 32) , it serves as an alpha(2)MR in a wide variety of species (for review see (33) ).

The LRP/alpha(2)MR rapidly removes from the circulation complexes between proteinases and alpha(2)M by specifically binding to a receptor recognition site on alpha(2)M. This recognition site on alpha(2)M is exposed by a conformational change, commonly referred to as activation of alpha(2)M (for review see (34) ). The conformational change is the result of a cleavage in the bait region of alpha(2)M by a protease that might become cross-linked to alpha(2)M via an internal thiolester of alpha(2)M. Here we report that OVR, believed to be the product of an ancestral gene involved in female reproduction, binds not only activated alpha(2)M, but also its native form. Analysis of coated vesicles derived from small vitellogenic oocytes and yolk from differently staged follicles suggests that alpha(2)M is a cytoplasmic constituent of chicken oocytes, which likely endocytose it via OVR.


EXPERIMENTAL PROCEDURES

Animals and Diets

White leghorn laying hens were purchased from Heindl (Vienna) and maintained as described(6) . Roosters (20-30 weeks old) were treated with 17alpha-ethinylestradiol dissolved in propylene glycol by injecting 10 mg/kg body weight into the breast muscle. After 72 h, blood was drawn from the wing vein for analysis of alpha(2)M levels. Adult female New Zealand White rabbits were used for raising antibodies(5) .

Isolation and Radiolabeling of alpha(2)M

Plasma alpha(2)M was purified from citrated plasma of laying hens essentially as described by Sottrup-Jensen et al.(35) . All steps were performed at 4 °C. Laying hen plasma was diluted 1:1 with 30 mM sodium phosphate, 150 mM NaCl, pH 7.4, and PEG 6000 was added to a final concentration of 4% (w/v). After stirring for 1 h, the precipitate was removed by centrifugation, and the supernatant was adjusted to 16% PEG; the efficacy of precipitation was assessed by SDS-PAGE of supernatants after 12% and 16% PEG precipitations (see ``Results''). After centrifugation, the precipitate was redissolved in 30 mM sodium phosphate, 150 mM NaCl, pH 7.4, 10 µg/ml soybean trypsin inhibitor and dialyzed overnight against running tap water. Precipitate was removed by centrifugation, and the supernatant was mixed with 0.1 volume of 0.2 M sodium phosphate, pH 6.0, and 0.05 volume of 3 M NaCl. Zn-charged iminodiacetic Sepharose (25-ml bed volume) was added, and the mixture was stirred for 1 h. The Zn-Sepharose was washed thoroughly with 20 mM sodium phosphate, 150 mM NaCl, pH 6.4, and packed into a column. Adsorbed alpha(2)M was eluted with 0.1 M sodium acetate, pH 5.0, concentrated by ultrafiltration, and gel filtrated on Sephacryl S-300. The pure alpha(2)M was concentrated to 5-10 mg/ml by ultrafiltration and stored at -20 °C or used immediately. alpha(2)M-trypsin complexes were prepared by incubating alpha(2)M with a 15-fold molar excess of trypsin for 1 min followed by the addition of the same amount of soybean trypsin inhibitor. Methylamine-activated alpha(2)M (alpha(2)MMA) was prepared by treating native alpha(2)M with 0.2 M methylamine for 2 h at 20 °C. Native alpha(2)M was radiolabeled using the lactoperoxidase method (36) to specific activities of 20-50 kcpm/ng. Labeled alpha(2)M-trypsin complexes were produced by reacting labeled native alpha(2)M with trypsin as described above.

Preparation of Oocyte Membranes, VTG, and VLDL

Oocyte membranes were prepared from previtellogenic follicles (4-6-mm diameter) and extracted with 1% Triton X-100 as described previously (6) . VTG and VLDL were prepared from laying hens or estrogen-treated roosters as described(6, 37) .

Electrophoresis and Transfer to Nitrocellulose

Gel electrophoresis under nondenaturing conditions was performed on 5% gels (100 min at 125 V) using the Tris borate system described by van Leuven et al.(38) . One-dimensional SDS-PAGE was performed according to Laemmli (39) on 4.5-18% gradient slab gels at 180 V for 60 min using the minigel system from Bio-Rad. Samples containing 10 mM dithiothreitol were heated for 3 min at 95 °C. Nonreducing samples were subjected to gel electrophoresis without prior heating. Samples containing proteolytic activity were added to excess preheated sample buffer and heated further at 95 °C for 3 min. The molecular masses of proteins were estimated with the broad range molecular mass standard (6-200 kDa) from Bio-Rad. Gels were stained with 3% Coomassie Blue (G-250) in 25% isopropyl alcohol, 10% acetic acid and destained in 10% acetic acid.

Electrophoretic transfer to nitrocellulose membranes (Hybond-C, Amersham Corp.) was performed in 20 mM Tris-HCl, 0.15 M glycine buffer, pH 8.4, for 90 min at 200 mA. After transfer, proteins were visualized by staining the membrane with Ponceau S (2 g/liter and 30 g/liter trichloroacetic acid) and rinsing with H(2)O. Nitrocellulose membranes used for Western blot analysis were blocked with 5% nonfat dry milk in PBS containing 0.1% Tween 20. Bound polyclonal antibodies were detected with horseradish peroxidase-conjugated protein A and an enhanced chemiluminescence (ECL) system (Renaissance system, DuPont NEN). Membranes were exposed on Reflection film (DuPont NEN) for the times indicated. Ligand blotting with I-alpha(2)M was performed as described for I-VLDL(6) , except that ligand-blotting buffer contained 1 mM phenylmethylsulfonyl fluoride, which was added immediately before use.

Antibodies

Purified chicken alpha(2)M was isolated by electroelution from a 4.5-18% gradient SDS-polyacrylamide gel and used for producing polyclonal antibodies in rabbits as described(5) . The IgG fraction was purified by protein A-Sepharose chromatography, and microimmunoaffinity purification of the IgG was performed as follows. alpha(2)M was subjected to SDS-PAGE and electrophoretically transferred onto nitrocellulose. The protein was visualized with Ponceau S and the area of the membrane containing alpha(2)M excised. The strip was blocked for 2 h in 2.5 ml of buffer A (25 mM Tris, 150 mM NaCl, 2 mM CaCl(2), pH 7.6) containing 5% bovine serum albumin. Then, 0.1 ml protein A-purified IgG (200 µg/ml) was added, and the incubation was carried on for another 2 h. The strip was washed extensively with buffer A without bovine serum albumin, and the bound IgG was eluted with 0.5 ml 0.1 M citric acid, pH 2.5, and neutralized immediately with 0.1 ml of 1 M Tris. Elution was repeated twice, and the fractions were combined and dialyzed against phosphate-buffered saline. Antibody against OVR was prepared as described(7) .

Analysis of Serum alpha(2)M Levels

Relative amounts of serum alpha(2)M were determined by Western blotting. Serum samples (0.1 or 0.05 µl/lane) were run on two 4-18% SDS-PAGE gradient gels under reducing conditions. One gel was then blotted electrophoretically onto nitrocellulose, and a Western blot using immunopurified anti-alpha(2)M antibody was performed as described above. The intensity of the bands was measured with a densitometer using the Image-Quant software package from Molecular Dynamics. The other gel was stained with Coomassie Blue, and the serum albumin band of corresponding samples was quantitated by densitometry and used to normalize the relative intensities obtained for alpha(2)M.

Analysis of Yolk Proteins from Follicles

Small yellow follicles (vitellogenic) were collected and the yolk obtained by extrusion. The yolk was diluted with 4 volumes of 5 mM HEPES, pH 7.4, 0.25 M sucrose, 1 mM EGTA, 0.5 mM MgCl(2), and centrifuged at 5,000 times g for 10 min. The supernatant was collected and centrifuged for 2 h at 400,000 times g. The supernatant was decanted, and the pellet was redissolved in MES buffer (0.1 M MES, pH 6.5, 1 mM EGTA, 0.5 mM MgCl(2), 3 mM NaN(3), 1 mM phenylmethylsulfonyl fluoride, 5 µM leupeptin) to give a final concentration of approximately 10 µg/µl. Alternatively, 400 µl of yolk diluted 1:1 with MES buffer was loaded on top of a gradient of 5-40% sucrose in 0.1 M MES and centrifuged for 20 h in a SW-40 rotor at 40,000 rpm. Fractions (0.5 ml) were collected starting from the top of the tube, and aliquots thereof were analyzed by SDS-PAGE and Western blotting as described above.

Cathepsin D Digestions

Chicken liver cathepsin D was obtained as described(40) . Purified alpha(2)M (30 µg) was incubated with 3 units of cathepsin D in 100 µl of 20 mM Tris-HCl, pH 5.5, at 37 °C for 20 h. Digestion was stopped by the addition of 1 µg of pepstatin A. As a control, 1 µg of pepstatin A was added to 30 µg of alpha(2)M before the addition of cathepsin D.

Coated Vesicles

Coated vesicles were prepared from the yolk of white (previtellogenic) and small yellow (vitellogenic) follicles using a ^1H(2)O/^2H(2)O, 8% sucrose gradient(41) . After homogenization in MES buffer the homogenate was centrifuged at 5,000 times g for 5 min. The pellet was resuspended again in MES buffer and centrifuged at 5,000 times g for 5 min. The supernatants from both centrifugations were combined and centrifuged at 100,000 times g for 1 h. The resulting pellet was suspended in MES buffer and centrifuged at 10,000 times g for 10 min. The resulting pellet was resuspended in MES buffer, and the centrifugation was repeated. The supernatants from the two centrifugations were combined and centrifuged at 100,000 times g for 1 h. The pellet was resuspended in 3 ml of MES buffer and centrifuged at 10,000 times g for 10 min; the pellet was again resuspended in 3 ml of MES buffer and centrifuged at 10,000 times g for 10 min. The combined supernatants (6 ml) were loaded on the top of 6 ml of 8% sucrose in ^2H(2)O and centrifuged at 80,000 times g for 2 h. The pellet, resuspended in 300 µl of MES buffer without phenylmethylsulfonyl fluoride and leupeptin, was centrifuged at 20,000 times g for 10 min. The supernatant was recovered and stored at 4 °C. The protein concentration of the resulting coated vesicle preparation was 1.5 mg/ml.

Uptake and Degradation of alpha(2)M by Transfected COS-7 Cells

Monolayers of COS-7 cells transiently transfected with pCDMCVR-1 (coding for full-length OVR), and control cells transfected with pCDM8 (vector without insert) (7) were incubated for 5 h in standard medium containing 2 mg/ml bovine serum albumin and the concentrations of radioiodinated ligand indicated in the figure legends. Assays of proteolytic degradation of I-labeled ligands in monolayers of cultured cells were determined according to the standard protocol for LDL(42) . Specific OVR-mediated degradation of alpha(2)M was calculated as the difference between the values obtained with OVR-expressing and mock transfected cells.

Other Methods

Protein concentrations were determined as described by Lowry et al.(43) . The protein concentration of samples containing lipoproteins or detergent was determined using a modified Lowry procedure(44) .


RESULTS

Purification of Chicken alpha(2)M

To initiate studies on the binding of alpha(2)M to oocyte membrane proteins and uptake into growing oocytes, we first purified it from laying hen plasma by modifications of the procedure described for human alpha(2)M(35) . Feldman and Pizzo (45) described an initial precipitation at 7% (w/v) PEG for the purification of chicken alpha(2)M, resulting in a significant reduction in yield. In our hands, initial precipitation at 4% (w/v) PEG led to a complete removal of fibrin and fibrinogen without significant loss of alpha(2)M. The second PEG precipitation was carried out at 16% (w/v) PEG, which increased the yield of chicken serum alpha(2)M by about 50% compared with that obtained with 12% (w/v) PEG routinely used for the precipitation of human alpha(2)M. After further chromatography on Zn-Sepharose and Sephacryl S-300, we obtained virtually pure alpha(2)M, as assessed by SDS-PAGE under reducing and nonreducing conditions (Fig. 1). It migrated as a single band with an apparent M(r) of 360,000 under nonreducing conditions, and with an apparent M(r) of 180,000 under reducing conditions, corresponding to the homodimer and monomer of native alpha(2)M, respectively. When compared with human alpha(2)M using nondenaturing electrophoresis (where native alpha(2)M, consisting of two noncovalently associated homodimers, migrated with a M(r) of 720,000), a significant difference between human and chicken alpha(2)M became evident. Whereas native human alpha(2)M (Fig. 1B, lane a) became transformed to the so-called fast form by small amines such as methylamine (lane b), native chicken alpha(2)M (lane d) did not undergo any conformational change detectable by electrophoresis (lane e). However, treatment with an excess of trypsin increased the mobility of chicken alpha(2)M (lane f) to the same extent as that observed in human alpha(2)M (lane c).


Figure 1: Electrophoretic analysis of purified chicken alpha(2)M. alpha(2)M was purified from laying hen plasma as described under ``Experimental Procedures'' and analyzed by 4.5-18% SDS-gradient PAGE (panel A) and nondenaturing PAGE (panel B). Panel A, 4 µg/lane purified chicken alpha(2)M (-, nonreducing; +, reducing). Positions of migration of standard marker proteins are shown on the left. Proteins were stained with Coomassie Brilliant Blue. Panel B, nondenaturing PAGE of slow and fast forms of purified human alpha(2)M (lanes a-c) and purified chicken alpha(2)M (lanes d-f). Lanes a and d, native alpha(2)M; lanes b and e, alpha(2)MMA; and lanes c and f, trypsin-treated alpha(2)M. 10 µg of protein/lane was applied and stained with Coomassie Brilliant Blue.



Estrogen Induction of Plasma alpha(2)M Levels in the Chicken

Pure chicken alpha(2)M was obtained by preparative SDS-PAGE under nonreducing conditions, and the eluted protein was used to produce specific antibodies. The anti-alpha(2)M IgG was immunopurified and shown to react equally well with reduced and nonreduced samples of chicken alpha(2)M in Western blots (data not shown). This IgG was used to determine relative concentrations of alpha(2)M in different chicken sera as described under ``Experimental Procedures.'' Fig. 2A shows a representative Western blot of sera derived from different animals. The relative intensities of the bands were measured and normalized using the intensity of the albumin band in Coomassie Blue-stained gels as described under ``Experimental Procedures'' and displayed graphically in Fig. 2B. Relative alpha(2)M levels in laying hens (30 weeks old) were three times higher than in immature hens (2 weeks old). Immature roosters (2 weeks old) had the same plasma concentration as immature hens, with slightly higher levels in mature roosters (30 weeks old). Upon estrogen administration to roosters, plasma levels rose about two to three times to levels similar to those in laying hens.


Figure 2: Analysis of relative alpha(2)M levels in serum samples. Panel A, 0.1 µl of the indicated plasma samples was separated on a 4.5-18% SDS-gradient polyacrylamide gel, and Western blotting was performed using microimmunopurified anti-alpha(2)M IgG as described under ``Experimental Procedures.'' Bands were visualized using the ECL system according to the manufacturer's instructions; exposure time was 3 min. IH, immature hen; LH, laying hen; IR, immature rooster; MR, mature rooster; ER, estrogenized rooster. Panel B, relative amounts of alpha(2)M in plasma were determined by densitometry of panel A, normalized to albumin levels as described under ``Experimental Procedures,'' and plotted in arbitrary units. Designations are as in panel A.



Binding of Native and Activated alpha(2)M to the Oocyte Receptor for VLDL and VTG

To study the interaction of alpha(2)M with OVR, we prepared oocyte membranes and performed ligand blotting experiments with purified alpha(2)M from chicken (Fig. 3A) and human serum (Fig. 3B). Radioiodinated trypsin-treated chicken alpha(2)M prominently labeled a 95-kDa band in oocyte membrane extracts (Fig. 3A, lane 1), corresponding to the VLDL/VTG receptor, as shown in lane 5 with an antibody prepared against a peptide derived from the cloned sequence of the receptor(7) . The binding of alpha(2)M was Ca-sensitive, since the addition of EDTA (final concentration, 20 mM) completely abolished it (lane 2). Surprisingly, native alpha(2)M, which is known not to bind to hepatic alpha(2)M receptors (for review see (33) ) and thus was intended to serve as control, also bound to the same receptor, although with lower affinity (lane 3); this binding too, was EDTA-sensitive (lane 4).


Figure 3: Ligand and Western blotting of oocyte membrane proteins. Oocyte membrane Triton X-100 extracts (35 µg of protein/lane) were subjected to electrophoresis on a 4.5-18% SDS-gradient polyacrylamide gel, transferred to nitrocellulose, and analyzed by ligand blotting (panel A) or a modified ligand blotting procedure as described under ``Experimental Procedures'' (panel B). Panel A, the blot was incubated with I-labeled trypsin-treated chicken alpha(2)M (alpha(2)M) (13 ng/ml, 56.800 cpm/ng) in the absence (lane 1) or presence (lane 2) of 20 mM Na(2)EDTA. Lane 3 (no Na(2)EDTA) and lane 4 (20 mM Na(2)EDTA) were incubated with I-labeled native chicken alpha(2)M (26 ng/ml, 56,800 cpm/ng). The autoradiograph was exposed for 44 h. Lane 5 is a Western blot using rabbit anti-OVR IgG (16 µg/ml) and horseradish peroxidase-protein A in conjunction with ECL. Exposure time was 2 min. Panel B, the blots were incubated with human trypsin-treated alpha(2)M (6 µg/ml) in the absence (lane 1) or presence (lane 2) of 20 mM Na(2)EDTA. Lane 3 (no Na(2)EDTA) and lane 4 (20 mM Na(2)EDTA) were incubated with native human alpha(2)M (12 µg/ml). In lane 5, the ligand was omitted as a control. Bound ligands were visualized with rabbit anti-human alpha(2)M IgG (30 µg/ml) as described under ``Experimental Procedures'' (lanes 1-5). Exposure time 1 min. Lane 6 was obtained as described for lane 5 in panel A.



To determine if the binding of native alpha(2)M to OVR was specific for the chicken ligand, we performed ligand blots with purified human alpha(2)M and chicken oocyte membrane extracts. In this set of experiments (Fig. 3B), we used a sandwich ligand blot technique in which bound ligands are visualized with specific antibodies against human alpha(2)M. This procedure eliminated the possibility that alpha(2)M became activated by the iodination process. As with the chicken protein, not only trypsin-treated human alpha(2)M (lane 1), but also the native form (lane 3) bound to the receptor in a Ca-dependent fashion (lanes 2 and 4). Importantly, when the procedure was performed without the addition of activated or native alpha(2)M, the antibody used for the second step did not cross-react with any protein in the oocyte membrane extract (lane 5). In Fig. 3B, lane 6, OVR is visualized with the specific antireceptor antibody. The use of human alpha(2)M also gave us the opportunity to check electrophoretically for any ``fast'' form present in the preparation used in the ligand blotting experiments. Even upon extreme overloading of the gel we could not detect any fast form in freshly prepared human alpha(2)M (cf. Fig. 1); thus, native alpha(2)M from both chicken and human serum bound to the chicken OVR.

When we tried to visualize the binding of chicken alpha(2)M to the receptor by the same procedure (i.e. without the labeling of chicken alpha(2)M), the anti-chicken alpha(2)M antibody cross-reacted with too many bands present in oocyte membrane extracts, making the results inconclusive.

The ability of chicken OVR to bind both native and activated alpha(2)M prompted us to perform experiments to determine whether the two forms and the major yolk precursor ligands VLDL and VTG bound to common sites on the receptor. In the first set of experiments (Fig. 4), we tested the ability of activated and native alpha(2)M to compete with the binding of activated alpha(2)M. As shown in Fig. 4A, binding of activated alpha(2)M can be competed for by both native and activated alpha(2)M. If native alpha(2)M was used as the labeled ligand (panel C), only the native form effectively displaced the ligand from the receptor. Under these experimental conditions, trypsin-treated alpha(2)M, even at almost 10,000-fold molar excess, had only little effect on the binding of native alpha(2)M (Fig. 4C, lane 5), suggesting that native and activated alpha(2)M bound to closely related or overlapping, but not identical, sites on the chicken OVR.


Figure 4: Cross-competition of alpha(2)M and alpha(2)M* with VLDL and VTG for the binding to OVR. Oocyte membrane Triton X-100 extract (35 µg of protein/lane) was subjected to electrophoresis on a 4.5-18% SDS-gradient polyacrylamide gel under nonreducing conditions, transferred to nitrocellulose, and ligand blotting was performed as described under ``Experimental Procedures.'' Panels A and B, the blots were incubated with I-alpha(2)M (7 ng/ml, 13,000 cpm/ng) with the following additions: panel A: lane 1, none; lane 2, 3 µg/ml alpha(2)M; lane 3, 60 µg/ml alpha(2)M, lane 4, 3 µg/ml alpha(2)M; lane 6, 60 µg/ml alpha(2)M; panel B: lane 1, none; lane 2, 7 µg/ml VTG; lane 3, 8 µg/ml VLDL; lane 4, 80 µg/ml VLDL. Panels C and D, the blots were incubated with native I-alpha(2)M (7 ng/ml, 13,000 cpm/ng) with the following additions: panel C: lane 1, none; lane 2, 3 µg/ml alpha(2)M; lane 3, 60 µg/ml alpha(2)M; lane 4, 3 µg/ml alpha(2)M; lane 6, 60 µg/ml alpha(2)M; panel D: lane 1, none; lane 2, 7 µg/ml VTG; lane 3, 8 µg/ml VLDL; lane 4, 80 µg/ml VLDL. Exposure times: panel A, 6 h; panel B, 2 days; panel C, 6 h; panel D, 2 days.



Next, we performed similar ligand binding competition assays using as competitors the hitherto established ligands of the oocyte receptor, VLDL and VTG(3) . Activated alpha(2)M could be displaced only to a small extent by VTG and VLDL (Fig. 4B), whereas binding of native alpha(2)M appeared to be more sensitive to competition by VLDL (Fig. 4D); a 10,000-fold molar excess of VLDL abolished the binding of native alpha(2)M. VTG appeared similarly effective, but we could not test its effect at the high concentrations used for VLDL, since VTG tends to precipitate at conditions used for ligand blotting (2 mM CaCl(2); 37). These results indicate that (i) the binding sites for native and activated alpha(2)M on the alpha(2)M receptor are closely related to the binding site(s) for VLDL and VTG; and (ii) VLDL as a competitor appears to discriminate between binding sites for native versus activated alpha(2)M.

alpha(2)M Is a Yolk Component

To see if the binding of alpha(2)M to OVR is coupled to uptake into oocytes, we tested yolk from growing follicles for the presence of alpha(2)M. Since we think that native alpha(2)M is taken up in vivo (see below) and the major yolk precursors (VLDL, VTG) compete in vitro for the binding of native alpha(2)M to OVR, alpha(2)M was not expected to be a major yolk protein. Therefore we prepared enriched fractions from total yolk. Sucrose gradient centrifugation, followed by immunoblotting, indeed demonstrated the presence of alpha(2)M in yolk (Fig. 5). Under nonreducing conditions, we observed three or four immunoreactive bands with our immunopurified IgG in the yolk fraction (Fig. 5, lane 3), one of which comigrated with alpha(2)M from plasma. Higher M(r) forms of alpha(2)M under nonreducing conditions have been shown, for human alpha(2)M, to represent differently cross-linked products of alpha(2)M with proteases(46) . We do not know whether the additional bands in lane 3 were the chicken yolk equivalents of these products. Under reducing conditions, where the plasma protein is monomeric (180 kDa; lane 2), the antibody specifically reacted with a 85-kDa protein in the yolk fraction (lane 4). This fragment corresponds to the 85-kDa fragment(s) produced by cleavage of the 180-kDa subunits of human alpha(2)M with concomitant binding to trypsin(47) . To test whether the 85-kDa band seen in yolk was indeed derived from alpha(2)M by partial proteolysis, we reacted chicken alpha(2)M with chicken cathepsin D purified from follicles(40) . The rationale for using cathepsin D was based on our previous findings that this protease is responsible for the partial proteolytic attack of apoB, the major apolipoprotein of VLDL, and of VTG, during their transport via OVR into the oocyte; it appears to be the sole enzyme involved in intraoocytic ligand processing(40, 48) . As demonstrated in lane 6 (panel B), chicken alpha(2)M became cleaved in vitro upon incubation with cathepsin D at pH 5.5, conditions shown previously to reproduce intraoocytic ligand proteolysis of apoB and VTG (40) . Cathepsin D-treated alpha(2)M migrated under reducing conditions as 85-kDa fragment(s) indistinguishable from the fragment(s) seen in yolk (lane 4). The same sample, analyzed under nonreducing conditions (lane 5), showed a set of bands at the position of one of the major bands in yolk (lane 3), not identical with native alpha(2)M (lane 7). Inasmuch as other immunoreactive products are undetectable under these conditions, the additional immunoreactive components detected in yolk (lane 3, see above) likely are complexes of alpha(2)M with other yolk components that are not produced by cathepsin D from pure ligand. When, for control purposes, the incubation with cathepsin D was carried out in the presence of pepstatin A, a specific inhibitor of the enzyme (lanes 7 and 8), no change in the migration of alpha(2)M was observed.


Figure 5: Analysis of alpha(2)M in chicken follicles. Panel A, Western blotting under nonreducing (lanes 1 and 3) and reducing (lanes 2 and 4) conditions of purified chicken alpha(2)M (lanes 1 and 2; 45 µg/lane) and an enriched fraction from yolk from vitellogenic follicles (lanes 3 and 4; 10 µg/lane) was performed using microimmunoaffinity-purified rabbit anti-chicken alpha(2)M IgG (45 µg/ml) as described under ``Experimental Procedures.'' Exposure time was 6 min. Panel B, analysis of cathepsin D-digested purified chicken alpha(2)M. alpha(2)M (50 µg) was incubated with 3 units of cathepsin D at pH 5.5 for 20 h at 37 °C (40) in the absence (lanes 5 and 6) or presence (lanes 7 and 8) of 1 µg of pepstatin A. Samples were analyzed by SDS-PAGE under nonreducing (lanes 5 and 7) and reducing conditions (lanes 6 and 8). The gel was stained with Coomassie Brilliant Blue.



alpha(2)M Is Present in Clathrin-coated Vesicles

If OVR mediates the uptake of alpha(2)M into the oocyte in vivo, it should be possible to colocalize alpha(2)M and the receptor in coated vesicles. Therefore, we prepared clathrin-coated vesicles from oocytes and tested them by Western blotting for the presence of alpha(2)M. As shown in Fig. 6, alpha(2)M is indeed present in these endocytic organelles. Under nonreducing conditions, alpha(2)M present in coated vesicles had an electrophoretic mobility indistinguishable from plasma alpha(2)M (lane 1). Under reducing conditions (lane 2), alpha(2)M in coated vesicles behaved as a mixture of intact (180 kDa) and cleaved (85 kDa) monomers comparable to those detected in total yolk (cf. Fig. 5). The same coated vesicle preparation contained OVR, as visualized by immunoblotting with anti-OVR IgG (lane 3). Thus, alpha(2)M was present, at least transiently, in receptor-containing endocytic vesicles, further strengthening the notion that alpha(2)M is taken up by the oocyte via receptor-mediated endocytosis.


Figure 6: Immunoblot analysis of alpha(2)M in clathrin-coated vesicles from chicken oocytes. Proteins from coated vesicles prepared from vitellogenic follicles (lanes 1 and 2, 50 µg/lane; lane 3, 20 µg) were separated on a 4.5-12% SDS-gradient polyacrylamide gel under nonreducing (lanes 1 and 3) or reducing (lane 2) conditions and transferred to nitrocellulose membranes. Lanes 1 and 2, Western blotting was performed with microimmunoaffinity-purifed rabbit anti-chicken alpha(2)M IgG (45 µg/ml); exposure time was 6 min. Lane 3, Western blotting was performed with rabbit anti-chicken OVR IgG (16 µg/ml); exposure time was 30 s.



Uptake and Degradation of alpha(2)M in COS-7 Cells Expressing OVR

The results presented above strongly suggest that alpha(2)M is a ligand for OVR. The receptor would be expected to mediate uptake and deposition of alpha(2)M into the yolk of growing oocytes, in analogy to VLDL and VTG. To support this notion further, we transiently transfected COS-7 cells with a cDNA construct encoding OVR and used these cells to study OVR-mediated degradation of alpha(2)M. This technique was originally established to assess the uptake of LDL by cells expressing the LDL receptor (42) and measures the degradation of endocytosed ligands in lysosomes of cultured cells. For our purpose, we could not use trypsin-treated alpha(2)M because even after careful gel chromatographic removal of excess trypsin, incubation of cell monolayers with this preparation resulted in their partial detachment from the culture dishes. We therefore used alpha(2)MMA, which, in ligand blots, bound to OVR equally well as trypsin-treated alpha(2)M did (data not shown). The data in Fig. 7demonstrate that COS-7 cells expressing OVR specifically degraded alpha(2)MMA in a concentration-dependent fashion, reaching saturation at approximately 15 µg/ml. For yet unknown reasons, levels of OVR, which lacks an O-linked sugar region, in transiently transfected cells were very low, (^2)resulting in the receptor-mediated degradation of no more than 10 ng of alpha(2)MMA/mg of cell protein during 5 h of incubation.


Figure 7: Degradation of alpha(2)MMA by COS-7 cells expressing OVR. COS-7 cells were transfected with a plasmid carrying the full-length cDNA encoding for chicken OVR or the empty plasmid as described under ``Experimental Procedures.'' On day 2 after transfection, cell monolayers received 2 ml of standard medium containing the indicated concentrations of I-alpha(2)MMA. After 5 h of incubation, degradation products secreted into the medium were measured as amount of trichloroacetic acid-soluble radioactivity recovered from the cell supernatant. No-cell blanks were subtracted from the values obtained for OVR-expressing and control cells. OVR-mediated degradation was calculated by subtracting the values for control cells from those obtained for OVR-expressing cells. Each value represents the average of triplicate determinations.




DISCUSSION

There are several interesting aspects to the present finding that OVR is positioned at the crossroads of lipoprotein and alpha(2)M metabolism in the laying hen. First, we can gain further insights into structure/function relationships of members of the LDL receptor gene family. Recent cloning of chicken OVR (7) has revealed that it is an eight-binding repeat relative of the LDL receptor and highly homologous to the mammalian VLDL receptor(8, 9, 10, 11, 12, 13) . Taking into account that OVR binds such structurally unrelated ligands as VLDL and VTG(3) , apoE(16) , and alpha(2)M, as shown here, it presents itself as a multifunctional receptor like its much larger relative, LRP/alpha(2)MR. LRP/alpha(2)MR is a multiple-domain protein that contains 31 complement-type binding repeats clustered in four distinct subdomains containing 2, 8, 10, and 11 of such repeats(19) , respectively. As these clusters are separated from each other by long epidermal growth factor precursor repeats, this large receptor can be envisioned as a head-to-tail arrangement of functionally independent domains. Possibly, such a cassette-like domain arrangement confers the observed multiple ligand capacity to LRP/alpha(2)MR. Recent attempts at molecular dissection of the ligand binding sites on LRP/alpha(2)MR support this hypothesis, e.g. in that activated alpha(2)M and plasminogen activator/plasminogen activator inhibitor-1 complexes bind to distinct regions of the receptor protein(49, 50) . Considering the simple structure of OVR (one set of eight repeats), structure/function analysis of this multifunctional protein will facilitate greatly the elucidation of minimal requirements for the binding of different ligands.

Second, the physiological implications of the identification of a second alpha(2)MR, which not only binds activated alpha(2)M like LRP/alpha(2)MR, but also interacts with native alpha(2)M, are of interest. We are aware that the experiments demonstrating the binding of native alpha(2)M have to be evaluated carefully, since activation of the native form, which might occur during isolation or labeling, could lead to binding of the protein to the receptor. Importantly, analysis on nondenaturing gels of freshly isolated chicken alpha(2)M did not detect the presence of any fast form of the protein (Fig. 1), which would have resulted from a bait region cleavage by a protease. However, chicken alpha(2)M treated with small amines does not shift in electrophoretic mobility as human alpha(2)M does, where such shift is interpreted as conversion to an activated form. Thus, we also used as a ligand purified native human alpha(2)M, in which any activated form would have been detected as the fast form. Significantly, preparations of native human alpha(2)M, devoid of fast form when analyzed on nondenaturing gel systems, strongly labeled the oocyte receptor in ligand blots. In addition, we were able to use a sandwich ligand-Western blotting procedure for studying the binding of human alpha(2)M to OVR, which avoids any alterations in the ligand possibly caused by labeling procedures. This cross-species experiment demonstrates not only that human alpha(2)M can interact with an avian receptor but also that the binding of native alpha(2)M to OVR is not a peculiarity of the chicken ligand, but is due to the properties of the receptor.

Since being in the electrophoretically ``slow'' form may not suffice to indicate the presence of native alpha(2)M, as suggested by van Leuven et al.(51) , we also addressed the binding of both activated and native forms of alpha(2)M in competition ligand binding studies. Experiments using activated and native alpha(2)M, VLDL, and VTG as unlabeled competitors demonstrated that binding of native alpha(2)M to OVR seems to be qualitatively different from that of activated alpha(2)M. In these in vitro experiments, activated alpha(2)M does not compete effectively with the binding of the native protein, and VLDL displaces native alpha(2)M completely, but activated alpha(2)M less efficiently. This finding is very similar to that described for cross-competition of VTG and VLDL to OVR (5, 52) . In that case, unlabeled VTG competed for the binding of labeled VTG and VLDL, whereas VLDL did not effectively block binding of VTG to OVR. In analogy to VLDL/VTG, we interpret the current findings to indicate that the binding site for activated alpha(2)M represents a substructure of the recognition site for native alpha(2)M. It is conceivable that the presence of native alpha(2)M in the binding site decreases the affinity for activated alpha(2)M, but the reverse appears not to be the case. Taken together, these results demonstrate for the first time the existence of a receptor for activated and native alpha(2)M.

In addition to finding an alpha(2)M receptor in the oocyte membrane, we could demonstrate that alpha(2)M is a component of chicken yolk. Its presence and colocalization with the receptor in clathrin-coated vesicles derived from chicken follicles are consistent with receptor-mediated transport of alpha(2)M from the plasma compartment into growing oocytes. This notion is supported by two additional findings. First, plasma concentrations of alpha(2)M are significantly increased upon estrogen administration, which is an important common property of serum-borne yolk precursors like VLDL, VTG, and riboflavin-binding protein(53) . Second, we used transformed COS-7 cells expressing OVR to demonstrate directly that alpha(2)M undergoes OVR-mediated endocytosis. For two reasons, these experiments were performed with alpha(2)MMA only. First, trypsin-activated alpha(2)M, even after careful gel chromatographic removal of excess trypsin, resulted in partial detachment of the cells from the culture dishes. Second, since native alpha(2)M becomes activated under incubation conditions (37 °C, 5 h) used for the degradation experiments, data obtained would not reflect uptake of native alpha(2)M.

Nevertheless, COS-7 cells expressing OVR specifically take up and degrade alpha(2)MMA, demonstrating endocytotic competence of OVR toward at least one conformational form of alpha(2)M. In this respect, a recently published result by Andreasen et al.(54) needs consideration. There, the purification of a VLDL receptor from bovine mammary gland was reported, and the same protein was demonstrated to be expressed in a human mammary carcinoma cell line. Using these cells, the authors could not show binding to and degradation of alpha(2)M via this protein. This contradictory finding could have several causes. First, the identity of this protein as the VLDL receptor was suggested based on amino-terminal protein sequencing only, leaving the possibility that it is a homologous protein, but in fact not the VLDL receptor. Second, binding of alpha(2)M could be restricted to the chicken representative of the VLDL receptor group, which is the key player in oocyte growth via receptor-mediated endocytosis of several yolk precursors. However, this explanation is rendered less likely by the very high degree of similarity of the primary structures of OVR and mammalian VLDL receptors. Third, the endogenous expression of the VLDL receptor in mammalian tissues and cultured cells appears to be extremely low. This is the major obstacle to establishing unequivocally the ligand binding specificity of this receptor in mammalian systems. The problem is confounded by the fact that most potential ligands also bind to LRP/alpha(2)MR, which is expressed abundantly in these cells. As shown here for alpha(2)M, chicken OVR, expressed at very high levels in follicles, has already demonstrated its advantages as a superior system to study physiological ligands of this new group of LDL receptor-related proteins.

At present, we do not know the exact conformational species of alpha(2)M which binds to the receptor in vivo and is transported into the oocyte. We have, however, obtained initial insights into this aspect by analysis of the content of clathrin-coated vesicles, which represent the earliest isolatable structures of the endocytic pathway. Coated vesicles from small vitellogenic follicles (5-6 mm in diameter) (4) contain a mixture of the 180-kDa form and the 85-kDa fragment of alpha(2)M. Since alpha(2)M is a homotetrameric protein, this result could indicate either the presence of a mixture of bait region cleaved and native alpha(2)M or of partially proteolyzed alpha(2)M. In an attempt to resolve this question, we isolated coated vesicles from previtellogenic follicles (1-2 mm in diameter), whose oocytes had not yet entered the rapid growth phase. Despite the low yield of vesicles from these follicles, we found that most of the immunoreactive material migrated as the 180-kDa subunit and that only traces of the 85-kDa fragment of alpha(2)M were present (data not shown). Since there is no indication that coated vesicles derived from previtellogenic or vitellogenic follicles are functionally or structurally different, we suggest that the cleaved form seen in preparations derived from vitellogenic follicles is most likely a contamination from bona fide yolk, i.e. endosomal yolk spheres (4 and references therein). Yolk spheres comprise the final, membrane-bound compartment for storage of endocytosed yolk proteins and make up the body of yolk (4) . Being the end product of vesicular endocytic activity, they contain the proteolytically processed form of yolk components, e.g. the cleaved form of alpha(2)M. In turn, the previtellogenic follicles we analyzed do not contain significant amounts of yolk, and vesicles prepared thereof are less likely contaminated with yolk spheres. In experiments not shown, we repeatedly washed the vesicle preparation from vitellogenic follicles and observed continuous loss of only the 85-kDa band.

Based on the properties of OVR, it is intriguing to speculate that growing oocytes preferentially take up native alpha(2)M, i.e. the prevalent form of this protein in plasma, consistent with a continuous flow from the plasma compartment into rapidly growing oocytes. Significant oocyte uptake of activated alpha(2)M would imply a mechanism that would locally, probably in the ovarian tissue, or systemically produce activated alpha(2)M in continuous fashion. To us it seems unlikely that continuous protease activation of alpha(2)M, a process underlying its proposed primary function in vertebrates, should be necessary for the transport of a yolk component into the oocyte. As a corollary, the oocyte, because of its abundance of OVR(5) , and not the liver, in which OVR is not expressed(7) , would serve as the major sink of functionally expended alpha(2)M, which is equally unlikely. However, we cannot rule out that there exists a specific mechanism in the chicken to produce transport-competent alpha(2)M, different from the activation by the reaction with proteases.

We envision two possible functions for alpha(2)M in the yolk. First, since partial proteolytic digestion of yolk precursors appears to be a prerequisite for their deposition in yolk, alpha(2)M may serve subsequent to endocytosis to inactivate cathepsin D within the yolk and during early embryogenesis. In this case, cathepsin D must be kept active prior to its reaction with other yolk precursors. Second, considering the efficacy of the uptake process to sustain the reproductive effort of the hen (12-15 g of yolk are produced every 25 h), the transport of alpha(2)M into the oocyte may serve as piggyback mechanism for a variety of other substances. Recently, we have described the receptor-mediated cotransport of riboflavin-binding protein and VTG(53) . Candidate molecules for transport via alpha(2)M are growth factors and other small cytokines, many of which have been shown to have high affinity for alpha(2)M (for review see (55) ). Such a transport system involving OVR, a multifunctional receptor capable of binding different ligands that can serve as carriers for other minor yolk precursors, seems to be an elegant and efficient way to meet the requirements of the rapidly growing female germ cell.

The fact that chicken OVR is highly homologous to mammalian VLDL receptors together with our finding that it also binds alpha(2)M have implications for the evaluation of the function(s) of this receptor in mammals, where this is still a matter of debate. The intriguingly high conservation of this protein in egg-laying species and mammals does point to a very important common function. In this respect, the recent discovery that significant levels of the VLDL receptor mRNA are present in placenta of man(12) , mouse (11) , and rat (56) might indicate that the mammalian VLDL receptor is part of a multifunctional transport machinery in the very tissue that continuously supplies the growing embryo with nutrients, vitamins, growth factors, and possibly other components. Therefore, the VLDL receptor could play a key role in mammalian reproduction similar to that demonstrated here for OVR in a nonplacental species.


FOOTNOTES

*
These studies were supported in part by Grants P-9040-MOB and P-9508-MOB from the Austrian Science Foundation (FWF) (to W. J. S. and J. N.) and by Grant EECBMH1-CT93-1088 from the European Community (to W. J. S.). The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore by hereby marked ``advertisement'' in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

§
Supported by the Danish Science Research Council.

Supported by a Lise Meitner postdoctoral fellowship from the Austrian Science Foundation (FWF).

**
To whom correspondence should be addressed: Dept. of Molecular Genetics, University and Biocenter Vienna, Dr. Bohr-Gasse 9/2, A-1030 Vienna, Austria. Tel.: 43-1-79515-2111; Fax: 43-1-79515-2900

(^1)
The abbreviations used are: (V)LDL, (very) low density lipoprotein; VTG, vitellogenin; OVR, oocyte very low density lipoprotein/vitellogenin receptor; apo, apolipoprotein; alpha(2)M, alpha(2)-macroglobulin; alpha(2)MR, alpha(2)M receptor; alpha(2)M*, protease-activated alpha(2)M; alpha(2)MMA, methylamine-treated alpha(2)M; LRP, LDL receptor-related protein; PEG, polyethylene glycol; PAGE, polyacrylamide gel electrophoresis; MES, 4-morpholineethanesulfonic acid.

(^2)
H. Bujo, personal communication.


ACKNOWLEDGEMENTS

We appreciate the excellent photographic work of Romana Kukina and the technical assistance of Lourdes Mola. Human alpha(2)M was kindly provided by Dr. Lars Sottrup-Jensen.


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