Symposium
Receptor-Mediated Lipoprotein Transport in Laying Hens1»2-3 JOHANNES
NIMPF AND WOLFGANG
J. SCHNEIDER4
Department of Biochemistry and Lipid and Lipoprotein Research Group, The University of Alberta, Edmonton, Alberta T6G 2S2, Canada tightly regulated by the well-established LDL receptor pathway (1).Although studies in mammalian systems show ubiquitous expression of this pathway, bio chemical evidence for or against an analogous prin ciple in birds is sparse. In light of the interesting physiology of egg-laying species, i.e., massive transport of VLDL and VTG into the oocyte on one side and cholesterol homeostasis in somatic cells on the other side, we have started to investigate the involvement of specific surface re ceptors in the regulation of these two separate but interrelated processes in the laying hen of Gollus domesticus.
ABSTRACT In laying hens, VLDL and vitellogenin (VTG) are secreted by the liver and eventually taken up by the growing oocyte via receptor-mediated endocytosis. Both macromolecules bind to the same receptor, termed the VLDL/VTG receptor, localized on the oocyte plasma membrane. Once taken up by the growing zy gote, apolipoprotein B, the major protein constituent of VLDL, is proteolytically cleaved by a chicken-specific cathepsin-D. Systemic cholesterol homeostasis in the chicken is maintained by expressing a different apoprotein B-specific receptor in somatic cells, which in terms of its function is very similar to the mammalian LDL receptor. The phenotype of the Restricted Ovulator hen, characterized by hereditary hyperlipidemia and the absence of egg laying, was identified as a lack of ex pression of functional VLDL/VTG receptors in the oocytes without affecting somatic apoprotein B receptors. J. Nutr. 121: 1471-1474, 1991.
THE CHICKEN OOCYTE RECEPTOR FOR VLDL
INDEXING KEY WORDS:
•receptors •oocyte •cholesterol •vitellogenin •chickens
Our initial efforts were directed toward the bio chemical characterization of a receptor molecule in
In oviparous species the developing embryo is abso lutely dependent on nutrients stored in the egg yolk. Most, if not all, yolk proteins are synthesized under the transcnptional control of estrogen in the liver, from where they are secreted into the blood stream. These macromolecules include the major components of the yolk, VLDL and vitellogenin (VTG),Svitaminbinding proteins such as riboflavin-, biotin-, and retinol-binding protein, and immunoglobulins. Once secreted into and transported by the blood to the ovary, these components are thought to be actively and specifically taken up by the growing oocytes. Existing evidence strongly suggests that at least VLDL and VTG are delivered to the oocytes by means of receptor-mediated endocytosis. Given this unidirec tional pathway of cholesterol and triglycéridefrom the liver to the oocytes in birds and other egg-laying species, one has to ask how cholesterol homeostasis is maintained and controlled in somatic cells. In mam mals, cholesterol homeostasis in extrahepatic cells is 0022-3166/91
$3.00 ©1991 American
Institute
of Nutrition.
'Symposium
presented
Downloaded from https://academic.oup.com/jn/article-abstract/121/9/1471/4754864 by University of Glasgow user on 03 April 2018
Poultry
Farms, Ltd., Smith Kline Beecham and Tyson Foods, Inc. 'Guest editor for this symposium was Pamela S. Hargis, Faculty of Nutrition, Department of Poultry Science, Texas Agricultural Experiment Station, The Texas A&M University System, College Station, TX. *The original research performed in the author's laboratory was supported by grants from the Medical Research Council of Canada and the Heart and Stroke Foundation of Canada and other support from the Alberta Foundation for Medical Research and the Medical Research Council of Canada. *To whom correspondence should be addressed. 'Abbreviations used: apo u, apolipoprotein VLDL-II; apo B, apoprotein B; M„molecular weight ratio,- RO, restricted VTG, vitellogenin.
Received 11 February 1991. Accepted 1471
as part of the 55th Annual
Nutrition Conference: Current Topics in Avian Lipid Metabolism and Biochemistry, given at the 74th Annual Meeting of the Feder ation of American Societies for Experimental Biology, Washington, DC, April 1, 1990, and supported by grants from Campbell Soup Company, Cuddy Farms, Inc., FJi Lilly and Company, Hoffman-La Roche, Holly Farms Foods, Inc., Perdue Farms, Inc., Pfizer, Pil grim's Pride Corporation, Rhône-Poulenc, Shaver Poultry Breeding
13 February 1991.
ovulator,-
1472
NIMPF AND SCHNEIDER
the plasma membrane of chicken oocytes that binds and internalizes VLDL. By means of ligand blotting and filter-binding assays, we found and characterized a 95-kDa protein in the oocyte plasma membrane that binds (2) and internalizes (3) VLDL. This binding is specific and is sensitive to EDTA, suramin, proteolytic enzymes and reductive methylation of the lysines in the protein portion of the ligand. Binding is mediated by the apoprotein B (apo B)moiety of VLDL. Apolipoprotein VLDL-n (apo H), which becomes a major part of these particles at the onset of egg laying, is not involved in receptor binding and internal ization of VLDL into the oocytes (4), although it plays an important role in the altered lipoprotein metabolism in the laying hen in comparison with roosters or immature hens. In experiments to define the speci ficity of the receptor binding, we found that the 95-kDa receptor protein also strongly binds mam malian lipoproteins containing apoprotein E, such as rabbit ß-VLDL. We demonstrated that this binding was indeed mediated by apoprotein E (5), an apoprotein that is not synthesized by birds (6). In addition to these functional similarities of the chicken oocyte receptor for VLDL with the mam malian LDL receptor, we discovered an immunological relationship between these two proteins. Polyclonal antibodies raised against purified bovine LDL receptor strongly cross-reacted with the 95-kDa protein from chicken oocyte membranes (2). Thus, the avian oocyte receptor for VLDL, despite its dif ferent molecular weight [the apparent molecular weight ratio (MJ of the mammalian counterpart is 35 kDa larger (7)], is surprisingly similar to the mam malian LDL receptor. Because other studies (3) showed that the 95-kDa protein not only binds VLDL in vitro, but also mediates its transport into oocytes in vivo, we investigated the fate of endocytosed VLDL. In mammals, receptor-mediated endocytosis of LDL leads to lysosomal degradation of the apo B-100 and hydrolysis of cholesterylester. The liberated cho lesterol serves cellular needs, inhibits cellular choles terol synthesis, and regulates downward the number of LDL receptors (1). In the chicken oocyte the en docytosed apo B undergoes partial proteolytical cleavage without affecting the composition or the size of the VLDL particle (8). This fragmentation seems to be catalyzed by a chicken-specific cathepsin-D. Be cause VLDL prepared from yolk exerts the same binding characteristics toward apo B-specific re ceptors in the chicken as native plasma-VLDL does, it is feasible that in this particular system VLDL is prepared to be stored and later used by the growing embryo via receptor-mediated endocytosis rather than being degraded as in mammalian somatic cells.
THE RESTRICTED
OVÃœLATOR HEN
The restricted ovulator (RO) hen was first described in 1974 (9) as a mutant chicken strain with hereditary Downloaded from https://academic.oup.com/jn/article-abstract/121/9/1471/4754864 by University of Glasgow user on 03 April 2018
hyperlipidemia and the lack of egg laying. Breeding studies suggest that this phenotype is due to a single, sex-linked gene defect (10). It has been suggested that the biochemical defect in RO hens may be an un paired transport of yolk precursors into the growing oocytes. Experiments in our laboratory to identify the exact defect demonstrated the absence of the previ ously characterized VLDL receptor in RO oocytes (3). Membrane-binding studies showed that ovarian mem branes from mutant hens did not bind VLDL in a specific and saturable manner as do membranes from their normal counterparts. Neither ligand blotting nor Western blotting experiments using radiolabeled VLDL or specific antibodies prepared against the chicken oocyte VLDL receptor revealed the 95-kDa protein in membrane preparations from RO ovaries. Finally, plasma clearance of intravenously injected radiolabeled VLDL was grossly reduced in RO hens. These results strongly support the notion that the absence of functional VLDL receptors from oocyte membranes represents the biochemical defect in the RO hen.
ROLE OF APOLIPOPROTEIN
VLDL-II
VLDL particles from laying hens contain large amounts of apo n in addition to apo B. Apolipoprotein VLDL-n, which is produced under the control of es trogen, is not involved in VLDL binding to its receptor in oocyte membranes as outlined above, but plays a crucial role in directing VLDL to the oocytes. ¿imammals, VLDL is secreted by the liver and undergoes rapid lipolysis by the action of lipoprotein lipase resulting in intermediate density lipoprotein and finally LDL. hi laying hens, VLDL does not un dergo any appreciable lipolysis en route from the liver to the oocytes. Furthermore, VLDL prepared from RO hens is apparently indistinguishable from normal VLDL, even though its half-life time in the circu lation is much longer than that of normal VLDL. Considering these facts, we have investigated the pos sibility that apo n functions as an inhibitor of lipoprotein lipase. Using rat myocytes and chicken granulosa cells as a source of lipoprotein lipase, apo n specifically inhibited lipoprotein lipase activity with a Kj of 40 mg/L (11). From these findings and the fact that apo n concentration in laying hen plasma is -400 mg/L, we conclude that the presence of apo U on VLDL ensures delivery of these triglyceride-rich par ticles to the oocyte membranes without substantial lipolysis.
THE OOCYTE RECEPTOR FOR VTG AND VLDL ARE THE SAME PROTEIN In addition to triglyceride-rich VLDL, plasma VTG constitutes the other major precursor of yolk compo nents in laying hens. Vitellogenin is a
SYMPOSIUM: AVIAN LIPID METABOLISM AND BIOCHEMISTRY
lipophosphoglycoprotein synthesized in the liver under the control of estrogen, secreted into the blood stream, and selectively taken up by growing oocytes. Using the same tools used for the characterization of the oocyte VLDL receptor, i.e., ligand blotting, we identified a protein in the oocyte plasma membrane that specifically binds VTG. The molecular weight of this protein (96 kDa) was very similar to that of the previously characterized VLDL receptor (12). Surpris ingly, the same polyclonal antibody against purified bovine LDL receptor, which cross-reacted with the VLDL receptor from chicken oocytes, immunoprecipitated VTG-binding activity from chicken oocyte membrane extracts. These findings, together with other biochemical similarities between the two proteins, prompted the idea that these two receptors are the same protein. In experiments to prove this hypothesis, we showed that binding activities for VLDL and VTG copurified on ligand affinity matrices (13). Furthermore, binding of VLDL and VTG was inhibited by the same antibody and both ligands com peted with each other for binding to the 95-kDa receptor. To further prove the point, we used the RO hen to obtain genetic evidence for the identity of these two receptors. As previously discussed, this mutant chicken strain carries a single gene defect responsible for the lack of expression of functional VLDL receptors. Using membranes prepared from RO ovaries we demonstrated not only that VLDL-binding activity is completely absent but also that VTG binding is grossly impaired. From these experiments we concluded that a single protein in oocyte mem branes binds both major yolk precursors, VLDL and VTG. These yolk precursors constitute >90% of the dry mass of yolk.
CHICKEN FIBROBLASTS EXPRESS A DIFFERENT APO B SPECIFIC RECEPTOR In parallel to the massive flux of VLDL and VTG from the liver to the growing oocytes in laying hens, there must be a way for somatic cells to indepen dently control and maintain cholesterol homeostasis. To address this problem we used cultured chicken embryo fibroblasts and carried out binding, intemalization and degradation studies in parallel with ligand and Western blotting experiments. The most in triguing finding was that fibroblasts express a dif ferent apo B specific receptor than do oocytes (14). This receptor has an apparent molecular weight of 130 kDa under non-reducing conditions and behaves very similarly to its mammalian counterparts in terms of regulating intracellular cholesterol levels. Interestingly, in contrast to the chicken oocyte receptor, this receptor does not bind ß-VLDL and does not cross-react with antibodies against the bovine Downloaded from https://academic.oup.com/jn/article-abstract/121/9/1471/4754864 by University of Glasgow user on 03 April 2018
LIPOPROTEIN
1473
RECEPTOR DICHOTOMY
IN THE LAVING HEN
Estrogen VTG
VLDL
i
Apo-ll
OOCYTES
SOMATIC
CELLS
FIGURE 1 Receptor-mediated pathways of vitellogeninand apolipoprotein B-containing particles in the laying hen. Estrogen stimulates the production in the liver of vitellogenin (VTG), which circulates as a dinier, and of VLDL, which contains apolipoprotein B (apo B) and apolipoprotein VLDL-n (apo H) as major protein components. Following secretion, VTG and VLDL become rapidly endocytosed by the growing oocyte via binding to a common receptor with an apparent molecular weight of 95,000 solely expressed in the oocyte. A small amount of VLDL becomes converted to LDL and is eventually taken up by somatic cells via endocytosis mediated by a different (M, - 130,000) receptor, which functions in systemic cholesterol homeostasis, in analogy to the mammalian LDL receptor.
LDL receptor. Because these findings point to similar ities but also differences between the somatic receptor in chicken and the mammalian LDL receptor on one hand and the oocyte receptor on the other hand, molecular cloning studies to delineate the molecular structure of the two different chicken re ceptors are underway. To determine whether the two different receptors in the hen are the product of two different genes, we again used the mutant RO hen, the phenotype of which we have shown to be due to the lack of expression of functional oocyte receptors for VLDL and VTG. We have been able to show that binding, uptake and degradation in fibroblasts cul tured from the skin of the mutant hens were exactly the same as in fibroblasts from normal hens. Further more, ligand blotting experiments confirmed the ex pression of the 130-kDa apo B specific receptor in fibroblasts from RO hens (unpublished data). These results clearly indicate that, in chickens, the two different apo B specific receptors are the product of different genes. In conclusion, Figure 1 schematically represents our current working model of the lipoprotein receptor
1474
NIMPF AND SCHNEIDER
dichotomy in the laying hen. During egg laying, massive amounts of VLDL and VTG are produced by the liver under the control of estrogen. Most of the VLDL, containing apo n in addition to apo B, escapes lipolysis by the protective action of apo n and be comes deposited by means of receptor-mediated endocytosis in the growing oocyte. The receptor respon sible for binding and internalization of VLDL is a 95-kDa protein that resides in the plasma membrane of growing oocytes. Surprisingly, binding and uptake of VTG, the second major yolk precursor, is mediated by the same protein, illustrating how economically the machinery required for the reproductive effort of the hen is designed. On the other hand, apo B specific receptors on somatic cells (130 kDa) differ from the oocyte receptor and maintain systemic lipoprotein homeostasis in analogy to the mammalian LDL receptor pathway. In RO hens, the failure to express functional oocyte receptors results in the inability to lay eggs and in the massive accumulation of lipids in the blood, and finally leads to severe atherosclerosis in the mutant hens.
LITERATURE CITED 1. Schneider, W. J. (1989) The low density lipoprotein receptor. Biochim. Biophys. Acta 988: 303-317. 2. George, R., Barber, D. L. &.Schneider, W. J. (1987) Characteri zation of the chicken oocyte receptor for low and very low density lipoproteins. J. Biol. Chem. 262: 16838-16847. 3. Nimpf, J., Radosavljevic, M. & Schneider, W. J. (1989) Oocytes from the mutant restricted ovulator hen lack receptor for very
Downloaded from https://academic.oup.com/jn/article-abstract/121/9/1471/4754864 by University of Glasgow user on 03 April 2018
low density lipoprotein. J. Biol. Chem. 264: 1393-1398. 4. Nimpf, J., George, R. &.Schneider, W. J. (1988) Apolipoprotein specificity of the chicken oocyte receptor for low and very low density lipoproteins: lack of recognition of apolipoprotein VLDL-n. J. Lipid Res. 29: 657-667. 5. Stevrer, E., Barber, D. L. &.Schneider, W. J. (1990)Evolution of lipoprotein receptors: The chicken oocyte receptor for very low density lipoprotein and vitellogenin binds the mammalian ligand, apolipoprotein E. J. Biol. Chem. 265: 19575-19581. 6. Hermier, D., Forgez, B. &. Chapman, M. J. (1985) A density gradient study of the lipoprotein and apolipoprotein distri bution in the chicken, Gallus domesticus. Biochim. Biophys. Acta 836: 105-118. 7. Daniel, T. O., Schneider, W. J., Goldstein, J. L. &.Brown, M. S. (1983)Visualization of lipoprotein receptors by ligand blotting. J. Biol. Chem. 258: 4606-^611. 8. Nimpf, J., Radosavljevic, M. &.Schneider, W. J. (1989) Specific postendocytic proteolysis of apolipoprotein B in oocytes does not abolish receptor recognition. Proc. Nati. Acad. Sci. USA 86: 906-910. 9. Ho, K.J., Lawrence, W. D., Lewis, L. A., Liu, L. B. &.Taylor, C. E. (1974) Hereditary hyperlipidemia in non-laying chickens. Arch. Pathol. 98: 161-172. 10. Jones, D. G., Briles, W. E. &.Schjeide, O. A. (1975)A mutation restricting ovulation in chickens. Poultry Sci. 54: 1780-1783. 11. Schneider, W. J., Carroll, R., Severson, D. L. &.Nimpf, J. (1990) Apolipoprotein VLDL-n inhibits lipolysis of triglyceride-rich lipoproteins in the laying hen. J. Lipid. Res. 31: 507-513. 12. Stifani, S., George, R. &.Schneider, W. J. (1988) Solubilization and characterization of the chicken oocyte vitellogenin receptor. Biochem. J. 250: 467-475. 13. Stifani, S., Barber, D. L., Nimpf, J. &.Schneider, W. J. (1990) A single chicken oocyte plasma membrane protein mediates uptake of very low density lipoprotein and vitellogenin. Proc. Nati. Acad. Sci. U.S.A. 87: 1955-1959. 14. Hayashi, K., Nimpf, J. &. Schneider, W. J. (1989) Chicken oocytes and fibroblasts express different apolipoprotein-Bspecific receptors. J. Biol. Chem. 264: 3131-3139.
Receptor-Mediated Lipoprotein Transport in Laying Hens1»2-3 JOHANNES
NIMPF AND WOLFGANG
J. SCHNEIDER4
Department of Biochemistry and Lipid and Lipoprotein Research Group, The University of Alberta, Edmonton, Alberta T6G 2S2, Canada tightly regulated by the well-established LDL receptor pathway (1).Although studies in mammalian systems show ubiquitous expression of this pathway, bio chemical evidence for or against an analogous prin ciple in birds is sparse. In light of the interesting physiology of egg-laying species, i.e., massive transport of VLDL and VTG into the oocyte on one side and cholesterol homeostasis in somatic cells on the other side, we have started to investigate the involvement of specific surface re ceptors in the regulation of these two separate but interrelated processes in the laying hen of Gollus domesticus.
ABSTRACT In laying hens, VLDL and vitellogenin (VTG) are secreted by the liver and eventually taken up by the growing oocyte via receptor-mediated endocytosis. Both macromolecules bind to the same receptor, termed the VLDL/VTG receptor, localized on the oocyte plasma membrane. Once taken up by the growing zy gote, apolipoprotein B, the major protein constituent of VLDL, is proteolytically cleaved by a chicken-specific cathepsin-D. Systemic cholesterol homeostasis in the chicken is maintained by expressing a different apoprotein B-specific receptor in somatic cells, which in terms of its function is very similar to the mammalian LDL receptor. The phenotype of the Restricted Ovulator hen, characterized by hereditary hyperlipidemia and the absence of egg laying, was identified as a lack of ex pression of functional VLDL/VTG receptors in the oocytes without affecting somatic apoprotein B receptors. J. Nutr. 121: 1471-1474, 1991.
THE CHICKEN OOCYTE RECEPTOR FOR VLDL
INDEXING KEY WORDS:
•receptors •oocyte •cholesterol •vitellogenin •chickens
Our initial efforts were directed toward the bio chemical characterization of a receptor molecule in
In oviparous species the developing embryo is abso lutely dependent on nutrients stored in the egg yolk. Most, if not all, yolk proteins are synthesized under the transcnptional control of estrogen in the liver, from where they are secreted into the blood stream. These macromolecules include the major components of the yolk, VLDL and vitellogenin (VTG),Svitaminbinding proteins such as riboflavin-, biotin-, and retinol-binding protein, and immunoglobulins. Once secreted into and transported by the blood to the ovary, these components are thought to be actively and specifically taken up by the growing oocytes. Existing evidence strongly suggests that at least VLDL and VTG are delivered to the oocytes by means of receptor-mediated endocytosis. Given this unidirec tional pathway of cholesterol and triglycéridefrom the liver to the oocytes in birds and other egg-laying species, one has to ask how cholesterol homeostasis is maintained and controlled in somatic cells. In mam mals, cholesterol homeostasis in extrahepatic cells is 0022-3166/91
$3.00 ©1991 American
Institute
of Nutrition.
'Symposium
presented
Downloaded from https://academic.oup.com/jn/article-abstract/121/9/1471/4754864 by University of Glasgow user on 03 April 2018
Poultry
Farms, Ltd., Smith Kline Beecham and Tyson Foods, Inc. 'Guest editor for this symposium was Pamela S. Hargis, Faculty of Nutrition, Department of Poultry Science, Texas Agricultural Experiment Station, The Texas A&M University System, College Station, TX. *The original research performed in the author's laboratory was supported by grants from the Medical Research Council of Canada and the Heart and Stroke Foundation of Canada and other support from the Alberta Foundation for Medical Research and the Medical Research Council of Canada. *To whom correspondence should be addressed. 'Abbreviations used: apo u, apolipoprotein VLDL-II; apo B, apoprotein B; M„molecular weight ratio,- RO, restricted VTG, vitellogenin.
Received 11 February 1991. Accepted 1471
as part of the 55th Annual
Nutrition Conference: Current Topics in Avian Lipid Metabolism and Biochemistry, given at the 74th Annual Meeting of the Feder ation of American Societies for Experimental Biology, Washington, DC, April 1, 1990, and supported by grants from Campbell Soup Company, Cuddy Farms, Inc., FJi Lilly and Company, Hoffman-La Roche, Holly Farms Foods, Inc., Perdue Farms, Inc., Pfizer, Pil grim's Pride Corporation, Rhône-Poulenc, Shaver Poultry Breeding
13 February 1991.
ovulator,-
1472
NIMPF AND SCHNEIDER
the plasma membrane of chicken oocytes that binds and internalizes VLDL. By means of ligand blotting and filter-binding assays, we found and characterized a 95-kDa protein in the oocyte plasma membrane that binds (2) and internalizes (3) VLDL. This binding is specific and is sensitive to EDTA, suramin, proteolytic enzymes and reductive methylation of the lysines in the protein portion of the ligand. Binding is mediated by the apoprotein B (apo B)moiety of VLDL. Apolipoprotein VLDL-n (apo H), which becomes a major part of these particles at the onset of egg laying, is not involved in receptor binding and internal ization of VLDL into the oocytes (4), although it plays an important role in the altered lipoprotein metabolism in the laying hen in comparison with roosters or immature hens. In experiments to define the speci ficity of the receptor binding, we found that the 95-kDa receptor protein also strongly binds mam malian lipoproteins containing apoprotein E, such as rabbit ß-VLDL. We demonstrated that this binding was indeed mediated by apoprotein E (5), an apoprotein that is not synthesized by birds (6). In addition to these functional similarities of the chicken oocyte receptor for VLDL with the mam malian LDL receptor, we discovered an immunological relationship between these two proteins. Polyclonal antibodies raised against purified bovine LDL receptor strongly cross-reacted with the 95-kDa protein from chicken oocyte membranes (2). Thus, the avian oocyte receptor for VLDL, despite its dif ferent molecular weight [the apparent molecular weight ratio (MJ of the mammalian counterpart is 35 kDa larger (7)], is surprisingly similar to the mam malian LDL receptor. Because other studies (3) showed that the 95-kDa protein not only binds VLDL in vitro, but also mediates its transport into oocytes in vivo, we investigated the fate of endocytosed VLDL. In mammals, receptor-mediated endocytosis of LDL leads to lysosomal degradation of the apo B-100 and hydrolysis of cholesterylester. The liberated cho lesterol serves cellular needs, inhibits cellular choles terol synthesis, and regulates downward the number of LDL receptors (1). In the chicken oocyte the en docytosed apo B undergoes partial proteolytical cleavage without affecting the composition or the size of the VLDL particle (8). This fragmentation seems to be catalyzed by a chicken-specific cathepsin-D. Be cause VLDL prepared from yolk exerts the same binding characteristics toward apo B-specific re ceptors in the chicken as native plasma-VLDL does, it is feasible that in this particular system VLDL is prepared to be stored and later used by the growing embryo via receptor-mediated endocytosis rather than being degraded as in mammalian somatic cells.
THE RESTRICTED
OVÃœLATOR HEN
The restricted ovulator (RO) hen was first described in 1974 (9) as a mutant chicken strain with hereditary Downloaded from https://academic.oup.com/jn/article-abstract/121/9/1471/4754864 by University of Glasgow user on 03 April 2018
hyperlipidemia and the lack of egg laying. Breeding studies suggest that this phenotype is due to a single, sex-linked gene defect (10). It has been suggested that the biochemical defect in RO hens may be an un paired transport of yolk precursors into the growing oocytes. Experiments in our laboratory to identify the exact defect demonstrated the absence of the previ ously characterized VLDL receptor in RO oocytes (3). Membrane-binding studies showed that ovarian mem branes from mutant hens did not bind VLDL in a specific and saturable manner as do membranes from their normal counterparts. Neither ligand blotting nor Western blotting experiments using radiolabeled VLDL or specific antibodies prepared against the chicken oocyte VLDL receptor revealed the 95-kDa protein in membrane preparations from RO ovaries. Finally, plasma clearance of intravenously injected radiolabeled VLDL was grossly reduced in RO hens. These results strongly support the notion that the absence of functional VLDL receptors from oocyte membranes represents the biochemical defect in the RO hen.
ROLE OF APOLIPOPROTEIN
VLDL-II
VLDL particles from laying hens contain large amounts of apo n in addition to apo B. Apolipoprotein VLDL-n, which is produced under the control of es trogen, is not involved in VLDL binding to its receptor in oocyte membranes as outlined above, but plays a crucial role in directing VLDL to the oocytes. ¿imammals, VLDL is secreted by the liver and undergoes rapid lipolysis by the action of lipoprotein lipase resulting in intermediate density lipoprotein and finally LDL. hi laying hens, VLDL does not un dergo any appreciable lipolysis en route from the liver to the oocytes. Furthermore, VLDL prepared from RO hens is apparently indistinguishable from normal VLDL, even though its half-life time in the circu lation is much longer than that of normal VLDL. Considering these facts, we have investigated the pos sibility that apo n functions as an inhibitor of lipoprotein lipase. Using rat myocytes and chicken granulosa cells as a source of lipoprotein lipase, apo n specifically inhibited lipoprotein lipase activity with a Kj of 40 mg/L (11). From these findings and the fact that apo n concentration in laying hen plasma is -400 mg/L, we conclude that the presence of apo U on VLDL ensures delivery of these triglyceride-rich par ticles to the oocyte membranes without substantial lipolysis.
THE OOCYTE RECEPTOR FOR VTG AND VLDL ARE THE SAME PROTEIN In addition to triglyceride-rich VLDL, plasma VTG constitutes the other major precursor of yolk compo nents in laying hens. Vitellogenin is a
SYMPOSIUM: AVIAN LIPID METABOLISM AND BIOCHEMISTRY
lipophosphoglycoprotein synthesized in the liver under the control of estrogen, secreted into the blood stream, and selectively taken up by growing oocytes. Using the same tools used for the characterization of the oocyte VLDL receptor, i.e., ligand blotting, we identified a protein in the oocyte plasma membrane that specifically binds VTG. The molecular weight of this protein (96 kDa) was very similar to that of the previously characterized VLDL receptor (12). Surpris ingly, the same polyclonal antibody against purified bovine LDL receptor, which cross-reacted with the VLDL receptor from chicken oocytes, immunoprecipitated VTG-binding activity from chicken oocyte membrane extracts. These findings, together with other biochemical similarities between the two proteins, prompted the idea that these two receptors are the same protein. In experiments to prove this hypothesis, we showed that binding activities for VLDL and VTG copurified on ligand affinity matrices (13). Furthermore, binding of VLDL and VTG was inhibited by the same antibody and both ligands com peted with each other for binding to the 95-kDa receptor. To further prove the point, we used the RO hen to obtain genetic evidence for the identity of these two receptors. As previously discussed, this mutant chicken strain carries a single gene defect responsible for the lack of expression of functional VLDL receptors. Using membranes prepared from RO ovaries we demonstrated not only that VLDL-binding activity is completely absent but also that VTG binding is grossly impaired. From these experiments we concluded that a single protein in oocyte mem branes binds both major yolk precursors, VLDL and VTG. These yolk precursors constitute >90% of the dry mass of yolk.
CHICKEN FIBROBLASTS EXPRESS A DIFFERENT APO B SPECIFIC RECEPTOR In parallel to the massive flux of VLDL and VTG from the liver to the growing oocytes in laying hens, there must be a way for somatic cells to indepen dently control and maintain cholesterol homeostasis. To address this problem we used cultured chicken embryo fibroblasts and carried out binding, intemalization and degradation studies in parallel with ligand and Western blotting experiments. The most in triguing finding was that fibroblasts express a dif ferent apo B specific receptor than do oocytes (14). This receptor has an apparent molecular weight of 130 kDa under non-reducing conditions and behaves very similarly to its mammalian counterparts in terms of regulating intracellular cholesterol levels. Interestingly, in contrast to the chicken oocyte receptor, this receptor does not bind ß-VLDL and does not cross-react with antibodies against the bovine Downloaded from https://academic.oup.com/jn/article-abstract/121/9/1471/4754864 by University of Glasgow user on 03 April 2018
LIPOPROTEIN
1473
RECEPTOR DICHOTOMY
IN THE LAVING HEN
Estrogen VTG
VLDL
i
Apo-ll
OOCYTES
SOMATIC
CELLS
FIGURE 1 Receptor-mediated pathways of vitellogeninand apolipoprotein B-containing particles in the laying hen. Estrogen stimulates the production in the liver of vitellogenin (VTG), which circulates as a dinier, and of VLDL, which contains apolipoprotein B (apo B) and apolipoprotein VLDL-n (apo H) as major protein components. Following secretion, VTG and VLDL become rapidly endocytosed by the growing oocyte via binding to a common receptor with an apparent molecular weight of 95,000 solely expressed in the oocyte. A small amount of VLDL becomes converted to LDL and is eventually taken up by somatic cells via endocytosis mediated by a different (M, - 130,000) receptor, which functions in systemic cholesterol homeostasis, in analogy to the mammalian LDL receptor.
LDL receptor. Because these findings point to similar ities but also differences between the somatic receptor in chicken and the mammalian LDL receptor on one hand and the oocyte receptor on the other hand, molecular cloning studies to delineate the molecular structure of the two different chicken re ceptors are underway. To determine whether the two different receptors in the hen are the product of two different genes, we again used the mutant RO hen, the phenotype of which we have shown to be due to the lack of expression of functional oocyte receptors for VLDL and VTG. We have been able to show that binding, uptake and degradation in fibroblasts cul tured from the skin of the mutant hens were exactly the same as in fibroblasts from normal hens. Further more, ligand blotting experiments confirmed the ex pression of the 130-kDa apo B specific receptor in fibroblasts from RO hens (unpublished data). These results clearly indicate that, in chickens, the two different apo B specific receptors are the product of different genes. In conclusion, Figure 1 schematically represents our current working model of the lipoprotein receptor
1474
NIMPF AND SCHNEIDER
dichotomy in the laying hen. During egg laying, massive amounts of VLDL and VTG are produced by the liver under the control of estrogen. Most of the VLDL, containing apo n in addition to apo B, escapes lipolysis by the protective action of apo n and be comes deposited by means of receptor-mediated endocytosis in the growing oocyte. The receptor respon sible for binding and internalization of VLDL is a 95-kDa protein that resides in the plasma membrane of growing oocytes. Surprisingly, binding and uptake of VTG, the second major yolk precursor, is mediated by the same protein, illustrating how economically the machinery required for the reproductive effort of the hen is designed. On the other hand, apo B specific receptors on somatic cells (130 kDa) differ from the oocyte receptor and maintain systemic lipoprotein homeostasis in analogy to the mammalian LDL receptor pathway. In RO hens, the failure to express functional oocyte receptors results in the inability to lay eggs and in the massive accumulation of lipids in the blood, and finally leads to severe atherosclerosis in the mutant hens.
LITERATURE CITED 1. Schneider, W. J. (1989) The low density lipoprotein receptor. Biochim. Biophys. Acta 988: 303-317. 2. George, R., Barber, D. L. &.Schneider, W. J. (1987) Characteri zation of the chicken oocyte receptor for low and very low density lipoproteins. J. Biol. Chem. 262: 16838-16847. 3. Nimpf, J., Radosavljevic, M. & Schneider, W. J. (1989) Oocytes from the mutant restricted ovulator hen lack receptor for very
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low density lipoprotein. J. Biol. Chem. 264: 1393-1398. 4. Nimpf, J., George, R. &.Schneider, W. J. (1988) Apolipoprotein specificity of the chicken oocyte receptor for low and very low density lipoproteins: lack of recognition of apolipoprotein VLDL-n. J. Lipid Res. 29: 657-667. 5. Stevrer, E., Barber, D. L. &.Schneider, W. J. (1990)Evolution of lipoprotein receptors: The chicken oocyte receptor for very low density lipoprotein and vitellogenin binds the mammalian ligand, apolipoprotein E. J. Biol. Chem. 265: 19575-19581. 6. Hermier, D., Forgez, B. &. Chapman, M. J. (1985) A density gradient study of the lipoprotein and apolipoprotein distri bution in the chicken, Gallus domesticus. Biochim. Biophys. Acta 836: 105-118. 7. Daniel, T. O., Schneider, W. J., Goldstein, J. L. &.Brown, M. S. (1983)Visualization of lipoprotein receptors by ligand blotting. J. Biol. Chem. 258: 4606-^611. 8. Nimpf, J., Radosavljevic, M. &.Schneider, W. J. (1989) Specific postendocytic proteolysis of apolipoprotein B in oocytes does not abolish receptor recognition. Proc. Nati. Acad. Sci. USA 86: 906-910. 9. Ho, K.J., Lawrence, W. D., Lewis, L. A., Liu, L. B. &.Taylor, C. E. (1974) Hereditary hyperlipidemia in non-laying chickens. Arch. Pathol. 98: 161-172. 10. Jones, D. G., Briles, W. E. &.Schjeide, O. A. (1975)A mutation restricting ovulation in chickens. Poultry Sci. 54: 1780-1783. 11. Schneider, W. J., Carroll, R., Severson, D. L. &.Nimpf, J. (1990) Apolipoprotein VLDL-n inhibits lipolysis of triglyceride-rich lipoproteins in the laying hen. J. Lipid. Res. 31: 507-513. 12. Stifani, S., George, R. &.Schneider, W. J. (1988) Solubilization and characterization of the chicken oocyte vitellogenin receptor. Biochem. J. 250: 467-475. 13. Stifani, S., Barber, D. L., Nimpf, J. &.Schneider, W. J. (1990) A single chicken oocyte plasma membrane protein mediates uptake of very low density lipoprotein and vitellogenin. Proc. Nati. Acad. Sci. U.S.A. 87: 1955-1959. 14. Hayashi, K., Nimpf, J. &. Schneider, W. J. (1989) Chicken oocytes and fibroblasts express different apolipoprotein-Bspecific receptors. J. Biol. Chem. 264: 3131-3139.
