Molecular characterization of the first avian LDL receptor

Abstract

Low levels of expression and sluggish sterol-mediated regulation have been likely reasons for the failure to molecularly characterize a bona fide LDL receptor (LDLR) in egg-laying species to date. The overall structure of the chicken LDLR, delineated here by cDNA cloning, has been conserved in evolution, since hallmark properties of mammalian LDLRs are already present in the avian protein.The chicken receptor appears to prefer LDL over VLDL as ligand, in compliance with its main role in providing lipoprotein-derived cholesterol for steroid production in ovarian follicular cells. This is also compatible with the fact that estrogen administration increased hepatic LDLR expression in roosters despite dramatically stimulated VLDL production. In cultured chicken embryo fibroblasts, expression of the receptor was induced by incubation with cholesterol synthesis inhibitors such as a statin. Furthermore, preincubation of induced cells with a specific anti-receptor antibody blocks LDL endocytosis, demonstrating that the receptor is ligand-endocytosis competent. Finally, the distribution of LDLRs among the extraoocytic cell populations lends support to a three-cell model for estrogen production within the ovarian follicle.In summary, the molecular characterization of the first avian LDLR reveals novel information about evolutionary, structural, and functional aspects of members of the supergene family of LDLR-related proteins. -Hummel, S., E. G. Lynn, A. Osanger, S. Hirayama, J. Nimpf, and W. J. Schneider. One of the main tasks of females of oviparous (egg-laying) species is to coordinate synthetic and metabolic pathways for the production of eggs, i.e., their reproductive effort. In the domesticated chicken ( Gallus gallus ), lipoprotein metabolism in the laying hen (LH) is under stringent hormonal control, which assures that the massive lipoprotein flow required for oocyte growth can be met without compromising systemic lipid homeostasis (1, 2). Chickens lack apolipoprotein B-48 (apoB-48) and apoE; furthermore, only apoA-I, but not apoA-II, is synthesized by this species. Our past studies in the avian system have shown that hepatically derived triglyceride-rich VLDL particles harboring apoB-100 and the unique protein apo-VLDL-II (3) as main functional surface components are directed toward two sets of receptors belonging to the supergene family of the LDL receptor (LDLR) and LDLR relatives (LRs). One set of LRs is expressed almost exclusively and at high levels in oocytes; these receptors recognize, in addition to apoB-100 of VLDL, the lipophosphoglycoprotein vitellogenin, which together with VLDL constitutes the vast majority of yolk. The other set of LRs is synthesized in cells and tissues other than the oocytes; receptors of this group bind apoB, but not vitellogenin, and are generally expressed at low levels compared with receptor genes directly involved in oocyte growth (2,4,5).The yolk precursor receptors thus far identified and characterized are the avian homolog of the mammalian socalled VLDLR (6), in c...

Extracellular Matrices of the Avian Ovarian Follicle

“…After induction with 2 mM isopropyl-1-thio-␤-D-galactopyranoside for 4 h, the proteins were purified and folded essentially as described (18). GST⅐RAP-Myc was purified using glutathione-Sepharose 4B (Amersham Biosciences).…”

“…GST⅐RAP-Myc was purified using glutathione-Sepharose 4B (Amersham Biosciences). As described previously (18), the pMAL-perl2-4 was refolded by dialysis in the presence of RAP-Sepharose. The bound and refolded protein was then eluted from the RAP-Sepharose with 0.5 ϫ TBS ϩ 2 mM CaCl 2 (TBS-C), 1 M NH 4 OH.…”

“…We utilized our recently developed system to express functionally folded clusters of LA repeats, as described for the chicken LDL receptor ligandbinding domain (18). To our knowledge, this domain has not been expressed previously without being linked to neighboring domain(s), nor has it been studied in terms of function (26,51,52).…”

“…I-VLDL was generated by the iodine monochloride method as described (18) and extensively dialyzed against 50 mM NaCl, 0.2 mM EDTA (pH 7.4) before use and stored at 4°C. Indicated concentrations represent the protein content.…”

“…Indicated concentrations represent the protein content. Mouse NIH3T3 cells were grown overnight on culture slides in medium supplemented with lipoprotein-deficient serum in the presence of 1 M NK104 to maximally induce LDL receptor expression (18) and then incubated for 1 h at 37°C with LDL alone or in the presence of perl2-4 at the indicated concentrations. Following incubation, the cells were fixed and permeabilized with methanol/acetone (1/1, by volume) at Ϫ20°C, incubated with rabbit anti-chicken apoB antiserum (1:500) (20) followed by 5 g/ml Alexa Fluor 568-labeled anti-rabbit IgG (Molecular Probes).…”