Proc. Natl. Acad. Sci. USA Vol. 86, pp. 906-910, February 1989 Cell Biology Specific postendocytic proteolysis of apolipoprotein B in oocytes does not abolish receptor recognition (lipoproteins/pepstatin A/endosome) JOHANNES NIMPF, MARKUS RADOSAVLJEVIC, AND WOLFGANG J. SCHNEIDER* Department of Biochemistry and Lipid and Lipoprotein Research Group, University of Alberta, Edmonton, AB, Canadi T6G 2H7 Communicated by Richard J. Havel, October 11, 1988 ABSTRACT Upon receptor-mediated transfer of plasma family ofglycophospholipoproteins with molecular masses of very low density lipoprotein (VLDL) particles into growing -240 kDa, which are degraded inside the oocyte to lipovi- chicken oocytes, their major apolipoprotein (apo) component, tellin(s) (the amino-terminal portion of vitellogenins), phos- apoB, is proteolytically cleaved. apoB fragmentation appears vitin(s) (the central, phosphoserine/serine-rich domain), and to be catalyzed by cathepsin D or a similar pepstatin A-sensitive carboxyl-terminal polypeptides termed phosvettes or lipovi- protease and results in the presence of a characteristic set of tellin(s) II. The specific breakdown of vitellogenin has been polypeptides on yolk VLDL particles. The nicks introduced suggested to constitute a regulatory step in the fusion into the apoB backbone during postendocytic processing occur between endocytic compartments in Xenopus oocytes (18). in yolk platelets and appear to prepare internalized VLDL for Lastly, it is known that chicken egg yolk does not contain storage in yolk. Since yolk VLDL binds to chicken receptors intact apolipoprotein B (apoB), the protein that directs specific for apoB-containing lipoproteins in identical fashion to VLDL particles to receptors on the oocyte plasma membrane plasma VLDL, the possibility exists that the developing embryo (1, 19-21). utilizes yolk VLDL as a nutrient by way of receptor-mediated Besides having a function in maintenance of a high rate of endocytosis. yolk formation, specific proteolysis may play a role in assuring the developing embryo access to macromolecular Specific cell-surface receptors are involved in the endocyto- nutrients, such as lipoproteins. Since this access might, at into least in part depend on receptor-mediated endocytosis, we sis of very low density lipoprotein (VLDL) particles have become interested in the binding behavior of VLDL growing oocytes of the laying hen (1). The massive import of particles stored in yolk. Here we report on a possible VLDL is part of the process of yolk deposition that assures mechanism for the processing of plasma VLDL by in- accumulation of sufficient amounts of nutrients in the oocyte traoocytic proteolysis and demonstrate that the proteolyzed for growth of the avian embryo. In addition to VLDL, most, yolk VLDL has identical receptor-binding characteristics as if not all, yolk components are believed to be transported the lipoprotein in the circulation. across the plasma membrane ofthe oocyte by way of specific receptors. Systems of receptor-mediated endocytosis may exist for transferrin (2, 3), various vitamin-binding proteins MATERIALS AND METHODS (4-12), and IgG (13, 14). Though direct biochemical demon- Materials. We obtained pepstatin A, thrombin (human stration ofoocyte receptors for most ofthese proteins has not plasma, no. T-6759), kallikrein (porcine pancreas, no. K- been reported, we have recently characterized the chicken 3627), cathepsin D (bovine spleen, no. C-3138), and bovine oocyte plasma membrane proteins responsible for the trans- serum albumin (no. P-5763) from Sigma; molecular mass port of VLDL (1) and vitellogenin, another major yolk standards from BRL; acrylamide, bisacrylamide, and glycine component (15). from Schwartz/Mann; nitrocellulose paper BA 85 from We are now concerned with the mechanisms by which the Schleicher & Schuell; Nuflow acetate membrane filters N25/ chicken embryo utilizes the nutrient stores of the oocyte 45 from Oxoid (Basingstoke, U.K.); goat anti-rabbit IgG (no. content for growth. Presumably, the lipoproteins VLDL and 0612-3151) from Cooper Biomedical; and Na'25I from Ed- vitellogenin are a major source not only for protein and monton Radiopharmaceutical Center (Edmonton, AB, Can- carbohydrate components but also for essential lipid building ada). Other materials were from previously reported sources blocks and energy. It appears that most of those plasma (1, 21). proteins that are concentrated in the oocyte through receptor- Animals and Diets. White Leghorn layers (8-18 months old) mediated endocytosis undergo specific proteolytic process- were obtained from a local poultry farm and maintained on ing following uptake from the plasma. This has been sug- layer mash fed ad libidum, with a light period of 12 hr. White gested for riboflavin-binding protein (16), which exists in Leghorn roosters (4-8 weeks old) were kindly provided by F. three closely related but distinct forms in the laying hen that Robinson (Department of Animal Sciences, The University can be isolated from plasma, egg white, and yolk. The egg of Alberta, Edmonton, AB, Canada) and maintained on white form is distinguished from the other two forms by its grower mash at the same conditions as the hens. We used different carbohydrate composition; the yolk protein is a adult female New Zealand White rabbits for raising antibod- truncated form of the plasma protein, lacking 11 or 13 amino ies. acid residues at the carboxyl terminus (5, 6). The carboxyl- Preparation and Analysis of Lipoproteins, Filtration Assay. terminal truncation in yolk riboflavin-binding protein has Lipoprotein fractions were isolated as described (21). VLDL been proposed to be elicited by specific intraoocytically from yolk (yVLDL) was isolated as follows: the yolk from localized protease(s) (6, 17). Another example of specific one freshly laid egg was separated carefully from the egg postendocytic processing is provided by the vitellogenins, a white and rinsed with ice-cold saline. The yolk membrane The publication costs of this article were defrayed in part by page charge Abbreviations: apo, apolipoprotein; (V)LDL, (very) low density payment. This article must therefore be hereby marked "advertisement" lipoprotein; pVLDL, plasma VLDL; yVLDL, yolk VLDL. in accordance with 18 U.S.C. §1734 solely to indicate this fact. *To whom reprint requests should be addressed. 906 Downloaded by guest on September 28, 2021 Cell Biology: Nimpf et al. Proc. Natl. Acad. Sci. USA 86 (1989) 907 was punctured with a needle and the yolk was squeezed out Table 1. Comparison of pVLDL and yVLDL composition and mixed with 5 vol of ice-cold buffer (20 mM Tris HCl/150 Relative % mM NaCI/0.2 mM EDTA/1 mM phenylmethylsulfonyl flu- oride/5 AM leupeptin, pH 8). The suspended yolk was Component pVLDL yVLDL subjected to ultracentrifugation at 150,000 x ga, for 12 hr at Protein 12.3 12.9 40C. The floating waxy yellow material was mixed with 10 vol Cholesterol, free + esterified 4.6 4.3 of buffer as above and subjected to a second centrifugation Phospholipid 16.8 17.0 under the same conditions. The yVLDL was recovered from Triglyceride 66.3 65.8 the top of the tube. Lipoproteins were radiolabeled with 1251 by the iodine monochloride method as described (1). All pVLDL and yVLDL were purified, and their content of protein, lipoproteins were extensively dialyzed against 0.15 M NaCl/ total cholesterol, phospholipid, and triglyceride was determined. 0.2 mM EDTA, pH 7.4, and stored at 40C. Lipoprotein g/ml and compared the composition (Table 1), morphology, concentrations are expressed in terms ofprotein content (22). and protein components (Fig. 1) ofthe particles. As shown in Triglyceride, cholesterol, and phospholipid contents of iso- Table 1, the relative amounts of triglyceride, cholesterol, lated VLDL fractions were determined with commercially phospholipid, and protein were essentially identical in the available assay kits. Oocyte membranes were prepared from two particles. These data suggested that pVLDL particles are small oocytes (3-15 mm in diameter) and extracted with octyl transported into the oocyte without major disruption of their glucoside as described (1). After dilution of the detergent to structural integrity, such as by lipolytic processes. This below its critical micellar concentration, the receptor protein notion was supported by morphological analysis of the was recovered by centrifugation and the pellet was used for isolated particles (Fig. 1 A and B)-namely, VLDL particles a solid-phase filtration assay as described (1, 23). of both plasma and yolk were spherical and uniform in size, Electron Microscopy. VLDL samples were negatively with a diameter of 38 + 0.8 nm (mean ± SEM, determined on stained with 2% sodium phosphotungstate (pH 7) and pho- samples of 400 particles from each source). These findings tographed in a Philips EM420 operated at 100 kV. The agree well with those of Burley et al. (20), who determined instrumental magnification was x60,000 and the low-dose mean particle diameters of 35 and 36 nm for pVLDL and unit was employed to minimize specimen damage. "yolk lipoprotein," respectively. Together with the obser- Electrophoretic and Blotting Procedures. One-dimensional vations ofGilbert and collaborators (27, 28), who showed that sodium dodecyl sulfate (SDS) gel electrophoresis was per- VLDL particles traverse the interstitial space of the granu- formed according to Laemmli (24) using a mini-gel system losa cell layer as well as the basal lamina unchanged, our data (mini Protean II slab cell) or a regular gel system (Protean II strongly suggest that intact pVLDL particles are transported slab cell) from Bio-Rad. Electrophoresis was conducted on into the oocyte. The accumulated evidence does not support gradient gels (4.5-18% polyacrylamide) at 200 V at room a previous suggestion (29) that lipoprotein lipase produced by temperature for 40-50 min for mini gels and at 35 mA per gel the granulosa cells might act on pVLDL in order to mediate at 10°C for 6-7 hr for regular gels (16 x 12 x 0.15 cm). the transport of VLDL-derived triglyceride fatty acids into Lipoprotein samples were delipidated by successive extrac- the oocyte.