Molecular cloning and functional characterization of chicken cathepsin D, a key enzyme for yolk formation.

Upon receptor-mediated endocytosis of very-low-density lipoprotein (VLDL) and vitellogenin into growing chicken oocytes, the protein moieties of these lipoproteins are proteolytically cleaved. Unlike the complete lysosomal degradation in somatic cells, enzymatic ligand breakdown in oocytes generates a characteristic set of polypeptides, which enter yolk storage compartments for subsequent utilization by the embryo. Here, we demonstrate directly that the catalyst for the intraoocytic processing of both apolipoprotein B and vitellogenin is cathepsin D. The enzyme was purified from oocytic yolk, preovulatory follicle homogenates, and liver by affinity chromatography. When plasma VLDL and vitellogenin were incubated with the purified enzyme, fragments indistinguishable from those found in yolk were generated from both precursors under identical, mildly acidic conditions. Amino-terminal sequencing of the pure enzyme demonstrated 88% identity with mammalian cathepsin Ds over 34 residues. On the basis of this information, a full-length clone specifying chicken preprocathepsin D was isolated from a chicken follicle cDNA library by screening with a human cathepsin D probe. Whereas previous studies have demonstrated that the receptors for lipoproteins in somatic cells and oocytes, respectively, of the chicken are the products of different genes, Southern and Northern blot hybridization experiments showed that the enzymes expressed in oocytes and liver are the product of a single gene, giving rise to a 3.3-kb transcript. The primary structure of the 335-residue mature protein suggests a high degree of conservation of known crucial features of aspartyl proteases; however, the absence of the so-called processing region and of an aromatic residue in a region thought to partake in catalysis raise questions with possible evolutionary implications.