IMMUNOLOGICAL IDENTIFICATION AND LOCALIZATION OF YEAST ASPARTIC PROTEASE 3-LIKE PROHORMONE-PROCESSING ENZYMES IN MAMMALIAN BRAIN AND PITUITARY

The novel aspartic proteases, yeast aspartic protease 3 and the mammal ian POMC-converting enzyme (PCE), can process prohormones at specific basic residue cleavage sites. We show that an antibody against yeast a spartic protease 3 (YAP3p) cross-reacted with purified bovine PCE on W estern blot, indicating structural homology between these two enzymes, but not with other aspartic proteases, such as renin or cathepsin D. A PCE-sized anti-YAP3p-immunoreactive band was detected on Western blo ts of bovine intermediate lobe where PCE activity has been found. YAP3 p antiserum also crossreacted with a protein of similar to 90 kDa from mouse hypothalamus and anterior pituitary, and bovine anterior pituit ary secretory granules. Distribution studies showed the presence of an ti-YAP3p-immunopositive cells in bovine pituitary and peptide-rich bra in regions, including the mouse arcuate nucleus and hippocampus and th e rat supraoptic nucleus, paraventricular nucleus, cortex, striatum, a nd reticular nucleus. In the bovine intermediate pituitary, a subpopul ation of cells was intensely stained with the YAP3p antiserum, and in combination with in situ hybridization, these cells were shown to cont ain POMC messenger RNA (mRNA). Only a subpopulation of cells was immun opositive for anti-YAP3p in bovine anterior pituitary, and most of the se cells were identified by double immunostaining with ACTH antiserum as corticotrophs. In situ hybridization in combination with immunocyto chemistry provided evidence for the localization of arginine vasopress in mRNA in YAP3p-immunopositive neurons in the rat supraoptic nucleus, whereas cholecystokinin mRNA was detected in YAP3p-immunopositive cel ls in the rat cortex and hippocampus. These results support the hypoth esis that YAP3p-like aspartic proteases, including PCE, play a role in prohormone processing in endocrine/neuroendocrine cells in vivo.