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 mammalian POMC-converting enzyme (PCE), can process prohormones at specific basic residue cleavage sites. We show that an antibody against yeast aspartic protease 3 (YAP3p) cross-reacted with purified bovine PCE on Western 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 blots of bovine intermediate lobe where PCE activity has been found. YAP3p antiserum also cross-reacted with a protein of approximately 90 kDa from mouse hypothalamus and anterior pituitary, and bovine anterior pituitary secretory granules. Distribution studies showed the presence of anti-YAP3p-immunopositive cells in bovine pituitary and peptide-rich brain regions, including the mouse arcuate nucleus and hippocampus and the rat supraoptic nucleus, paraventricular nucleus, cortex, striatum, and reticular nucleus. In the bovine intermediate pituitary, a subpopulation of cells was intensely stained with the YAP3p antiserum, and in combination with in situ hybridization, these cells were shown to contain POMC messenger RNA (mRNA). Only a subpopulation of cells was immunopositive for anti-YAP3p in bovine anterior pituitary, and most of these cells were identified by double immunostaining with ACTH antiserum as corticotrophs. In situ hybridization in combination with immunocytochemistry provided evidence for the localization of arginine vasopressin mRNA in YAP3p-immunopositive neurons in the rat supraoptic nucleus, whereas cholecystokinin mRNA was detected in YAP3p-immunopositive cells in the rat cortex and hippocampus. These results support the hypothesis that YAP3p-like aspartic proteases, including PCE, play a role in prohormone processing in endocrine/neuroendocrine cells in vivo.
Cawley
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Section on Cellular Neurobiology, National Institute of Child Health and Human Development, National Institutes of Health, Bethesda, Maryland 20892, USA.
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D000818:Animals; D016282:Aspartic Acid Endopeptidases / Q000032:analysis* / Q000276:immunology; D001483:Base Sequence; D015153:Blotting, Western; D001921:Brain / Q000201:enzymology*; D002417:Cattle; D003429:Cross Reactions; D003594:Cytoplasmic Granules / Q000201:enzymology; D019084:Fluorescent Antibody Technique, Indirect; D005656:Fungal Proteins / Q000032:analysis*; D007150:Immunohistochemistry; D008297:Male; D008322:Mammals; D051379:Mice; D008815:Mice, Inbred Strains; D009928:Organ Specificity; D010902:Pituitary Gland / Q000201:enzymology*; D016133:Polymerase Chain Reaction; D043484:Proprotein Convertases; D015347:RNA Probes; D051381:Rats; D017207:Rats, Sprague-Dawley; D012441:Saccharomyces cerevisiae / Q000201:enzymology*
D005656:Fungal Proteins; D015347:RNA Probes; D043484:Proprotein Convertases; D016282:Aspartic Acid Endopeptidases