Novel aspartyl proteinase associated to fat body histolysis during Ceratitis capitata early metamorphosis.

ABSTRACT

During larva to adult transition, the larval fat body of the Medfly (Ceratitis capitata) progressively disintegrates to be replaced by the adult one, after imago ecdysis. Here we show that a temporal correlation exists among the microscopy images of fat body progressive disintegration, the activation of fat body lysosomes (as judged by acid phosphatase activity), and the activity of a novel fat body aspartyl proteinase. The enzyme was purified and partially characterized. This proteinase exhibited a wide range of acid isoforms with isoelectric points from 5.6 to 7.3, an optimum pH of 3.0 for hemoglobin digestion, and was completely inhibited by pepstatin A. The apparent molecular weight was estimated (42 +/- 1 kDa) and the protein was characterized as N-glycosylated, judging from affinity to Concanavalin A. From the biochemical characteristics, the enzyme that we called "Early Metamorphosis Aspartyl Proteinase" (EMAP) appears to be similar to mammalian Cathepsin D. However, the N-terminal sequence of EMAP showed no similarity with any known animal Cathepsins and exhibited an important instability to neutral and alkaline pH. This feature seems to be a peculiar characteristic of insect aspartyl proteinases. The temporal activity profile of EMAP during metamorphosis correlated well with the microscopy images of fat body cell autolytic death. Our data support the notion that EMAP is a metamorphosis-specific lysosomal proteinase, mostly expressed during larval fat body histolysis.