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.

Intestinal aspartate proteases TiCatD and TiCatD2 of the haematophagous bug Triatoma infestans (Reduviidae): Sequence characterisation, expression pattern and characterisation of proteolytic activity

“…Data significantly different to the previous pH value are indicated by * (P 0.05), ** (P 0.01) and *** (P 0.001). and Ixodus ricinus, and are with about 43 kDa also congruent with the molecular mass of these enzymes (Terra et al, 1988;Cho et al, 1991;Cho and Raikhel, 1992;Rabossi et al, 2004;Gui et al, 2006;Sojka et al, 2008), except for the cathepsin D from Tribolium castaneum in which the molecular mass at 22 kDa is apparently smaller. The cathepsin D-like proteases from Aedes aegypti and R. prolixus have first been detected as native enzymes with molecular masses of 80 and 88 kDa, respectively, which seem to be homodimers respectively composed of two 40 and two 44 kDa subunits (Terra et al, 1988;Cho and Raikhel, 1992).…”

Purification and characterization of two cysteine peptidases of the Mediterranean fruit fly Ceratitis capitata during metamorphosis

“…In the Medfly, we described a temporal correlation between metamorphosis progress and the overall lysosomal proteolytic activity, catalyzed by aspartic and cystein proteinases (Rabossi and Quesada-Allué, 2000). This process involves the sequential dissociation of fat body adipocytes, followed by nuclear and cellular lysis (Rabossi et al, 2004). The aspartic proteinase appears at the onset of pupation, simultaneously with the cystein peptidases shown here.…”

The steroid hormone 20-hydroxyecdysone upregulated the protein phosphatase 6 for the programmed cell death in the insect midgut

“…Midgut remodeling includes a series of physiological events. The most critical are the larval midgut PCD and the formation of a new imaginal midgut by intestinal stem cells (Parthasarathy and Palli 2007;Rabossi et al 2004). Midgut PCD causes the larval midgut to condense to the yellow body, thus leading to decomposition to provide recycled material for the formation of the new midgut.…”