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

Abstract

In holometabolous insects, there is a complete body remodeling from larva to adult. We determined in Ceratitis capitata that the transition from pre-pupa to pupa, 40 to 48 h after puparium formation (h APF), is a key moment of metamorphosis; when salivary glands, intestine, fat body, and muscles are in different stages of cell death. At 44-46 h APF, muscles from segments 1-3 (thoracic region) appeared fully disintegrated, whereas posterior muscles just started death processes. To understand some of the biochemical events eventually involved in histolytic processes during early metamorphosis, two cysteine peptidases coined "Metamorphosis Associated Cysteine Peptidase" (MACP-I and MACP-II) were purified to homogeneity from 40-46-h APF insects. Both enzymes were inhibited by Ep-475, a specific inhibitor of papain-like cysteine-peptidases. MACP-I is a single chain protein with an apparent molecular mass of 80 kDa and includes several isoforms with pI values of pH 6.25-6.35, 6.7, and 7.2. The enzyme has an optimum pH of 5.0 and its pH stability ranges from pH 4.0 to 6.0. The molecular weight and N-terminal sequence suggest that MACP-I might be a novel enzyme. MACP-II is an acidic single chain protein with a pI of pH 5.85 and an apparent molecular mass of 30 kDa. The enzyme is labile with a maximum stability in the pH range of 4.0 to 6.0 and an optimum pH among 5.0 to 6.0. MAPCP-II characteristics suggest it is a cathepsin B-like enzyme.

Ecdysone Receptor Agonism Leading to Lethal Molting Disruption in Arthropods: Review and Adverse Outcome Pathway Development

“…1C) have been identified in insects and crustaceans, such as trypsin-like serine proteases, cysteine proteases, carboxypeptidases and metalloproteases. [147][148][149][150][151][152][153][154][155][156][157][158][159][160][161] The Mfps are responsible for digestion of Cps by cleavage of the peptide bonds. More importantly, they may function as proteolytic activators of chitinase precursors.…”

“…Multiple types of molting fluid proteases (Mfp, Figure C) have been identified in insects and crustaceans, such as trypsin-like serine proteases, cysteine proteases, carboxypeptidases, and metalloproteases. − The Mfps are responsible for digestion of Cps by cleavage of the peptide bonds. More importantly, they may function as proteolytic activators of chitinase precursors. ,, The major serine proteases characterized in the molting fluid were found to be negatively regulated by the 20E titer, such as serine protease meta fission product-1 (Mfp-1) in the tobacco hornworm ( Manduca sexta ) , and trypsin-like protease 2 (Tlp2) in the cotton bollworm Helicoverpa armigera .…”

Peptidomics as a Tool to Assess the Cleavage of Wine Haze Proteins by Peptidases from Drosophila suzukii Larvae

“…These enzymes favor alkaline pH conditions and temperatures of 30–60 °C [ 48 , 49 , 50 ]. In contrast, cysteine peptidases have an optimal pH range of 4–6 [ 51 , 52 ] and they complement serine peptidases in insect nutrition [ 42 ]. Among the D. suzukii larval peptidases we identified, γ-glutamyltranspeptidase 1 (XP_016943864.1), caspase-3 (XP_016923550.1), and cathepsin L1 (XP_016943011.1) are cysteine peptidases, which in Drosophila species have been associated with apoptosis and the digestion of cytoplasmic components.…”

Chymotrypsin-like peptidases from Tribolium castaneum: A role in molting revealed by RNA interference

“…The molting fluid fills the exuvial space between the old and the new cuticle and exhibits chitinolytic and proteolytic activities that facilitate degradation of the inner layers of the old cuticle as part of the molting process before ecdysis. Several peptidases have been identified in insect molting fluids, including trypsin-like serine peptidases, carboxypeptidases, cysteine peptidases and metallopeptidases such as aminopeptidases (Dong et al, 2007;Liu et al, 2006Liu et al, , 2009Ote et al, 2005;Rabossi et al, 2008;Samuels et al, 1993a,b;Sui et al, 2009;Wei et al, 2007).…”