Proteasomal activities in the claw muscle tissue of European lobster, Homarus gammarus, during larval development

Abstract

Decapod crustaceans grow discontinuously and gain size through complex molt processes. The molt comprises the loss of the old cuticle and, moreover, substantial reduction and re-organization of muscles and connective tissues. In adult lobsters, the muscle tissue of the massive claws undergoes significant atrophy of 40-75% before ecdysis. The degradation of this tissue is facilitated by calcium-dependent proteases and by the proteasome, an intra-cellular proteolytic multi-enzyme complex. In contrast to the adults, the involvement of the proteasome during the larval development is yet not validated. Therefore, we developed micro-methods to measure the 20S and the 26S proteasomal activities within mg-and sub-mg-quantities of the larval claw tissue of the European lobster, Homarus gammarus. Within the three larval stages (Z1-3) we distinguished between sub-stages of freshly molted/hatched (post-molt), inter-molt, and ready to molt (pre-molt) larvae. Juveniles were analyzed in the post-molt and in the inter-molt stage. The trypsin-like, the chymotrypsin-like, and the peptidyl-glutamyl peptide hydrolase activity (PGPH) of the 20S proteasome increased distinctly from freshly hatched larvae to pre-molt Z1. During the Z2 stage, the activities were highest in the post-molt animals, decreased in the inter-molt animals and increased again in the pre-molt animals. A similar but less distinct trend was evident in the Z3 stages. In the juveniles, the proteasomal activities decreased toward the lowest values. A similar pattern was present for the chymotrypsin-like activity of the 26S proteasome. The results show that the proteasome plays a significant role during the larval development of lobsters. This is not only reflected by the elevated activities, but also by the continuous change of the trypsin/ chymotrypsin-ratio which may indicate a shift in the subunit composition of the proteasome and, thus, a biochemical adjustment to better cope with elevated protein turnover rates during larval development.

The proteasomes of two marine decapod crustaceans, European lobster (Homarus gammarus) and Edible crab (Cancer pagurus), are differently impaired by heavy metals

“…The fraction that we separated by glycerol-gradient centrifugation contained most likely both, the 20S and the 26S population of the proteasome. Previous studies on crustaceans showed that both forms of the proteasome are active under physiological conditions (Mykles, 1998(Mykles, , 1999aGötze and Saborowski, 2011). Therefore, the use of a mixed population of the proteasome appears suitable for our study.…”

Pitfalls in invertebrate proteasome assays

“…Instead, short fluorophore-labeled peptides are commonly used, which are less specific and can therefore also be targeted by other proteolytic enzymes (Rosser et al, 1993;Garcia et al, 1998;Michael et al, 2005). Of particular importance when preparing crude tissue extracts is avoiding contamination with digestive enzymes from the gut or digestive glands of the animals, which may entirely mask the innate proteasomal activity of the sample (Götze and Saborowski, 2011a;A.B., personal observation). So, when starting work with new species the degree of cross-reactivity needs to be determined in crude extracts of uncontaminated samples.…”

“…For example, the proteasome is thought to play an important role in claw muscle degradation during the molting process of adult and larval lobsters (Mykles, 1999b;Götze and Saborowski, 2011a). Bivalves were established as new model organisms to investigate physiological processes such as stress tolerance and ageing (Cherkasov et al, 2006;Ivanina et al, 2008;Philipp and Abele, 2010).…”