Phenoloxidase specific activity in the red swamp crayfishProcambarus clarkii

Abstract

The prophenoloxidase (proPO) system is considered an important mechanism of innate defence in arthropods. This enzymatic cascade has been studied in crustaceans such as the crayfishes Astacus astacus and Pacifastacus leniusculus and is located inside the haemocytes. An initial characterisation of this system in the commercially important red swamp crayfish Procambarus clarkii is described. The P. clarkii proPO system was activated by trypsin and also by zymosan A. This activation was calcium ion dependent. The calcium ion concentration also a#ected the background activation of the system and at 5 mM was highest as measured by the ability of phenoloxidase to oxidise L-3,4-dihydroxyphenylalanine. The e#ect of calcium ions appears to be related to the activation of an endogenous serine protease, but other calcium ion-dependent factors can also a#ect proPO activation. Lipopolysaccharides (LPS), glycolipids found in the outer leaflet of the outer membrane of Gram-negative bacteria, were also able to activate the proPO system in P. clarkii after a significant lag time of 25 to 30 min. However, LPS derivatives (deacylated LPS, lipid A and -D-GlcNAc-[1<6]-D-GlcNAc) were not able to activate the enzymatic cascade in P. clarkii. Activation of the proPO system in other crayfishes by LPS has been shown to be mediated by serine protease-like enzymes. The observed e#ect of LPS and LPS derivatives on the activation of the P. clarkii proPO system suggests that a protease activity triggered by these molecules may be mediated through the recognition of a ''complete'' LPS molecule (polysaccharide and lipid A). The intermolecular recognition of LPS by a putative endogenous serine protease zymogen might explain the lag time observed in proPO activation. Academic Press Limited

Flow cytometric analysis of crayfish haemocytes activated by lipopolysaccharides

“…This result agrees with the recognized endotoxic properties of lipid A in LPS [29,30], and the observed reduction of toxicity of deacylated LPS that still possesses potent inflammatory properties in humans [31,32]. The reaction of live haemocytes to LPS Rc, and LPSdex is similar to the reported effects of these molecules on the proPO activation from cell lysates, where LPS Rc was effective in activating the system and LPSdex was not [12]. This activation might be mediated by an endogenous haemocyte proteinase [13].…”

“…As structural differences in LPS molecules have been shown to effect the activation of proPO from HLS [12], haemocytes were incubated with the LPS derivatives LPS Rc (1 mg ml ÿ1 ) and LPSdex (10 mg ml ÿ1 ) (Fig. 3).…”

“…Phenoloxidase (PO)-specific activity was determined as described before [12], by using a volume of 50 ml from each TS. To render proPO active, trypsin (1 ml of a 10 mg ml ÿ1 solution) was included in duplicate sets of TS.…”

“…Documented as being important in the recognition and defence system of arthropods [11], the prophenoloxidase activating system ( proPO) can be converted to active phenoloxidase (PO) by LPS [12,13]. Activation of proPO from crayfish haemocyte lysates (HLS) requires lipid A acting in concert with some core polysaccharides of LPS because detoxified LPS (LPSdex), lipid A, or its sugar backbone b-D-GlcNAc-[1/6]-D-GlcNAc (compound X) acting alone failed to render proPO active [12]. This pattern of activation suggests that the polysaccharide portion of LPS is important in conditioning the endotoxic activity of lipid A of LPS in crayfish.…”

“…Based on the observations of proPO activation by LPS in crayfish haemocyte lysates [12,13,16], a better delineation of the effect of LPS on the cellular activation of crayfish haemocytes is needed. Flow cytometry was used to assess the patterns of cell activation.…”