Extracellular glycerophospholipid:cholesterol acyltransferase from Aeromonas salmonicida: activation by serine protease

Abstract

Extracellular haemolytie activities of Aeromonas salmonicida ssp. salmonicida to salmon red blood cells were shown to be due to different forms of the mcmbranc-activc enzyme glyccrophospholipid:choicstcrol acyltransferase {GCAT). About 10% of the total haemolytie activity was due to a high molecular mass complex of LPS and GCAT (mol. mass > 1000kDa), containing 35-50% neutral sugars and 1-5-2-0% protein. Some haemolytie activity (30-40% of total), corresponding to 5O-70kDa by gel filtration, also contained GCAT-aetivity and may represent aggregated forms of GCAT. However, about 50% or more of the haemol>tic activity was due to a protein of 26kDa free GCAT. Rabbit antibodies to GCAT neutralized the haemolytie activity of both GCAT and GCAT-LPS. A transposon-produced serine protease negative mutant of the same A. salmonicida strain showed reduced haemolytie -activity. The mutant produced a 38-kDa GCAT preform of low haemolytie activity. The proform was processed by autogenous serine protease to a highly haemolytie 26-kDa molecule with pi 6-3. similar to GCAT of the parent strain. The weakly haemolytie GCAT-LPS analogue of the mutant strain did not contain detectable amounts of the 26-kDa molecule and was not activated by proteases.

Inhibition of hemolytic activity of Aeromonas salmonicida GCAT in rainbow trout red blood cells by a monoclonal antibody

“…A 'nicking mechanism' forms the enzymatically active form of GCAT (31.7 kDa). The 3 bands found with esterolytic activity correspond with findings of Eggset et al (1994) describing the same properties of various GCAT forms using a protease deficient strain treated with a serine protease. The reactivity of the MAbs, in particular 7D2, with GCAT from crude ECP (Lachmann et al 1998), and on nitrocellulose after native PAGE, does not clearly exhibit the site of the epitope on the 3 parts of the molecule, but it appears not to be on the 3.7 kDa peptide excised by proteases.…”

“…salmonicida in a complex with lipopolysaccharide (LPS) has been characterized as a major lethal toxin for salmonids (Titball & Munn 1981, Buckley 1982, Lee & Ellis 1989, 1990, Arnesen et al 1993, Røsjø et al 1993, Eggset et al 1994. It was suggested subsequently that free GCAT contributes only very little to the toxicity of the pathogen because of its tendency to form aggregates with extracellular polysaccharides (Bricknell et al 1997).…”

Serum anti-LPS antibody production by rainbow trout (Oncorhynchus mykiss) in response to the administration of free and liposomally-incorporated LPS fromAeromonas salmonicida

“…The A-layer protein (Olivier et al, 1985;Thuvander et al, 1993), glycerophospholipid: cholesterol acyltransferase (GCAT) in its free form (Lee & Ellis, 1991) or in a complex with LPS (GCAT/LPS) (Lee & Ellis, 1990), an extracellular serine protease Eggset et al, 1994), and iron-regulated outer membrane proteins (IROMPS) (Hirst & Ellis, 1994) have been recognised as important protective antigens of A. salmonicida. Despite the ability of LPS and other antigens from A. salmonicida to elicit good antibody production in fish (Cipriano & Pyle, 1985;Lund et al, 1991;Thuvander et al, 1993), the ability of these components alone to stimulate substantial levels of protective immunity remains ambiguous.…”

Zinc Metalloproteinase ProA Directly Activates Legionella pneumophila PlaC Glycerophospholipid:cholesterol Acyltransferase

“…Cysteines Cys-343, Cys-388, Cys-415, and Cys-427 Are Essential for PlaC-associated PLA and GCAT Activities-It has been shown that the GCAT activity of A. salmonicida SatA requires activation by a bacterial serine proteinase (48). Additionally, it was published that trypsin is able to activate A. hydrophila SatA GCAT expressed in A. salmonicida by means of processing the protein between two cysteines (Cys-225 and Cys-281), forming a disulfide bridge (49).…”