Apolipoprotein A-I, an antimicrobial protein in Oncorhynchus mykiss: Evaluation of its expression in primary defence barriers and plasma levels in sick and healthy fish
Introduction
Besides the well-known role of HDL in reverse cholesterol transport and lipid metabolism, much evidence has accumulated in the recent years that involve this lipoprotein and its major apolipoprotein ApoA-I in diverse protective roles in mammals such as anti-atherosclerotic, anti-oxidant, anti-inflammatory and anti-thrombotic activities [1], [2], [3], [4], [5], [6], [7]. Among these protective functions, those related with primary defence seem to be extremely relevant, in fact, the proteomic analysis of mammalian HDL has recently demonstrated that it contains at least 56 associated proteins, including several proteins of the immune and complement system [8]. Moreover it has been suggested that HDL would constitute a platform for the assembly of different components that participate in innate immunity [9]. Although innate immunity constitutes the first line of defence in all vertebrates, it is widely recognized that it is critical for lower vertebrates, such as fish, because their acquired immunity is less developed than in mammals [10]. In this context, it was shown that HDL not only constitutes the most abundant plasma protein in the fish Cyprinus carpio (carp) but also that its major apolipoproteins (ApoA-I and ApoA-II), display potent antimicrobial activity in the micromolar range against Gram positive and Gram negative bacteria, including some fish pathogens [11], [12].
It was also reported that in this teleost fish both apolipoproteins are expressed not only in the liver but also in the epidermal layer where they are apparently secreted as part of nascent HDL particles to the mucus [11]. This finding is very relevant, especially taking in consideration that the mucus layer is in direct contact with the aquatic environment and therefore constitutes the first and most extended defensive barrier against pathogen invasion. In addition, it has been recently reported that ApoA-I could participate in the regulation of fish complement [13] and in the epithelial integrity of gill cell culture in trout [14].
Here it is shown that Oncorhynchus mykiss ApoA-I is not only an antimicrobial protein abundantly expressed in the most important epithelial and mucosal barriers, such as epidermis, gills, and intestinal mucosa, but also that its liver expression and plasma concentration does not decrease during the acute phase, as is the case in mammals [15], [16], [17]. These results confirm our previous findings in the carp and gives additional support to the idea of a critical role for HDL in innate immunity in teleost fish.
Section snippets
Chemicals
Chemicals were purchased from Sigma Chemical Co. (USA), if not otherwise specified. The reagents and enzymes used in molecular biology techniques were obtained from Invitrogen, except for Taq polymerase and RNasin® that were purchased from Promega.
Fish
Non-vaccinated rainbow trout (O. mykiss) were obtained from different fish farms (Province of Valdivia, Chile). One group of experimental fish was healthy and never exposed to disease while the other group corresponded to untreated diseased fish with
Purification of trout plasma HDL and apolipoprotein A-I
As shown in Fig. 1A, to isolate HDL from trout plasma by affinity chromatography on Affi-Gel-Blue-agarose, it was necessary to include an additional washing step with the chaotropic agent, NaSCN, to eliminate most contaminants previous to elution of HDL with ANS. Although this additional step resulted in some loss of Apo-AI from HDL particles (Fig. 1A inset, lane 2), most of it was recovered in the eluted fraction and the contamination with other proteins was negligible (Fig. 1A inset, lane 3).
Discussion
Apolipoprotein A-I, the major protein of HDL, has been extensively studied in mammals because of its important roles in lipid metabolism and anti-atherogenic properties [27]; however, only recently important defensive functions have been reported for this apolipoprotein in mammals and lower vertebrates [1], [2], [3], [4], [5], [6], [7], [8], [9], [10], [11], [12], [13], [14], [28].
As previously shown for carp HDL and its major apolipoproteins [11], [12], it was found that trout ApoA-I also
Acknowledgements
This research was supported by grant 1050637 from FONDECYT and S-2004-40 from the Dirección de Investigación y Desarrollo—Universidad Austral de Chile. We also would like to thank Cultivos Marinos Chiloe Ltda. for their generous donation of fish.
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