Purification of properoxinectin, a myeloperoxidase homologue and its activation to a cell adhesion molecule Xionghui Lin a , Lage Cerenius a, ⁎ , Bok Luel Lee b , Kenneth Söderhäll a a Department of Comparative Physiology, Uppsala University, Norbyvägen 18A, SE-752 36 Uppsala, Sweden b College of Pharmacy, Pusan National University, Jangjeon Dong, Kumjeong Ku, Busan, 609-735, Korea Received 18 April 2006; received in revised form 5 June 2006; accepted 13 June 2006 Available online 25 July 2006 Abstract Peroxidases are important mediators of innate immune reactions throughout the animal kingdom. In many arthropods a myeloperoxidase homologue, peroxinectin, is known to function as a cell adhesion factor and an opsonin. Here, we report in the freshwater crayfish Pacifastacus leniusculus the isolation of properoxinectin, inactive in cell adhesion, and we also show that properoxinectin is produced in the mature blood cells whereas the hematopoietic tissue contains very little of this protein. Both properoxinectin and peroxinectin are catalytically active as peroxidases, at least when using low molecular weight substrates. The extracellular processing of properoxinectin into an active cell adhesion protein was found to involve proteolytic steps shared with the prophenoloxidase activating system to yield catalytically active phenoloxidase. Thus, the regulation of activities by two ancient metalloproteins, both potentially producing highly toxic substances aimed at pathogens, is carried out by limited proteolysis. The proteolytic processing is triggered in the presence of microbial compounds such as beta-glucans or lipopolysaccharide after the release of properoxinectin and prophenoloxidase activating serine proteinases from the blood cells. © 2006 Elsevier B.V. All rights reserved. Keywords: Peroxidase; Prophenoloxidase; Serine proteinase; Hemocyte 1. Introduction The animal peroxidase family or myeloperoxidase family [1] contains heme proteins with important roles in innate immunity, cell–cell interactions, hormone processing and for several other biological functions. To this group belongs myeloperoxidases, lactoperoxidases, eosinophil peroxidase and others that throughout the animal kingdom perform important innate immune reactions by producing compounds with antimicrobial activity by hydrogen peroxide-dependent reactions. Myeloper- oxidase creates an environment within the phagolysosome that will aid in killing endocytosed pathogens by its production of hypohalous acids and other toxic compounds [2,3]. Myeloper- oxidase may be released from the cells and the released enzyme can damage also the host by catalyzing tyrosyl radical formation, tyrosine peroxide, lipoprotein oxidation, etc., and, consequently, it is likely that its processing and activity need to be tightly controlled [4]. Outside the cell myeloperoxidase binds to integrins on leukocytes [5], an event that amplifies the neutrophil immune response [6]. Different intracellular proteolytic proces- sing events and glycosylation steps are required to produce the different forms of mammalian myeloperoxidase, but compar- ably little is known about the formation and function of the this enzyme outside the cells [4]. Some myeloperoxidases have a mosaic structure with a peroxidase domain located towards the carboxy-terminal and one or a few amino-terminal domains mediating other biological activities, e.g., cell–matrix and protein–protein interactions [7,8]. Among such mosaic perox- idases present in both vertebrates and invertebrates are peroxidasin first described in Drosophila melanogaster [7] and peroxinectin originally found in a freshwater crayfish, Pacifastacus leniusculus [9,10]. Peroxinectins have now been described in, e.g., D. melanogaster [11], Penaeus monodon Biochimica et Biophysica Acta 1770 (2007) 87 – 93 www.elsevier.com/locate/bbagen Abbreviations: proPO-system, prophenoloxidase activating system; PPAE, prophenoloxidase activating enzyme; HLS, hemocyte lysate supernatant; Hpt, hematopoietic tissue; CPBS, crayfish phosphate-buffered saline; TOF, time-of- flight; PVDF, polyvinylidene fluoride; BSA, bovine serum albumin; ABTS, 2, 2′-azino-di(3-ethylbenzthiazoline-6-sulfonate); CD, cytoplasmic domain ⁎ Corresponding author. E-mail address: Lage.Cerenius@ebc.uu.se (L. Cerenius). 0304-4165/$ - see front matter © 2006 Elsevier B.V. All rights reserved. doi:10.1016/j.bbagen.2006.06.018