Short communication Molecular characterization and expression analysis of the channel catﬁsh cathepsin D genes Tingting Feng a, b , Hao Zhang b , Hong Liu b , Zunchun Zhou b , Donghong Niu b , Lilian Wong b , Huseyin Kucuktas b , Xiaolin Liu a , Eric Peatman b , Zhanjiang Liu b, * a College of Animal Science and Technology, Northwest A&F University, Yangling, Shaanxi 712100, China b The Fish Molecular Genetics and Biotechnology Laboratory, Department of Fisheries and Allied Aquacultures and Program of Cell and Molecular Biosciences, Aquatic Genomics Unit, Auburn University, Auburn, AL 36849, USA article info Article history: Received 25 February 2011 Received in revised form 14 April 2011 Accepted 15 April 2011 Available online 1 May 2011 Keywords: Catﬁsh Cathepsin D Infection Innate immunity Antimicrobial abstract Cathepsin D is a lysosomal aspartic proteinase that participates in various degradation functions of the cell. In this study, we characterized the cathepsin D genes in channel catﬁsh and found two genes encoding catﬁsh cathepsin D, referred to as cathepsin D1 and D2 genes. These two genes are highly similar in genomic structure and organization, sharing a moderate level of amino acid sequence simi- larity (56%). Genomic Southern analysis suggested the presence of a single copy of each of the cathepsin D1 and D2 genes. Phylogenetic analysis provided strong evidence that two cathepsin D genes are present in most of the teleost lineage, with cathepsin D2 likely having been lost in some higher vertebrate lineages. The catﬁsh cathepsin D1 and D2 genes are expressed in virtually all the 11 tested tissues (brain, gill, heart, head kidney, trunk kidney, intestine, liver, muscle, skin, spleen, and stomach) on the transcript level, but appear to exhibit greater levels of expression in immune-related tissues and organs. Upon infection with Edwardsiella ictaluri, the expression of the catﬁsh cathepsin D genes showed the most signiﬁcant changes in liver and head kidney, with time points and magnitude of transcript changes varying between the two genes. We additionally examined bacterially-mediated changes of expression in gill, intestine, and trunk kidney. The fact that bacterial infection can induce expression of the cathepsin D genes and that they appeared to be expressed naturally at higher levels in immune-related organs may suggest that they are an important component of the innate immune response of catﬁsh against bacterial infections. Ó 2011 Elsevier Ltd. All rights reserved. 1. Introduction Lysosomes are membrane-bound cytoplasmic organelles that serve as major degradative compartments in eukaryotic cells [1]. They play an important role in maintaining cellular homeostasis. Endogenous and exogenous macromolecules can be delivered to the lysosome through the biosynthetic and endocytic pathways [2]. Waste materials and cellular debris are degraded in lysosomes through acid hydrolysis. Proteases of the cathepsin family are among the most thoroughly studied lysosomal hydrolases [3]. There are over a dozen cathepsin proteases in a typical animal cell, and they are distinguished by their substrate speciﬁcities. For instance, Cathepsin A and G are serine proteases; cathepsin B, C, F, H, K, L1, L2, O, S, W, and Z are cysteine proteases; while cathepsin D and E are aspartyl proteases. Cathepsin D is a 40 kDa protein with an isoelectric point of 6.95, whereas cathepsin E is a dimeric peptide each having a molecular mass of 40 kDa with an isoelectric point of 4.6 and 4.65 [4]. With the exception of cathepsin K, which works extracellularly after secretion by osteoclasts during bone resorption, most cathepsin proteases are within the lysosomes. Cathepsin D plays an important role in the lysosomal-mediated degradation of proteins [5]. It has a broad peptide bond speci- ﬁcity similar to pepsin and has been shown to be involved in various physiological pathways, such as intracellular catabolic proteolysis [6,7], extracellular proteolysis and processing, secretion and activation of enzymes and hormones [8]. Cathepsin D has been cloned and sequenced in a number of mammalian species [9,10], and in several species of ﬁsh such as tilapia (Tilapia nilotica  Tilapia aurea) [11], rainbow trout (Onco- rhynchus mykiss) [12], Antarctic iceﬁsh (Chionodraco hamatus) [13], seabream (Sparus aurata) [14], zebraﬁsh (Danio rerio) [15], carp (Cyprinus carpio) [16], herring (Clupea harengus) [17], pufferﬁsh * Corresponding author. Tel.: þ1 334 844 8727; fax: þ1 334 844 4694. E-mail address: zliu@acesag.auburn.edu (Z. Liu). Contents lists available at ScienceDirect Fish & Shellﬁsh Immunology journal homepage: www.elsevier.com/locate/fsi 1050-4648/$ e see front matter Ó 2011 Elsevier Ltd. All rights reserved. doi:10.1016/j.fsi.2011.04.006 Fish & Shellﬁsh Immunology 31 (2011) 164e169