Molecular characterization of a novel pattern recognition protein from nonspecific cytotoxic cells: Sequence analysis, phylogenetic comparisons and anti-microbial activity of a recombinant homologue

Abstract

Nonspecific cytotoxic cells (NCC) are the first identified and most extensively studied killer cell population in teleosts. NCC kill a wide variety of target cells including tumor cells, virally transformed cells and protozoan parasites. The present study identified a novel evolutionarily conserved oligodeoxynucleotide (ODN) binding membrane protein expressed by channel catfish (Ictalurus punctatus) NCC. Peptide fingerprinting analysis of the ODN binding protein (referred to as NCC cationic anti-microbial protein-1/ncamp-1) identified a peptide that was used to design degenerate primers. A catfish NCC cDNA library was used as template with these primers and the PCR-amplified product was sequenced. The translated sequence contained 203 amino acids (molecular mass of 22,064.63 Da) with characteristic lysine rich regions and a pI=pH 10.75. Sequence comparisons of this protein indicated similarity to zebrafish (51.2%) histone family member 1-X and (to a lesser extent) to trout H1. A search of EST databases confirmed that ncamp-1 is also expressed in various tissues of channel catfish as well as zebrafish. Inspection for signature repeats in ncamp-1 and comparisons with histone-like peptides from different species indicated the presence of multiple lysine based motifs composed of AKKA or PKK repeats. The novel protein was cloned, expressed in E. coli and the recombinant was used to generate rabbit anti-serum. The recombinant ncamp-1 bound GpC and CpG ODNs and was detected with homologous anti-ncamp-1 polyclonal antibodies. Western blots of NCC membranes using anti-ncamp-1 serum detected a 29 kDa protein. Binding competition experiments demonstrated that anti-ncamp-1 antibodies and GpC bound to the same protein on NCC. Two different truncated forms of ncamp-1 as well as the full-length recombinant protein exhibited anti-microbial activity. The present study demonstrated the expression by NCC of a new membrane protein that may participate in the recognition of bacterial DNA and as such participate in innate anti-microbial immune responses in teleosts.

Introduction

Bacterial DNA initiates inflammatory responses and is responsible for development of innate immunity in mammals [1], [2], [3], [4]. Bacterial DNA binding to phagocytic cells and antigen presenting cells [3] via toll-like receptor 9 (TLR9) and possibly scavenger receptors suggest a direct participation of prokaryotic DNA in exacerbating inflammatory responses [1]. These actions are principally attributed to the presence of relatively high concentrations (compared to eukaryotic DNA) of unmethylated CpG dinucleotide motifs. Studies have shown a wide diversity of functional oligodeoxynucleotide (ODN) sequences. Dinucleotide versus single base composition [5]; different structural configurations (G-tetrads/guanosine quadraplexes) [6], [7]; as well as the most effective ‘backbone’ (hexose-phosphate) derivatizations (phosphodiester versus phosphorothioate) [8] have all been reported to influence effector cell activity in different animal species.

There are several different classes/types of DNA binding proteins on mammalian cells represented by a limited number of germ-line encoded receptors that are expressed predominantly on antigen presenting cells (with low levels of expression on T-cells and NK cells). These proteins are referred to as pattern recognition receptors (PRR) [9], [10] and they bind to pathogen associated molecular pattern ligands (PAMP) of microbial origin. The PAMPS include LPS, peptidoglycan, certain lipoproteins, CpG oligodeoxynucleotides (ODNs), etc. The most widely distributed of the PRR that bind ODNs are the Toll-like receptor 9, Mac-1 (CD 11b/CD18) [11], [12], [13], [14], [15] and Scavenger receptor-A [16], [17]. ODN binding to PRR may cause either activation or inhibitory responses depending on the ODN concentration and target cell type. In addition, ligation of PRR in vivo by ODNs may also produce different pathways of immunoregulation such as autoimmunity, Th1 bias activation, etc. [18], [19], [20].

Studies of anti-bacterial innate cellular responses in teleosts provides an interesting evolutionary/phylogenetic model for mechanisms of defense. In higher vertebrates, the (direct) binding and functional activation of NK cells by bacterial DNA has not been defined and the expression of DNA binding proteins on these cells in many cases remains controversial. Non-specific cytotoxic cells (NCC) directly bind to and are activated by ODNs [21]. In the present study an ODN binding protein from NCC was identified, sequenced and a recombinant homologue was expressed. This protein has sequence similarity to histones and in recombinant form has anti-bacterial activity.

Histone proteins have been previously detected in membrane preparations from human leukocytes [22], [23]; monocytes [24]; a T-cell line (HPB-ALL) [25]; and an H1-like protein has been described from neuronal cells [26]. A 30 kDa DNA receptor was identified [27], [28] on the membrane of human leukocytes. Evidence for the association of cell-derived and/or cell membrane histone H1 as a participant in anti-bacterial innate immunity has also been provided by studies of human ileal mucosal extracts [29] and human ulcerative colitis (UC) [30]. In both cases H1 was either released from villus epithelial cells [29] or was associated with a serum marker for UC [30]. In addition, Raji cells express 14, 17, 18, 33 and 34 kDa DNA binding proteins [31]. These histone or histone-like proteins were described as being responsible for the binding, endocytosis and degradation of exogenous DNA. Interestingly, the thyroglobulin receptor on the cell surface of J774 (mouse) macrophages is an H1 protein [32].

Teleosts appear to be the most abundant source [33], [34], [35], [36], [37], [38] for model studies of the role of histone-like proteins in anti-microbial immunity. These studies have demonstrated that histones and histone-like proteins exist in a wide variety of teleost tissues and they exhibit anti-microbial and anti-parasitic activities. Here, we demonstrate the expression of a novel cationic ODN binding protein (ncamp-1) on NCC membranes. The expressed recombinant ncamp-1: bound to ODN; was used to produce polyclonal anti-serum; and had anti-bacterial activity. The membrane expression of ncamp-1 was confirmed by flow cytometry and by Western blots of NCC membrane lysates using the anti-ncamp-1 anti-serum. Finally, the recombinant ncamp-1 killed Gram positive and Gram negative bacteria. We postulate that ncamp-1 may comprise a new class of pattern recognition proteins involved in recognition of bacterial DNA and amplification of teleost innate immunity.