F-type lectin from serum of the Antarctic teleost fish Trematomus bernacchii (Boulenger, 1902): Purification, structural characterization, and bacterial agglutinating activity

Highlights

• F lectin isolated and characterized from emerald rock cod Trematomus bernacchii serum.

• Bacterial recognition protein activity at Antarctic temperatures from Notothenoid

• Adaptation of fish FTLs in host-pathogen interactions at low temperature environment

Abstract

The increasing availability of sequenced genomes has enabled a deeper understanding of the complexity of fish lectin repertoires involved in early development and immune recognition. The teleost fucose-type lectin (FTL) family includes proteins that preferentially bind fucose and display tandemly arrayed carbohydrate-recognition domains (CRDs) or are found in mosaic combinations with other domains. They function as opsonins, promoting phagocytosis and the clearance of microbial pathogens.

The Antarctic fish Trematomus bernacchii is a Perciforme living at extremely low temperatures (−1.68 °C) which is considered a model for studying adaptability to the variability of environmental waters. Here, we isolated a Ca⁺⁺-independent fucose-binding protein from the serum of T. bernacchii by affinity chromatography with apparent molecular weights of 32 and 30 kDa under reducing and non-reducing conditions, respectively. We have characterized its carbohydrate binding properties, thermal stability and potential ability to recognize bacterial pathogens. In western blot analysis, the protein showed intense cross-reactivity with antibodies specific for a sea bass (Dicentrarchus labrax) fucose-binding lectin. In addition, its molecular and structural aspects, showing that it contains two CRD-FTLs confirmed that T. bernacchii FTL (TbFTL) is a bona fide member of the FTL family, with binding activity at low temperatures and the ability to agglutinate bacteria, thereby suggesting it participates in host-pathogen interactions in low temperature environments.

Introduction

Although teleost fish express most components of typical mammalian adaptive immune responses, it is well-established that a significant portion of their immediate response against infectious challenges is carried out by innate immune defence mechanisms. Accordingly, during the past few years, interest in the structural-functional aspects of fish innate immune responses, particularly lectins, toll-like receptors and complements, has grown at an exponential rate (Magnadóttir, 2006; Vasta et al., 2011; Cammarata et al., 2016; Elumalai et al., 2019; Sahoo, 2020). Lectins are carbohydrate-binding proteins present in essentially all living organisms and are involved in immune responses as well as in other biological processes. Most lectins are multivalent proteins capable of recognizing and binding carbohydrate moieties by specific domains (carbohydrate-recognition domains; CRDs) (Drickamer, 1999); they also participate in various biological functions (Kuhlman et al., 1989; Cooper et al., 1994), including innate and adaptive immune responses (Arason, 1996; Drickamer and Dodd, 1999; Vasta and Ahmed, 2008). Because lectins may display CRDs in combination with other domains, they not only recognize carbohydrates on the surfaces of potential pathogens, but also mediate several effector functions, including the opsonization of microbial pathogens, immobilization, and agglutination, as well as complement pathway and phagocyte activation (Russell and Lumsden, 2005). Based on the presence of conserved amino acid sequence motifs in their CRDs, their calcium requirements and structural folding, animal lectins have been classified into several families, such as C-, P-, I-, and L-type lectins, galectins, pentraxins and others (Arason, 1996; Vasta et al., 2004). Among these, the F-type lectin family is characterized by the binding of fucose, a unique sequence motif in its CRD (F-type CRD), and a novel structural fold (Odom and Vasta, 2006; Bianchet et al., 2010; Bianchet et al., 2002). The F-type CRD can be associated with other domains (pentraxin, C-type lectin, or “sushi” domains), yielding complex multidomain proteins (Vasta et al., 2004).

Lectins from most families, including C- and R-type lectins and galectins, have been isolated from tissues and organs of teleost fish species, and various defence functions have been identified, including opsonization, enhancement of intracellular respiratory burst, and activation of bactericidal activity (Arason, 1996; Jensen et al., 1997; Listinsky et al., 1998; Suzuki et al., 2003; Russell and Lumsden, 2005; Cammarata et al., 2016). F-type lectins (FTLs) have also been identified and characterized from the serum of several teleost fishes, such as eels (Anguilla sp.), striped bass (Morone saxatilis), gilt-head bream (Sparus aurata), the European bass (Dicentrarchus labrax) (Cammarata et al., 2016). Although F-type lectins have been proposed to mediate roles as molecular recognition factors in innate immunity, the experimental evidence is fragmentary and the detailed mechanisms of their activity have not been fully elucidated (Vasta et al., 2004).

Among teleost fish, the Arctic and Antarctic species present unique research opportunities because of the extreme environments they live in. Notothenioidei is a sub-order within the Perciformes that has radiated to become the most numerous and widespread fish taxon in the Antarctic region, where it encompasses at least 90% of the biomass of the fishes populating this environment (Wells and Eastman, 1994). During the past few years, studies have placed emphasis on their environmental adaptations, particularly on their ability to avoid freezing and survive at extremely low temperatures (−1.68 °C) (Devries, 1982). Furthermore, because the Perciformes are a closely related groups, the physiological consequences of ecological adaptations between species can be evaluated without the difficulties posed by comparing fishes with different phylogenetic backgrounds. In this regard, they constitute an excellent study model for investigating structural and functional adaptations of proteins to low temperatures. For example, biochemical features, such as protein amino acid differences among species, have been useful tools for investigating evolutionary processes and relationships, and for identifying events associated with speciation (Macdonald et al., 1988).

Trematomus bernacchii, an Antarctic notothenioid fish, has been the species of choice as a model system for investigations focused on adaptability to climate change and environmental contamination, particularly the potential warming and acidification of environmental waters, as well as its detoxification capabilities for environmental pollutants (Santovito et al., 2012; Laura et al., 2017). Studies have been carried out on immunoglobulin genes, individuating structural and functional modifications and adaptations to the low temperature of the Southern Ocean (Oreste and Coscia, 2004; Coscia et al., 2010; Coscia et al., 2011).

Unlike immunoglobulins, lectins do not generate diversity in recognition by genetic recombination and, therefore, additional interest has arisen in the germline-encoded diversity of the lectin repertoires, including their allelic variation or polymorphisms, the presence of tandem gene duplications and multigene families, the formation of chimeric structures by exon shuffling, additional mechanisms for expanding the ligand recognition spectrum by alternative splicing, and the structural basis for the potential “plasticity” of their carbohydrate binding sites (Vasta et al., 2011).

Our interest in this species has centred on its innate immune recognition capacity mediated by lectins in these extreme low-temperature conditions. In this study, we initiated the characterization of a two-CRD, 32 kDa FTL (TbFTL) that we isolated from T. bernacchii serum, including its carbohydrate binding properties, thermal stability, structural aspects as compared to available FTL structures and potential ability to recognize bacterial pathogens. Our results confirmed that is a bona fide member of the FTL family, has binding activity at low temperatures and can agglutinate bacteria, thereby suggesting its potential participation in host-pathogen interactions in the Antarctic low temperature aquatic environment.