An ancient and variable mannose-binding lectin from the coral Acropora millepora binds both pathogens and symbionts

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Summary

Corals form the framework of the world's coral reefs and are under threat from increases in disease and bleaching (symbiotic dysfunction), yet the mechanisms of pathogen and symbiont recognition remain largely unknown. Here we describe the isolation and characterisation of an ancient mannose-binding lectin in the coral Acropora millepora, which is likely to be involved in both processes. The lectin (‘Millectin’) was isolated by affinity chromatography and was shown to bind to bacterial pathogens as well as coral symbionts, dinoflagellates of the genus Symbiodinium. cDNA analysis of Millectin indicate extensive sequence variation in the binding region, reflecting its ability to recognise various mannose-like carbohydrate structures on non-self cells, including symbionts and pathogens. This is the first mannose-binding lectin to show extensive sequence variability as observed for pattern recognition proteins in other invertebrate immune systems and, given that invertebrates rely on non-adaptive immunity, is a potential keystone component of coral defence mechanisms.

Introduction

Scleractinian corals are sessile marine invertebrates that form the framework of coral reefs and sustain an enormous biodiversity. However, coral reefs are experiencing increased levels of stress from a range of factors including diseases and bleaching—loss of their algal symbionts (Symbiodinium) [1], [2], [3]. There has been a rapid increase in investigation of coral diseases and stress responses over the last decade, yet study of the scleractinian coral immune system remains in its infancy. Little is known about the molecular mechanisms underlying pathogen defence and symbiont uptake in corals despite their inherent association with coral disease and bleaching. A fundamental feature of the innate immune system of all metazoans is the ability to identify non-self utilising pattern recognition receptors (PRRs) to recognise and bind surface structures on external foreign entities [4]. A PRR binding event can initiate signalling cascades that induce a downstream immune response or immediate reactions such as agglutination of invading cells and opsonisation for phagocytosis [4], [5]. In addition, agglutination and phagocytosis assist in symbiont uptake by animals [6], [7]. We therefore hypothesised that keystone elements of non-self recognition in both coral immune responses and symbiotic processes may be found amongst the pattern recognition proteins. The interest in invertebrate PRRs has also increased over the last few years as it has become evident that invertebrates may utilise an alternative adaptive immune system involving highly variable PRRs [8], [9], [10], [11], [12], [13]. An important group of PRRs in vertebrates and invertebrates are the lectins, proteins that can recognise carbohydrate structures on non-self cells [14], [15], [16]. Mannans, consisting of multiple densely packed terminal mannose residues, are abundant carbohydrate entities on the surface of non-metazoan cells and function as key targets for several lectins involved in immunity, including that of the well-studied vertebrate mannose-binding lectin (MBL) [17]. Bioinformatics studies on expressed genes have found that corals appear to have high ancestral complexity and have many genes in common with vertebrates [18]. Some of these genes do not appear in many other invertebrates, suggesting extensive gene loss in these taxa [18]. Hence, studies into coral immunity may provide novel information on the evolution of the early innate immune system of animals. One example reflecting the presence of unexpected immune-related genes in corals is the discovery of complement C3 [19], [20]. This molecule interacts with MBL in a complex system in vertebrates and a more simplified system in higher invertebrates, further fuelling the interest to investigate coral lectins with preference for mannose. We used mannose affinity chromatography to isolate a protein from the coral Acropora millepora that we have named Millectin, which is the first functional PRR to be described in a scleractinian coral. The protein binds both pathogens and algal symbionts, suggesting lectins may have been co-opted from an ancient innate immune system into a role in selecting and maintaining the photosynthetic endosymbionts in reef-building corals. Millectin is an ancient relative of the collectins MBL and SP-A and has extensive sequence variation in the binding region, providing insight into the function and evolution of the innate immune system.

Section snippets

Materials

A. millepora used in this study was collected from the reef flat on Heron Island, Great Barrier Reef, Australia (23°27′12″S, 151°55′47″E). Fragments of 10×10 cm were transported live to the laboratory where protein extractions were performed. Coral fragments destined for RNA extraction and subsequent cDNA library construction were snap frozen in liquid nitrogen immediately after collection and stored at −80 °C. Bacterial isolates used in this study all came from the Aquatic Animal Health

Isolation, purification and amino acid sequencing of Millectin

Eluates from the mannose affinity chromatography yielded purified aliquots of mannose-binding protein. The subunit structure of the protein was determined by SDS-PAGE in which two bands of 16 and 17 kDa were detected (Figure 1). N-terminal sequencing of these bands confirmed that they possessed identical N-terminal amino acid sequence (GILENIEKAVDPCGDV). The size variation is likely to represent glycosylated (17 kDa) and non-glycosylated (16 kDa) subunit isoforms, reflected by sequence variation

Discussion

As the ability to distinguish non-self from self is one of the most crucial and important aspects of an immune system, we considered this to be the logical starting point for investigating immune responses in an organism for which functional data are scarce. In doing so, we have isolated and identified a PRR that is of interest from a range of perspectives including a potential role in symbiosis, diversity in invertebrate immune receptors and the evolution of players in the innate immune

Acknowledgements

This work was supported in part by grants from the PADI Foundation, the Australian Coral Reef Society and the ARC Centre of Excellence for Coral Reef studies (to OHG). We thank E. Sampayo for providing Symbiodinium cultures and knowledge of PAUP, and J. Hill and T. Ainsworth for assistance with the Zeiss LSM 510 Meta. We also thank J. Roff and J. Davy for valuable comments.

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