Understanding the settlement of Balanus amphitrite through the molecular and structural characterisation of the settlement-inducing protein complex

Abstract

Many barnacle species are gregarious and their cypris larvae display a remarkable ability to explore surfaces before committing to permanent attachment. The cuticular tissue of adult barnacles contains an α2-macroglobulin-like glycoprotein contact cue referred to as the settlement-inducing protein complex (SIPC) which increases the attractiveness of surfaces and signals cyprids to settle. Despite decades of research into marine fouling and the development of anti-fouling systems, detailed knowledge of the biochemical and structural composition of marine pheromone cues is poor. This cue is key to barnacle gregarious settlement and represents an attractive target for custom synthesis of antagonistic surfaces. Using the tropical acorn barnacle Balanus amphitrite, this project endeavoured to characterise the structure and glycan moiety of the SIPC. The SIPC active fraction was purified by ion exchange chromatography and gel filtration and detected through SDSPAGE gel antibody immunoblotting. The carbohydrate structure was characterised using a combination of hydrophilic interaction liquid chromatography with fluorescence detection (HILIC-fluorescence) and exoglycosidase digestions. This provided evidence of predominantly oligomannose glycans with the occurrence of monofucosylated oligomannose glycans in lower proportions. The characterisation of high mannose glycosylation is supported by observations on the effect of mannose in solution increasing settlement in B. amphitrite cypris larvae. Protocols to create surface bound carbohydrate-functionalised polymers were successfully developed and confirmed using X-ray photoelectron spectroscopy, ellipsometry and contact angle measurements. These surfaces were shown to act as SIPC mimics, cueing settlement on contact. The SIPC is known to contain seven potential N-glycosylation sites. Additional work using transmission electron microscopy and atomic force microscopy has further enhanced understanding of the glycoprotein structure. Obtaining complete structural characterisation of the SIPC remains a goal that has the potential to inspire solutions to the age-old problem of barnacle fouling.