Characterization of Two 20kDa-Cement Protein (cp20k) Homologues in Amphibalanus amphitrite

Abstract

The barnacle, Amphibalanus amphitrite, is a common marine fouling organism. Understanding the mechanism of barnacle adhesion will be helpful in resolving the fouling problem. Barnacle cement is thought to play a key role in barnacle attachment. Although several adult barnacle cement proteins have been identified in Megabalanus rosa, little is known about their function in barnacle settlement. In this study, two homologous 20k-cement proteins (cp20k) in Amphibalanus amphitrite, named Bamcp20k-1 and Bamcp20k-2, were characterized. The two homologues share primary sequence structure with proteins from other species including Megabalanus rosa and Fistulobalanus albicostatus. The conserved structure included repeated Cys domains and abundant charged amino acids, such as histidine. In this study we demonstrated that Bamcp20k-1 localized at the α secretory cells in the cyprid cement gland, while Bamcp20k-2 localized to the β secretory cells. The differential localizations suggest differential regulation for secretion from the secretory cells. Both Bamcp20k-1 and Bamcp20k-2 from cyprids dissolved in PBS. However, adult Bamcp20k-2, which was dominant in the basal shell of adult barnacles, was largely insoluble in PBS. Solubility increased in the presence of the reducing reagent Dithiothreitol (DTT), suggesting that the formation of disulfide bonds plays a role in Bamcp20k-2 function. In comparison, Bamcp20k-1, which was enriched in soft tissue, could not be easily detected in the shell and base by Western blot and easily dissolved in PBS. These differential solubilities and localizations indicate that Bamcp20k-1 and Bamcp20k-2 have distinct functions in barnacle cementing.

Sequence basis of Barnacle Cement Nanostructure is Defined by Proteins with Silk Homology

“…Finally, discrete points of similarity revealed several other pairs including two 105 kDa proteins (Table 1). Other known homologues (AaCP100/AaCP11434 and AaCP20-1/AaCP20-237) were found to verify the similarity matrix.…”

“…Analysis of transcript sequences collected from the basal membranes of barnacles A. amphitrite and T. japonica confirmed cement proteins are produced in tissues contained just above the baseplate343637. Both studies performed on A. amphitrite have revealed two additional cement proteins that share primary structure and amino acid chemistry with existing sequences, suggesting that barnacles produce cement proteins in specialized subfamilies3437. However, transcriptomic studies have revealed few new proteins in addition to those identified more than a decade ago.…”

Structural characterization of tick cement cones collected from in vivo and artificial membrane blood-fed Lone Star ticks (Amblyomma americanum)

“…These proteins self-assemble into crystalline repeats which are bound together by disulfide bond formation, glycosylation, or cation interactions (Winkler and Kaplan, 2000). Barnacles also secrete an adhesive cement which is used to firmly adhere to their substrate however, barnacle cement differs from the other adhesives listed here as the protein composition of barnacle cement contains cysteine repeats (He et al, 2013) rather than a large number of glycine repeats. However, the two-cement process of barnacles (Burden et al, 2012) mimics the two-cement composition of tick cement.…”

Barnacle biology before, during and after settlement and metamorphosis: a study of the interface

“…While the patchy material was first observed with the nucleic acid stains, the protein stains suggest the material was mainly proteinaceous and at least some proteins were phosphorylated. Cyprid adhesive and adult glues contain a large fraction of protein (Kamino and Shizuri, 1998;Urushida et al, 2007;He et al, 2013;Gohad et al, 2014). Phosphoprotein was of particular interest as it was found in the cyprid adhesive plaque (Gohad et al, 2014) and is a critical component of other marine adhesives such as mussel (Waite and Qin, 2001;Flammang et al, 2009).…”