Cloning and expression of a cDNA encoding an insoluble matrix protein in the gastroliths of a crayfish, Procambarus clarkii

Abstract

In the crayfish Procambarus clarkii, the gastroliths are formed as a paired structure in the stomach during the premolt period, and contain calcium carbonate and a small amount of an organic matrix. In this investigation, a cDNA encoding an insoluble matrix protein was isolated from P. clarkii. The open reading frame encoded 505 amino acid residues including two unique repeated sequences. The N-terminal half of the amino acid sequence, which included 10-amino-acid repeats, exhibited a high degree of similarity to that of involucrin, a protein synthesized in human keratinocytes. Northern blot analysis revealed that mRNA encoding the matrix protein is specifically expressed in the gastrolith discs during the premolt period in which the gastroliths formed. In the gastrolith discs, levels of expression of this mRNA correlated increases in weights of the gastroliths concomitant with their formation. Organ culture of the gastrolith discs suggested that expression of mRNA in the discs is induced by molting hormone, 20-hydroxyecdysone. These results reinforced the relationship between the matrix protein and formation of the gastroliths. Functional analysis showed that the protein inhibits calcium carbonate crystallization in a solution system, suggesting that the protein plays a role in the calcification of the gastroliths.