DISTRIBUTION OF L-DOPA-CONTAINING PROTEINS INVOLVED IN OYSTER SHELL FORMATION

Marine bivalve organisms such as Mytilus edulis L. (blue mussel), Crassostrea virginica (eastern oyster), and Mercenaria mercenaria (hard clam) produce structural proteins and peptides that are critical in adhesive strategies and shell biomineralization. The unique properties of these proteins and peptides derive from the amino acid composition. One unique amino acid is of singular importance: L-3,4-dihydroxyphenylalanine (L-dopa), which is a key component in the enzymatically catalyzed sclerotization (or cross-linking) of these proteins. Dopa-containing proteins play a role in formation of the insoluble organic matrix that is an essential component in shell biomineralization; as such, L-dopa can be considered a biomarker for identification and localization of shell formation proteins. The distribution and role of precursor L-dopa proteins in the tissues of the organism is still a source of debate within the biomineralization community.

The focus of this research was to determine the distribution and possible role of L-dopa containing proteins involved in the process of biomineralization in the eastern oyster, Crassostrea virginica.  Three organismal compartments were identified as possible sources of L-dopa protein pre-cursors: hemocytes, cell-free hemolymph, and mantle tissue.  Nascent shell material was also surveyed for L-dopa content.  Hemolymph was harvested from the adductor muscle of notched oysters and hemocytes were subsequently collected via hemolymph centrifugation.  Mantle tissue was collected along a transect from ventral shell margin to umbo.  Nascent shell material was collected from un-notched oysters and also from oysters that had repaired the notch.  Amino acid composition of samples that Samples were acid-hydrolyzed in vacuo and amino acid composition was determined via anion exchange chromatography with pulsed amperometric detection.  L-dopa was found in all samples, but varied depending on sample type, and for notched oysters was related to the time since notching.  Higher levels of L-dopa were found in hemocytes collected 24-28 hours post-notching possibly indicating an induction of resources for notch repair. Levels of L-dopa were consistently found in hemolymph irrespective of notching indicating a constitutive level L-dopa protein production.  L-dopa was found in higher levels in nascent shell and concentrations declined as the shell aged indicating that the proteins had become cross-linked.  These data support the premise that L-dopa-containing proteins are involved in oyster shell formation and that they are distributed among several components of the system.  The components involved - hemocytes, hemolymph, mantle tissue - allow for the rapid movement of precursor proteins to the site of shell formation whether it is for normal shell formation or for shell repair.