Purification and characterization of a novel calcium-binding protein from the extrapallial fluid of the mollusc, Mytilus edulis.
In the bivalve mollusc Mytilus edulis shell thickening occurs from the extrapallial (EP) fluid wherein secreted shell matrix macromolecules are thought to self-assemble into a framework that regulates the growth of CaCO(3) crystals, which eventually constitute approximately 95% of the mature shell. Herein is the initial report on the purification and characterization of a novel EP fluid glycoprotein, which is likely a building block of the shell-soluble organic matrix. This primary EP fluid protein comprises 56% of the total protein in the fluid and is shown to be a dimer of 28,340 Da monomers estimated to be 14.3% by weight carbohydrate. The protein is acidic (pI = 4.43) and rich in histidine content (11.14%) as well as in Asx and Glx residues (25.15% total). The N terminus exhibits an unusual repeat sequence of histidine and aspartate residues that occur in pairs: NPVDDHHDDHHDAPIVEHHD approximately. Ultracentrifugation and polyacrylamide gel electrophoresis demonstrate that the protein binds calcium and in so doing assembles into a series of higher order protomers, which appear to have extended structures. Circular dichroism shows that the protein-calcium binding/protomer formation is coupled to a significant rearrangement in the protein's secondary structure in which there is a major reduction in beta-sheet with an associated increase in alpha-helical content of the protein. A model for shell organic matrix self-assembly is proposed.
Hattan
S J
SJ
Departments of Chemistry and Biochemistry & Molecular Biology, University of New Hampshire, Durham, New Hampshire 03824, USA.
Laue
T M
TM
Chasteen
N D
ND
D000595:Amino Acid Sequence; D000596:Amino Acids / Q000032:analysis; D000818:Animals; D049872:Bivalvia; D002135:Calcium-Binding Proteins / Q000737:chemistry / Q000302:isolation & purification*; D002850:Chromatography, Gel; D002852:Chromatography, Ion Exchange; D002942:Circular Dichroism; D004591:Electrophoresis, Polyacrylamide Gel; D013058:Mass Spectrometry; D008969:Molecular Sequence Data; D008970:Molecular Weight; D014357:Trypsin / Q000737:chemistry
D000596:Amino Acids; D002135:Calcium-Binding Proteins; D014357:Trypsin