Date of Award

Fall 2005

Project Type

Dissertation

Program or Major

Chemistry

Degree Name

Doctor of Philosophy

First Advisor

N Dennis Chasteen

Abstract

The major protein component of the extrapallial (EP) fluid of the mollusc Mytilus edulis, named the EP protein, has been previously isolated and partially characterized. It has been proposed to play an important role in shell mineralization because of its intriguing property of Ca2+ -binding induced self-assembly.

This dissertation reports on the characterization of the primary structure of the EP protein by RT-PCR and cDNA sequencing methods. The EP protein is comprised of 213 amino acids post cleavage of a signal peptide of 23 amino acids, and is rich in His, Glu and Asp residues. The consensus site of N-glycosylation "NHTE" and the intramolecular disulfide bond of the protein have been characterized also. Sequence comparisons reveal that the EP protein highly resembles a heavy metal binding protein and a histidine-rich glycoprotein both from the hemolymph of Mytilus edulis. The predicted domain profile and amino acid composition suggest that its N-terminus may be involved in calcium binding. The abundance of histidine residues of the protein may account for its heavy metal binding property. It is conceivable that the EP protein plays different roles in Mytilus edulis, as a heavy metal detoxification protein, a Ca2+-transport and a shell matrix protein.

The characterization and analysis of shell matrix proteins by 2-D gel electrophoresis coupled with MALDI-TOF mass spectrometry is discussed. Tropomyosin and paramyosin have been identified in the shell matrix, but no evidence for the EP protein has been found by the proteomic methods.

The post-translational modification (particularly glycosylation and phosphorylation) analysis of the EP protein by enzymatic digestion and MALDI-TOF mass spectrometry suggests that protein phosphorylation is not the major source of the charge heterogeneity on the EP protein. Also, the N-linked glycan of the EP protein is proposed to possess one or more phosphodiester linkages that are susceptible to lambda-protein phosphatase cleavage.

The protein component mixture of the EP fluid was found to inhibit calcium carbonate crystallization with a novel in vitro method. This approach could be applied to study functions of individual protein in the fluid.

Finally, the byssal threads and plaques from Mytilus galloprovincialis were investigated by electron paramagnetic resonance spectroscopy and shown to contain semiquinone radicals and Fe(III). In addition, Cu(II) and a second radical (g = 2.007) were found in cultivated threads.

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