Environmental Marine Biotechnology: Mussel Plasma Histidine-Rich Glycoprotein (HRG)-Biomarker, Key to Metal Transport, Novel Natural Product

Lead Pi: William Robinson · 7/2000 - 6/2002

Project Personnel: Chrys Chryssostomidis, Gordon Wallace, Manackam Sugumaran

Project number: 2000-RB-53

Objectives:We propose to establish mussel HRG as a biomarker of metal exposure and toxicity. We will characterize HRG’s role in metal transport and metabolism, and asses HRG as a novel natural product chelator. We recently reported the first isolation and purification of Histidine-Rich Glycoprotein (HRG) from an invertebrate- the marine mussel Mytilus edulis. Present at high concentrations in mussel blood plasma (~60% of total protein concentrations). HRG strongly binds metals such as Cd (6 high affinity sites per molecule with K = 7.65; 10 low affinity sites with K = 5.41); preliminary data indicates that it also binds Ca and is probably the main transport protein for Ca in mussel blood. Based on reported binding affinities for Zn, Cd, Hg, Ni, and Cu for mammalian HRG, it is highly probably the contaminant metals displace Ca from mussel HRG. This would make mussel HRG an excellent biomarker for metal exposure. Because HRG’s role in Ca transport would be disrupted by contaminant metals, HRG would also be an excellent biomarker for metal toxicity. Methodology:We will use/advance state-of-the-art molecular/cellular biology and metal analytical techniques throughout this study, combining the expertise of three investigators (aquatic toxicology, molecular biology/protein biochemistry, and inorganic geochemistry). We have already worked out Immobilized Metal ion Affinity Chromatography (IMAC) techniques to isolate and purify mussel HRG. We will determine which metals bind to HRG, examine the displacement of Ca from HRG by other metals, and determine binding strengths of HRG for Ca and other metals (titration experiments with stable metal isotopes and competitive equilibrium studies) by taking advantage of the extremely low detection limits provided by Inductively Coupled Plasma Emission Spectroscopy-Mass Spectrometry (ICP-MS). Using Reverse Transcriptase-PCR, we will develop cDNA probes for mussel HRG mRNA that will be used along with the HRG antibodies we will produce, to determine which tissues of the mussel express and produce HRG, which other bivalve species contain HRG, whether expression or HRG concentrations vary temporally or spatially (Northern plot, ELISA, Fluorescent immunocytochemistry), and whether HRG can be induced by laboratory exposure to metal. Using a 3′ and 5′ Rapid Amplification of cDNA Ends (RACE) strategy, we will determine the entire HRG cDNA sequence. This will allow us to compare both the cDNA sequence and the deduced amino acid sequence with that of mammalian HRG. Having characterized HRG as a biomarker, we will propose a methodology for its routine use. Rationale:Aside from our understanding of intracellular metallothionein regulation of some metals, we know very little about how marine invertebrates transport and ‘metabolize’ metals. We use marine mussels as bioindicators of ecosystem health, and have collected abundant data on metal and organic contaminant whole body burdens in this species over the past 20 years. However, we cannot adequately interpret this data, because we lack the basic understanding of how metals are processes in these animals. Our proposed studies will fill in a major missing piece of knowledge- how metals are transported in bivalve blood for delivery to internal organs. These studies will allow us to better interpret mussel body burden data, and better monitor the environmental utilizing the current ‘Mussel Watch’ approach.The discovery of metallothionein in 1957 by Margoshes and Vallee initiated an avalanche of studies in intracellular meta-regulation in marine organisms that is continuing today. We hypothesize that HRG will prove to be the next major component of the mussel’s metal processing machinery to be investigated and which will spur studies for years to come.