Year

2008

Degree Name

Master of Science

Department

School of Biological Sciences - Faculty of Science

Abstract

Alpha-2-macroglobulin (α2M) is a 720 kDa glycoprotein consisting of four identical (180 kDa) subunits and is the major representative of the α-macroglobulin group of plasma proteins, present at high concentrations in human plasma.&#;α2M is best known for its ability to inhibit a broad spectrum of proteases which it accomplishes using a unique “trapping” method. Protease trapping induces α2M to adopt an activated conformation which exposes a binding site for the low density lipoprotein receptor (LRP), facilitating clearance of the complexes from the body. α2M has been ascribed many biological roles which extend beyond simple protease inhibition including immune regulation, mediation of the inflammatory response via cytokine binding and more recently chaperone activity. α2M has been shown to inhibit the heat-induced precipitation of proteins in vitro through the formation of stable complexes. The work outlined in this study further characterises the chaperone activity of α2M under conditions of heat and oxidative stress and establishes the relationship between this and its role as a protease inhibitor.

When present at physiological concentrations, α2M was found to inhibit the oxidation-induced precipitation of lysozyme (lys). In a preliminary study, it was shown that α2M forms stable, soluble complexes with heat-stressed proteins. In the current study, native agarose gel electrophoresis and immunoprecipitation analyses were used to demonstrate that α2M also forms stable, soluble complexes with oxidised proteins. Removal of α2M from human plasma was found to significantly increase the level of plasma protein precipitation under conditions of heat and oxidative stress. Proteins co-purifying with α2M from human plasma (following incubation at either 43 °C or room temperature for 72 h) were analysed by mass spectrometry; this identified fibrinogen as a putative endogenous chaperone client protein of α2M. It was also shown that protease-mediated activation of &#;2M abolishes the chaperone activity, but that native &#;2M is able to form soluble complexes with heat stressed proteins and then subsequently become activated by protease trapping. Oxidation of (chaperone-inactive) protease bound α2M was shown to restore chaperone activity but not the protease inhibitor function. These behaviours provide an alternative means for generating &#;2M/stressed protein/protease complexes which could be cleared in vivo by LRP-mediated cellular uptake and degradation.

The ability of α2M/stressed protein complexes to bind to cell surface receptors was investigated using JEG-3, Hep-G2, and U937 cell lines and granulocytes derived from whole human blood. α2M/CS complexes had limited ability to bind to LRP expressed on the surface of JEG-3 cells. However, preliminary results indicated that activation of α2M (α2M*) and α2M/stressed protein complexes (α2M*/CS) with trypsin resulted in subsequent binding to the surface of JEG-3 cells. Native α2M/CS complexes were found to bind to granulocytes and Hep-G2 cells via unidentified, non-LRP receptors.

Collectively, the results presented here further establish α2M as a potent extracellular chaperone with the ability to protect proteins from heat and oxidation-induced stress. α2M appears likely to have a dual role in vivo, as a protease inhibitor and as an extracellular chaperone, the first identified mammalian protein with both activities. The evidence suggests that it may function as part of an extracellular quality control system for protein folding important in the control of inflammation and protein conformational disorders (PCDs) such as Alzheimer's disease and type II diabetes. The pathology of PCDs has been linked to the development of extracellular deposits of misfolded proteins. This thesis provides evidence supporting the hypothesis that α2M binds to misfolded extracellular proteins to keep them soluble and mediates their cellular uptake and subsequent degradation. Future advances in understanding of extracellular protein folding quality control are likely to provide novel insights into the mechanisms underpinning the development of serious human diseases and identify opportunities for the development of new therapies.

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Unless otherwise indicated, the views expressed in this thesis are those of the author and do not necessarily represent the views of the University of Wollongong.