Abstract
Alpha-2-macroglobulin is an abundant secreted protein that is of particular interest because of its diverse ligand binding profile and multifunctional nature, which includes roles as a protease inhibitor and as a molecular chaperone. The activities of alpha-2-macroglobulin are typically dependent on whether its conformation is native or transformed (i.e. adopts a more compact conformation after interactions with proteases or small nucleophiles), and are also influenced by dissociation of the native alpha-2-macroglobulin tetramer into stable dimers. Alpha-2-macroglobulin is predominately present as the native tetramer in vivo; once purified from human blood plasma, however, alpha-2-macroglobulin can undergo a number of conformational changes during storage, including transformation, aggregation or dissociation. We demonstrate that, particularly in the presence of sodium chloride or amine containing compounds, freezing and/or lyophilization of alpha-2-macroglobulin induces conformational changes with functional consequences. These conformational changes in alpha-2-macroglobulin are not always detected by standard native polyacrylamide gel electrophoresis, but can be measured using bisANS fluorescence assays. Increased surface hydrophobicity of alpha-2-macroglobulin, as assessed by bisANS fluorescence measurements, is accompanied by (i) reduced trypsin binding activity, (ii) increased chaperone activity, and (iii) increased binding to the surfaces of SH-SY5Y neurons, in part, via lipoprotein receptors. We show that sucrose (but not glycine) effectively protects native alpha-2-macroglobulin from denaturation during freezing and/or lyophilization, thereby providing a reproducible method for the handling and long-term storage of this protein.
Highlights
Alpha-2-macroglobulin (α2M) is an ancient component of the innate immune system that is highly conserved in animal species separated by over half a billion years of evolution
As a commonly used strategy for longterm storage of proteins involves freezing [51], the protein was analyzed after storage in phosphate buffered saline (PBS)/ Az at -20°C for 10 days; less than 50% of the purified α2M was found to migrate to a position corresponding to native α2M when assessed by native polyacrylamide gel electrophoresis (PAGE), the remainder of the protein migrated to a position corresponding to transformed α2M (Fig 1B)
Analysis of α2M by native PAGE after rapid freezing in liquid nitrogen followed by lyophilization from PBS/Az revealed that a heterogeneous mixture of species was present, which migrated at positions corresponding to native, transformed and aggregated α2M (Fig 1C)
Summary
Alpha-2-macroglobulin (α2M) is an ancient component of the innate immune system that is highly conserved in animal species separated by over half a billion years of evolution. These include native and transformed [23, 24]; slow and fast, due to the enhanced mobility of the compact form of α2M as assessed by native gel electrophoresis [2, 20]; native and activated [5, 16, 25] and active (or functional) and inactivated [26, 27] The latter two naming systems can cause confusion given that in the compact state α2M may be considered “activated” in reference to its newly acquired ability to bind to LRP [5] or α2M may be considered “inactivated” in terms of its ability to trap proteases [26, 27]. This has enabled us to identify a suitable method for the long-term preservation of α2M in its native state
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