The α-macroglobulin proteinase inhibitors (αMs) are a family of proteins with the unique ability to inhibit a broad spectrum of proteinases. Whereas monomeric, dimeric and tetrameric αMs have been identified in vertebrates, all invertebrate αMs characterized so far have been dimeric. This paper reports the isolation and characterization of a tetrameric αM from the tropical planorbid snail Biomphalaria glabrata. The sequence of 18 amino acids at the N-terminus indicates homology with other αMs. The subunit mass of approx. 200 kDa was determined by matrix-assisted laser desorption/ionization time-of-flight mass spectrometry and SDS/PAGE. The quaternary structure was determined by sedimentation equilibrium centrifugation and native pore-limit electrophoresis. Evidence for a thioester is provided by the fact that methylamine treatment prevents the autolytic cleavage of the snail αM subunit and results in the release of 4 mol of thiols per mol of snail αM. The snail αM inhibited the serine proteinase trypsin, the cysteine proteinase bromelain and the metalloproteinase thermolysin. The spectrum of proteinases inhibited, together with the demonstration of steric protection of the proteinase active site and a ‘slow to fast’ conformational change after reacting with trypsin, all suggest that the inhibitory mechanism of the snail αM is similar to the ‘trap mechanism’ of human α2-macroglobulin.
Purification and characterization of a tetrameric α-macroglobulin proteinase inhibitor from the gastropod mollusc Biomphalaria glabrata
