Purification and characterization of α₂-macroglobulin from the white shrimp (Penaeus vannamei)

Abstract

α₂-Macroglobulin (α₂M) is a broad-spectrum protease-binding protein abundant in plasma from vertebrates and several invertebrate phyla. This protein was purified from cell-free hemolymph of the white shrimp, Penaeus vannamei, using Blue-Sepharose and Phenyl-Sepharose chromatography. The shrimp α₂M is a 380 kDa protein, a homodimer of two apparently identical subunits of ∼180 kDa linked by disulphide bridges. The amino acid sequence of the N-terminus is similar to the Limulus α₂M counterpart. The shrimp α₂M has a wide inhibition spectrum against different proteinase types including trypsin, leucine amino peptidase, chymotrypsin, elastase and papain. The secondary structure of shrimp α₂M is mainly beta-sheet (36%), with a characteristic minimum elipticity at 217 nm. Evidence for a thiolester-mediated inhibition mechanism of proteases by α₂M was provided by inactivation with methylamine.

Introduction

α₂-Macroglobulin (α₂M) has been defined as a ubiquitous high molecular weight proteinase inhibitor. Although most of the biochemical studies have been done with human α₂M, this protein has been detected in mammals, birds, reptiles, amphibians and fishes. The α₂M has also been identified and characterized in insects (Brivio et al., 1992) and mollusk (Thogersen et al., 1992, Yigzaw et al., 2001) as well as crustaceans including freshwater crayfish (Hergenhahn et al., 1988, Stöcker et al., 1991), lobster (Spycher et al., 1987), and horseshoe crab (Armstrong et al., 1991, Iwaki et al., 1996).

Active-site proteinase inhibitors are usually reactive against a limited variety of enzymes; however, α₂M is notably promiscuous, reacting with members of all classes of proteases (Sottrup-Jensen et al., 1989, Quigley et al., 1991, Iwaki et al., 1996). In addition, α₂M does not block the catalytic site, but physically traps the proteinases inside a pocket or cage and sterically restricts the substrates access. Molecules small enough to diffuse into the cage are actively hydrolyzed, whereas for large proteins the access to the active site is restricted and are, thus, protected from degradation.

The molecular trap model was proposed by Barrett and Starkey (1973), and postulates that the proteinases cleave one or more sensitive peptide bonds located in a defined ‘bait’ region of the α₂M molecule (Barrett and Starkey, 1973, Armstrong and Quigley, 1999). This leads to conformational changes of the α₂M and the physical trapping of the proteinase within the α₂M cage, without affecting the proteinase catalytic site. After binding and entrapment of the proteinase, a receptor recognition site is exposed (Debanne et al., 1975, Imber and Pizzo, 1981, Kaplan et al., 1981), which permits the clearance of the α₂M-proteinase complex from the circulatory system (Imber and Pizzo, 1981, Melchior et al., 1995) followed by degradation. Blood cells appear to be involved in this process (Armstrong and Quigley, 1999). Evidence for the conformational change of α₂M upon proteinase binding or methylamine modification derives from electron microscopy (Svehag et al., 1967), gel electrophoresis (Barrett et al., 1979), analytical ultracentrifugation (Gonias et al., 1982) and circular dichroism (Bjork and Fish, 1982, Feldman and Pizzo, 1984) analyses.

Although most of the biochemical studies have focused on human α₂M, its role in immunity and the importance of regulation of proteolysis in invertebrates may have particular importance (Kanost, 1999). Several proteinases are involved in the activation of the coagulation process (Muta et al., 1990, Muta et al., 1995, Roth and Levin, 1992) and the prophenoloxidase activation system (Aspán et al., 1990, Hernández-López et al., 1996, Söderhäll and Cerenius, 1998, Wang et al., 2001) in arthropods. Thus, proteinase inhibitors are required to control the proteinase activity in physiological range and to avoid tissue damage. α₂M has been purified from some crustaceans (Spycher et al., 1987, Hergenhahn et al., 1988, Stöcker et al., 1991), but not from marine penaeids. In this paper we report the purification and characterization of α₂M from the Pacific white shrimp, Penaeus (=Litopenaeus) vannamei.