Molecular cloning of Limulus α₂-macroglobulin

The American horseshoe crab Limulus polyphemus contains α₂-macroglobulin (α₂M) in the hemolymph plasma and hemocytes. α₂M from Limulus shows many of the typical characteristics of mammalian α₂M, including the presence of an internal thiol-ester, reactivity with a diversity of endopeptidases, a unique proteinase-trapping mechanism, and reactivity with the mammalian α₂M receptor. Additionally, Limulus α₂M has the unique property that it regulates the limulin-based hemolytic system of the plasma. A cDNA encoding Limulus α₂M has been obtained from a hemocyte cDNA library. The open reading frame encodes an N-terminal signal sequence of 25 amino acid residues and a mature protein of 1482 residues. The entire amino acid sequence is similar to those of the mammalian α₂Ms (28-29% identity) and contains common features found in mammalian α₂Ms, a bait region, an internal thiol-ester site, and a receptor-binding domain. However, the N-terminal portion (positions 24-105) has no sequence similarity with those of mammalian α₂Ms, and it is structurally related to that of the human complement factor C8γ chain, consistent with a role for Limulus α₂M in host defense. The component sugar analysis of Limulus α₂M showed the existence of a complex type of oligosaccharide chain similar to those of mammalian α₂M. However, unlike mammalian α₂M, no sialic acid was detected in Limulus α₂M and it contained approximately 3 mol/mol N-acetylgalactosamine, suggesting the presence of O-linked sugar chains, which have not been found in mammalian α₂M. Expression of α₂M was detected in hemocytes, but not in hepatopancreas, heart, stomach, intestine, coxal gland, brain and skeletal muscle. Furthermore, immunoblotting of large and small granules of the hemocytes with antiserum against α₂M indicated the presence of the α₂M in large granules. Trypsin-treated Limulus α₂M, but not the native α₂M, displaced methylamine-treated human ¹²⁵I-α₂M from the human α₂M receptor with a K(d) of 30 nM, suggesting conservation of the proteinase-clearance mechanisms between mammalian and arthropod evolutionary lineages.