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.
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