title: Molecular cloning of Limulus alpha 2-macroglobulin.
abstract: The American horseshoe crab Limulus polyphemus contains alpha 2-macroglobulin (alpha 2M) in the hemolymph plasma and hemocytes. alpha 2M from Limulus shows many of the typical characteristics of mammalian alpha 2M, including the presence of an internal thiol-ester, reactivity with a diversity of endopeptidases, a unique proteinase-trapping mechanism, and reactivity with the mammalian alpha 2M receptor. Additionally, Limulus alpha 2M has the unique property that it regulates the limulin-based hemolytic system of the plasma. A cDNA encoding Limulus alpha 2M 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 alpha 2Ms (28-29% identity) and contains common features found in mammalian alpha 2Ms. 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 alpha 2Ms, and it is structurally related to that of the human complement factor C8 chain, consistent with a role for Limulus alpha 2M in host defense. The component sugar analysis of Limulus alpha 2M showed the existence of a complex type of oligosaccharide chain similar to those of mammalian alpha 2M. However, unlike mammalian alpha 2M, no sialic acid was detected in Limulus alpha 2M and it contained approximately 3 mol/mol N-acetylgalactosamine, suggesting the presence of O-linked sugar chains, which have not been found in mammalian alpha 2M. Expression of alpha 2M 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 alpha 2M indicated the presence of the alpha 2M in large granules. Trypsin-treated Limulus alpha 2M, but not the native alpha 2M, displaced methylamine-treated human 125I-alpha 2M from the human alpha 2M receptor with a Kd of 30 nM, suggesting conservation of the proteinase-clearance mechanisms between mammalian and arthropod evolutionary lineages.
authors.affiliation: Department of Biology, Faculty of Science, Kyushu University, Fukuoka, Japan.
authors.forename: D
authors.initials: D
authors.lastname: Iwaki
authors.forename: S
authors.initials: S
authors.lastname: Kawabata
authors.forename: Y
authors.initials: Y
authors.lastname: Miura
authors.forename: A
authors.initials: A
authors.lastname: Kato
authors.forename: P B
authors.initials: PB
authors.lastname: Armstrong
authors.forename: J P
authors.initials: JP
authors.lastname: Quigley
authors.forename: K L
authors.initials: KL
authors.lastname: Nielsen
authors.forename: K
authors.initials: K
authors.lastname: Dolmer
authors.forename: L
authors.initials: L
authors.lastname: Sottrup-Jensen
authors.forename: S
authors.initials: S
authors.lastname: Iwanaga
mesh_terms: D000595:Amino Acid Sequence; D000818:Animals; D001483:Base Sequence; D001665:Binding Sites; D001667:Binding, Competitive; D001777:Blood Coagulation; D002451:Cell Compartmentation; D003001:Cloning, Molecular; D003185:Complement C8 / Q000737:chemistry; D018076:DNA, Complementary / Q000235:genetics; D015870:Gene Expression; D006023:Glycoproteins / Q000737:chemistry; D006737:Horseshoe Crabs / Q000235:genetics*; D006801:Humans; D026503:Low Density Lipoprotein Receptor-Related Protein-1; D008969:Molecular Sequence Data; D012333:RNA, Messenger / Q000235:genetics; D011971:Receptors, Immunologic / Q000378:metabolism; D016415:Sequence Alignment; D017386:Sequence Homology, Amino Acid; D000511:alpha-Macroglobulins / Q000737:chemistry / Q000235:genetics*
chemical_list: D003185:Complement C8; D018076:DNA, Complementary; D006023:Glycoproteins; D026503:Low Density Lipoprotein Receptor-Related Protein-1; D012333:RNA, Messenger; D011971:Receptors, Immunologic; D000511:alpha-Macroglobulins