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Localization of carbohydrate attachment sites and disulfide bridges in limulus alpha(2)-macroglobulin - evidence for two forms differing primarily in their bait region sequences

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Overview

authors

  • Husted, L. B.
  • Sorensen, E. B.
  • Armstrong, P. B.
  • Quigley, James
  • Kristensen, L.
  • Sottrup-Jensen, L.

publication date

  • 2002

journal

  • Journal of Biological Chemistry  Journal

abstract

  • The primary structure determination of the dimeric invertebrate alpha(2)-macroglobulin (alpha(2)M) from Limulus polyphemus has been completed by determining its sites of glycosylation and disulfide bridge pattern. Of seven potential glycosylation sites for N-linked glycosylation, six (Asn(275), Asn(307), Asn(866), Asn(896), Asn(1089), and Asn(1145)) carry common glucosamine-based carbohydrates groups, whereas one (Asn(80)) carries a carbohydrate chain containing both glucosamine and galactosamine. Nine disulfide bridges, which are homologues with bridges in human alpha(2)M, have been identified (Cys(228)-Cys(269), Cys(456)-Cys(580), Cys(612)-Cys(799), Cys(657)-Cys(707), Cys(849)-Cys(876), Cys(874)-Cys(910), Cys(946)-Cys(1328), Cys(1104)-Cys(1155), and Cys(1362)-Cys(1475)). In addition to these bridges, Limulus alpha(2)M contains three unique bridges that connect Cys(361) and Cys(382), Cys(1370) and Cys(1374), respectively, and Cys(719) in one subunit with the same residue in the other subunit of the dimer. The latter bridge forms the only interchain disulfide bridge in Limulus alpha(2)M. The location of this bridge within the bait region is discussed and compared with other alpha-macroglobulins. Several peptides identified in the course of determining the disulfide bridge pattern provided evidence for the existence of two forms of Limulus alpha(2)M. The two forms have a high degree of sequence identity, but they differ extensively in large parts of their bait regions suggesting that they have different inhibitory spectra. The two forms (Limulus alpha(2)M-1 and -2) are most likely present in an approximately 2:1 ratio in the hemolymph of each animal, and they can be partially separated on a Mono Q column at pH 7.4 by applying a shallow gradient of NaCl.

subject areas

  • Amino Acid Sequence
  • Animals
  • Asparagine
  • Binding Sites
  • Carbohydrates
  • Chromatography, Gel
  • Chromatography, High Pressure Liquid
  • Chromatography, Ion Exchange
  • Cloning, Molecular
  • Cysteine
  • DNA, Complementary
  • Disulfides
  • Electrophoresis, Polyacrylamide Gel
  • Glycosylation
  • Hemolymph
  • Horseshoe Crabs
  • Humans
  • Hydrogen-Ion Concentration
  • Methylamines
  • Molecular Sequence Data
  • Polymerase Chain Reaction
  • Protein Binding
  • Protein Structure, Tertiary
  • Sequence Homology, Amino Acid
  • Sodium Chloride
  • Trypsin
  • alpha-Macroglobulins
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Identity

International Standard Serial Number (ISSN)

  • 0021-9258

Digital Object Identifier (DOI)

  • 10.1074/jbc.M208236200

PubMed ID

  • 12218066
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Additional Document Info

start page

  • 43698

end page

  • 43706

volume

  • 277

issue

  • 46

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