Localization of Carbohydrate Attachment Sites and Disulfide Bridges in Limulus α2-Macroglobulin

The primary structure determination of the dimeric invertebrate α 2 -macroglobulin (α 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 α 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 α 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 α 2 M. The location of this bridge within the bait region is discussed and compared with other α-macroglobulins. Several peptides identified in the course of determining the disulfide bridge pattern provided evidence for the existence of two forms of Limulus α 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 α 2 M-1 and -2) are most likely present in an ∼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.