Reversible and irreversible cross-linking of immunoglobulin heavy chains through their carbohydrate residues.
After periodate oxidation and incubation with a dihydrazide, cross-linking of the two heavy chains of immunoglobulins G from several species proceeds specifically through their oligosaccharides. We have used malonic acid dihydrazide, adipic acid dihydrazide and dithiodipropionic acid dihydrazide. The last compound is introduced in this work as a cleavable-carbohydrate-specific cross-linker. It was found that in rabbit and human immunoglobulins the degree of cross-linking was strongly dependent on the oxidation conditions but only very weakly dependent on the concentration and size of the dihydrazides. Papain cleavage of the cross-linked rabbit IgG indicated that the cross-linking occurred predominantly, if not exclusively, in the Fc region, probably through the two glycans linked to Asn-297 in the CH2 domain of each of the two heavy chains. The immunoglobulins from sheep, pig, goat and guinea pig show a comparable cross-linking pattern, indicating that the sugar chains from these immunoglobulins have a spatial structure closely related to that of rabbit and human IgG. When dithiodipropionic acid dihydrazide was used as the cross-linker, the cross-link could be cleaved by mercaptoethanol.
Heimgartner
U
U
Department of Biotechnology, Swiss Federal Institute of Technology, Zürich.
Kozulić
B
B
Mosbach
K
K
D000818:Animals; D003432:Cross-Linking Reagents / Q000494:pharmacology*; D006041:Goats; D006168:Guinea Pigs; D006801:Humans; D007074:Immunoglobulin G; D007143:Immunoglobulin Heavy Chains; D009844:Oligosaccharides; D010084:Oxidation-Reduction; D011817:Rabbits; D012756:Sheep; D013552:Swine
D003432:Cross-Linking Reagents; D007074:Immunoglobulin G; D007143:Immunoglobulin Heavy Chains; D009844:Oligosaccharides