Analysis of the assembly mechanism for multimeric lamprey VLRB antibodies

Abstract Instead of the immunoglobulin-type antigen receptors in jawed vertebrates, the surviving jawless vertebrates use variable lymphocyte receptors (VLRs) that are constructed with variable leucine-rich repeat (LRR) sequences to recognize antigens. Two VLR genes in lamprey and hagfish are somatically assembled during lymphopoiesis using a gene conversion-like mechanism in which highly diverse LRR flanking sequences are copied in serial fashion to construct mature VLRs. Lamprey have been shown to secrete polymeric VLRB antibodies that consist of 8-12 identical disulfide-linked subunits. The overall structure of VLRB antibodies resembles that of IgM antibodies in that both are composed of 4-6 dimers. Previous studies have shown that eight cysteines in the C-terminal hydrophobic region are involved in assembly of the multivalent VLRB antibodies through formation of disulfide bonds. We have characterized a panel of VLRB Cys to Ser mutants using site-directed mutagenesis to determine which cysteines are used for disulfide bond formation and VLRB polymerization. Two or more disulfide bonds were found to be essential for VLRB dimerization and polymerization. Our data further suggest that the carboxy-terminal cysteines 5, 6, 7, and 8 form the cornerstone for VLRB dimerization and polymerization, whereas cysteines 1, 2, 3, and 4 contribute to the stability of the polymer structure.