Disulfide bond formation of heterodimer and heterotrimer of human laminin-332 coiled-coil domains

Abstract

Laminin (LM) α, β, and γ chains were connected by disulfide bonds at the C-and N-termini of the LM coiled-coil (LCC) domain to form heterodimers and heterotrimers. At the Cterminus of LCC domain, one disulfide bond is formed to connect β and γ chains while it was unclear how disulfide bond pattern is formed to connect α, β, and γ chains at the Nterminus of LCC domain. Using an insect cell-free translation system, we succeeded to produce heterotrimers of LCC domain of human LM-332. To analyze disulfide bond formation at the N-terminus of LCC domain, we mutated cysteines of LCC domains into alanines by site-directed mutagenesis and co-expressed these mutants in an insect cell-free translation system. Mutation of a single cysteine at the N-terminus of LCC domain of one chain caused the failure of disulfide bond formation of heterotrimers. However, mutation of cysteines at the N-terminus of LCC domain of two different chains recovered the disulfide bond formation of heterotrimers with different efficiencies. These results suggest that the disulfide bond patterns at the N-terminus of human LM-332 LCC domains are not specific. ARTICLE INFO ABSTRACTEditor: