THE PLASMA AND CYTOPLASMIC FORMS OF HUMAN GELSOLIN DIFFER IN DISULFIDE STRUCTURE

Gelsolin is a widely distributed actin binding protein that regulates actin filament length. It exists in both an intracellular and an extra cellular form that is derived from a single gene by alternative splici ng. Both forms contain the six homologous domains that are responsible fur function. Little is known regarding differences between the forms . We have used a combination of cysteine-specific modification with 3- vinylpyridine, HPLC peptide mapping methods, and mass spectrometry to analyze the disulfide structures of human plasma and cytoplasmic gelso lin. Of the five Cys residues in the human gelsolin sequence, all were present in the free thiol form in human cytoplasmic gelsolin while on ly three of them were foe thiols in the human plasma form. Cys residue s 188 and 201 in domain 2 of plasma gelsolin were disulfide linked. Re combinant human plasma gelsolin that had been expressed intracellularl y In Escherichia call and as a secreted protein from Cos green monkey cells was also investigated. The E. coli product lacked the disulfide but could be converted to the plasma-like structure with mild oxidatio n while the mammalian product formed tile correct disulfide prior to i solation. Structural differences were also detected by limited proteol ysis with plasmin. The differences in proteolytic susceptibility were also due to perturbations in domain 2. These studies demonstrate that the intracellular and extracellular gelsolins are structurally distinc t and suggest that at least some of the preparations of recombinant ge lsolin that are being used to study structure/function may be improper ly folded. The experiments also demonstrate a general method fur the l ocation of disulfide bonds in proteins.