THIOREDOXIN DOMAIN NON-EQUIVALENCE AND ANTI-CHAPERONE ACTIVITY OF PROTEIN DISULFIDE-ISOMERASE MUTANTS IN-VIVO

Coexpression of the enzyme, protein disulfide isomerase (PDI), has bee n shown to increase soluble and secreted IgG levels from baculovirus-i nfected insect cells (Hsu, T.-A., Watson, S., Eiden, J. J., and Betenb augh, M. J. (1996) Protein Expression Purif. 7, 281-288), PDI is known to include catalytic active sites in two separate thioredoxin-like do mains, one near the amino terminus and another near the carboxyl termi nus, To examine the role of these catalytic active sites in enhancing immunoglobulin solubility, baculovirus constructs were utilized with c ysteine to serine mutations at the first cysteine of one or both of th e CGHC active site sequences, Trichoplusia ni insect cells were coinfe cted with a baculovirus vector coding for IgG in concert with either t he wild-type human PDI virus, amino-terminal mutant (PDI-N), carboxyl- terminal mutant (PDI-C), or mutant with both active sites altered (PDI -NC), Western blot analysis revealed that both immunoglobulins and PDI protein were expressed in the coinfected cells, To evaluate the effec t of the PDI variants on immunoglobulin solubility and secretion, the infected cells were labeled with S-35-amino-acids for different period s, and the soluble immunoglobulins were immunoprecipitated from clarif ied cell lysates and culture medium using anti-IgG antibodies, Only co infections with the wild-type PDI and PDI-N mutant led to increased im munoglobulin solubility and higher IgG secretion, In contrast, infecti on with the PDI-C and PDI-NC variants actually lowered immunoglobulin solubility levels below those achieved with a negative control virus, Immunoprecipitation with anti-PDI antibody revealed that heterologous PDI-C and PDI-NC were insoluble, even though PDI-N and wild-type PDI p rotein were detected in soluble form, The capacity for PDI-N to increa se immunoglobulin solubility whereas the PDI-C mutant lowered solubili ty indicates that the amino-and carboxyl-terminal thioredoxin domains of PDI are functionally distinct in vivo following mutations to the ac tive site, Furthermore, mutations at the active site of the carboxyl-t erminal thioredoxin domain result in PDI variants that can act as anti -chaperones of immunoglobulin solubility in vivo as has been observed in vitro for lysozyme aggregation by wild-type PDI and PDI mutants (Pu ig, A., and Gilbert, H. F. (1994) J. Biol. Chem. 269, 7764-7771).