E. Eldering et al., COOH-TERMINAL SUBSTITUTIONS IN THE SERPIN C1 INHIBITOR THAT CAUSE LOOP OVERINSERTION AND SUBSEQUENT MULTIMERIZATION, The Journal of biological chemistry, 270(6), 1995, pp. 2579-2587
The region COOH-terminal to the reactive center loop is highly conserv
ed in the serine protease inhibitor (serpin) family, We have studied t
he structural consequences of three substitutions (Val(451) --> Met, P
he(455) --> Ser, and Pro(476) --> Ser) found in this region of C1 inhi
bitor in patients suffering from hereditary angioedema, Equivalent sub
stitutions have been described in alpha 1-antitrypsin and antithrombin
m. The mutant C1 inhibitor proteins were only partially secreted upon
transient transfection into COS-7 cells and were found to be dysfunct
ional, Immunoprecipitation of conditioned media demonstrated that in t
he intact, uncleaved form they all bind to a monoclonal antibody which
recognizes specifically the protease-complexed or reactive center-cle
aved normal C1 inhibitor, A second indication for an intrinsic conform
ational change was the increased thermostability compared to the norma
l protein. Furthermore, gel filtration studies showed that the Val(451
) --> Met and Pro(476) --> Ser mutant proteins, and to a lesser extent
Phe(455) --> Ser, were prone to spontaneous multimerization. Finally,
a reduced susceptibility to reactive center cleavage by trypsin was o
bserved for all three mutants, and the cleaved Val(451) --> Met and Pr
o(476) --> Ser mutants failed to adopt the conformation recognized by
a cleavage-specific monoclonal antibody, Investigation of plasmas of p
atients with the Val(451) --> Met or Pro(476) --> Ser substitutions sh
owed that these dysfunctional proteins circulate at low levels and are
recognized by the complex-specific antibody. These results strongly i
ndicate a conformational change as a result of these carboxyl-terminal
substitutions, such that anchoring of the reactive center loop at the
COOH-terminal side is not achieved properly. We propose that this res
ults in over-insertion of the loop into beta-sheet A, which subsequent
ly leads to multimerization.