Prevention of domain swapping inhibits dimerization and amyloid fibril formation of cystatin C: use of engineered disulfide bridges, antibodies, and carboxymethylpapain to stabilize the monomeric form of cystatin C.

Maria Nilsson,Zbigniew Grzonka,Mariusz Jaskolski,Patrik Önnerfjord,Gunilla Westermark,Sylwia Rodziewicz-Motowidlo,Anders Grubb,Veronica Lindström,Xin Wang,Robert Janowski

doi:10.1074/jbc.m402621200

Abstract

Amyloidogenic proteins like cystatin C and prion proteins have been shown to form dimers by exchange of subdomains of the monomeric proteins. This process, called "three-dimensional domain swapping," has also been suggested to play a part in the generation of amyloid fibrils. One variant of cystatin C, L68Q cystatin C, is highly amyloidogenic, and persons carrying the corresponding gene suffer from massive cerebral amyloidosis leading to brain hemorrhage and death in early adult life. The present work describes the production of two variants of wild type and L68Q cystatin C with disulfide bridges at positions selected to inhibit domain swapping without affecting the biological function of the four cystatin C variants as cysteine protease inhibitors. The capacity of the four variant proteins to form dimers was tested and compared with that of wild type and L68Q cystatin C. In contrast to the latter two proteins, all four protein variants stabilized by disulfide bridges were resistant toward the formation of dimers. The capacity of the two stabilized variants of wild type cystatin C to form amyloid fibrils was investigated and found to be reduced by 80% compared with that of wild type cystatin C. In an effort to investigate whether exogenous agents could also suppress the formation of dimers of wild type and L68Q cystatin C, a monoclonal antibody or carboxymethylpapain, an inactivated form of a cysteine protease, was added to systems inducing dimerization of wild type and L68Q cystatin C. It was observed that catalytic amounts of both the monoclonal antibody and carboxymethylpapain could suppress dimerization.

Highlights

Amyloidogenic proteins like cystatin C and prion proteins have been shown to form dimers by exchange of subdomains of the monomeric proteins
To investigate whether suppression of this flexibility would suppress the ability of the molecules to form dimers and fibrils, an attempt was made to produce two variants of each protein stabilized by an extra disulfide bridge for each variant
To create the second type of stabilized monomeric wt and L68Q cystatin C molecules, the mutations were selected to introduce a disulfide bridge between the ␣-helix and the ␤5-strand, which separate during the process of three-dimensional domain swapping of cystatin C [7]

Summary

Location of disulfide bond wt cystatin C

The crystallographic model of the wt cystatin C dimer explains why the mutated protein is less stable and undergoes domain swapping dimerization much more [7]. Because the altered residue is located in the central strand (␤3) of the ␤-sheet and is covered by the ␣-helix, where it occupies a hydrophobic pocket formed by residues Val-31 and Tyr-34, the longer and hydrophilic glutamine side chain causes destabilization of the molecular ␣/␤ interface, leading to structural rearrangements and increased probability of partial unfolding. The objective of the present work has been to produce two variants of both wt and L68Q cystatin C with disulfide bridges at positions selected to inhibit domain swapping without affecting the biological function of the four cystatin C variants and to investigate whether this would result in suppression of dimerization and fibril formation. In additional experiments investigating whether exogenous agents could inhibit the formation of dimers of wt and L68Q cystatin C, a monoclonal antibody and carboxymethylpapain were tested and both were shown to suppress the dimerization process

EXPERIMENTAL PROCEDURES

RESULTS

AFCSFQIYAVPWQGTCTLSK ϩ STCQDA ϩ ALDCAVGEYNK

DISCUSSION