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.
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.
Nilsson
Maria
M
Department of Clinical Chemistry, University Hospital, S-22185 Lund, Sweden.
Wang
Xin
X
Rodziewicz-Motowidlo
Sylwia
S
Janowski
Robert
R
Lindström
Veronica
V
Onnerfjord
Patrik
P
Westermark
Gunilla
G
Grzonka
Zbigniew
Z
Jaskolski
Mariusz
M
Grubb
Anders
A
D000682:Amyloid / Q000737:chemistry*; D000911:Antibodies, Monoclonal / Q000737:chemistry; D055316:Cystatin C; D015891:Cystatins / Q000737:chemistry*; D019281:Dimerization; D004220:Disulfides; D004305:Dose-Response Relationship, Drug; D004587:Electrophoresis, Agar Gel; D004926:Escherichia coli / Q000378:metabolism; D006801:Humans; D007700:Kinetics; D013058:Mass Spectrometry; D008854:Microscopy, Electron; D016297:Mutagenesis, Site-Directed; D010206:Papain / Q000737:chemistry; D011487:Protein Conformation; D017433:Protein Structure, Secondary; D017434:Protein Structure, Tertiary; D019032:Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization; D013997:Time Factors; D014357:Trypsin / Q000494:pharmacology
D000682:Amyloid; D000911:Antibodies, Monoclonal; C525030:CST3 protein, human; D055316:Cystatin C; D015891:Cystatins; D004220:Disulfides; D014357:Trypsin; C028596:S-carboxymethyl-papain; D010206:Papain