Amyloid fibril formation by human stefin B in vitro: immunogold labelling and comparison to stefin A.
The mechanism by which proteins form amyloid fibrils is of high interest to the scientific community as its understanding could resolve questions relevant to conformational diseases. The structural and energetic basis of the process is still largely unknown. The main controversial issue is the co-existence of several protein conformations. Three models for the mechanism of protein fibrillogenesis have been proposed which need to be tested by experiments. In this report, amyloid fibrils grown from human stefin B (type I cystatin) are described. This physiologically relevant protein readily forms fibrils in vitro, in contrast to the homologue--human stefin A--which forms fibrils under extreme conditions only. In order to specifically label stefin B fibrils in vitro, rabbit polyclonal antibody and mouse monoclonal antibody A6/2 against human stefin B were used for immunogold labelling. Samples were examined by transmission electron microscopy. Fibrils of stefin B were strongly labelled using polyclonal antibody and Protein A gold, whereas no positive reaction was observed with monoclonal antibody A6/2.
Zerovnik
Eva
E
Department of Biochemistry and Molecular Biology, Institute Jozef Stefan, Ljubljana, Slovenia.
Zavasnik-Bergant
Valentina
V
Kopitar-Jerala
Natasa
N
Pompe-Novak
Marusa
M
Skarabot
Miha
M
Goldie
Kenneth
K
Ravnikar
Maja
M
Musevic
Igor
I
Turk
Vito
V
D000682:Amyloid / Q000737:chemistry / Q000378:metabolism* / Q000648:ultrastructure; D000911:Antibodies, Monoclonal / Q000378:metabolism; D055312:Cystatin A; D055313:Cystatin B; D015891:Cystatins / Q000378:metabolism*; D006801:Humans; D006863:Hydrogen-Ion Concentration; D007150:Immunohistochemistry / Q000379:methods; D018625:Microscopy, Atomic Force; D008854:Microscopy, Electron; D011485:Protein Binding; D011487:Protein Conformation; D017510:Protein Folding; D011994:Recombinant Proteins / Q000737:chemistry
D000682:Amyloid; D000911:Antibodies, Monoclonal; C524994:CSTB protein, human; C501463:Csta1 protein, mouse; C524992:Cstb protein, mouse; D055312:Cystatin A; D015891:Cystatins; D011994:Recombinant Proteins; C026407:CSTA protein, human; D055313:Cystatin B