Instability of the amyloidogenic cystatin C variant of hereditary cerebral hemorrhage with amyloidosis, Icelandic type.
A cystatin C variant with L68Q substitution and a truncation of 10 NH2-terminal residues is the major constituent of the amyloid deposited in the cerebral vasculature of patients with the Icelandic form of hereditary cerebral hemorrhage with amyloidosis (HCHWA-I). Variant and wild type cystatin C production, processing, secretion, and clearance were studied in human cell lines stably overexpressing the cystatin C genes. Immunoblot and mass spectrometry analyses demonstrated monomeric cystatin C in cell homogenates and culture media. While cystatin C formed concentration-dependent dimers, the HCHWA-I variant dimerized at lower concentrations than the wild type protein. Amino-terminal sequence analysis revealed that the variant and normal proteins produced and secreted are the full-length cystatin C. Pulse-chase experiments demonstrated similar levels of normal and variant cystatin C production and secretion. However, the secreted variant cystatin C exhibited an increased susceptibility to a serine protease in conditioned media and in human cerebrospinal fluid, explaining its depletion from the cerebrospinal fluid of HCHWA-I patients. Thus, the amino acid substitution may induce unstable cystatin C with intact inhibitory activity and predisposition to self-aggregation and amyloid fibril formation.
Wei
L
L
Departments of Pharmacology and Pathology, New York University Medical Center, New York, New York 10016, USA.
Berman
Y
Y
Castaño
E M
EM
Cadene
M
M
Beavis
R C
RC
Devi
L
L
Levy
E
E
D000595:Amino Acid Sequence; D000686:Amyloidosis / Q000235:genetics*; D000818:Animals; D016657:Cerebral Amyloid Angiopathy / Q000235:genetics*; D002543:Cerebral Hemorrhage / Q000235:genetics*; D003470:Culture Media; D055316:Cystatin C; D015891:Cystatins / Q000235:genetics* / Q000378:metabolism; D019281:Dimerization; D010450:Endopeptidases / Q000378:metabolism; D006801:Humans; D006868:Hydrolysis; D051379:Mice; D008969:Molecular Sequence Data; D011480:Protease Inhibitors / Q000494:pharmacology; D011499:Protein Processing, Post-Translational; D014162:Transfection
C525030:CST3 protein, human; C525031:Cst3 protein, mouse; D003470:Culture Media; D055316:Cystatin C; D015891:Cystatins; D011480:Protease Inhibitors; D010450:Endopeptidases