Instability of the Amyloidogenic Cystatin C Variant of Hereditary Cerebral Hemorrhage with Amyloidosis, Icelandic Type

Abstract

A cystatin C variant with L68Q substitution and a truncation of 10 NH 2 -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.

Checking the conformational stability of cystatin C and its L68Q variant by molecular dynamics studies: Why is the L68Q variant amyloidogenic?

“…The longest distance between the and interface in the L68Q mutant in comparison with wt cystatin C suggests that the L68Q molecule displays a larger solvent accessible surface than that of the native protein. Therefore, the secreted wt cystatin C protein is probably more resistant to extracellular proteolysis, whereas the L68Q cystatin C variant is quickly degraded in vitro by proteases (Wei et al, 1998). It is not well known whether L68Q cystatin C variant is truncated in vivo (Bjarnadottir et al, 2001;Gerhartz and Abrahamson, 2002).…”

Hydrogen Peroxide Induces Lysosomal Protease Alterations in PC12 Cells

“…Changes in mRNA and protein expression of CYSC have been associated with oxidative stress [26]. Furthermore, changes in CYSC expression have been seen in various models of neuronal injuries such as PD [27][28][29], AD [30,31], ALS [32,33], heritable cerebral hemorrhage with amyloidosis of the Icelandic type angiopathy (HCHWA-I) [34], and transient forebrain ischemia [35]. However, the significance between the association of CYSC with CATB and CATD in neurodegenerative disorders and neuronal injury remains unknown.…”

Cystatin C in aging and in Alzheimer’s disease

“…This primary sequence is indicative of a secreted protein, and accordingly, most newly synthesized CysC is secreted from a cell (Barka et al, 1992; Chapman et al, 1990; Paraoan et al, 2001; Tavera et al, 1992; Wei et al, 1998; Zucker-Franklin et al, 1987). Cell surface CysC has been demonstrated in various cells (Calkins et al, 1998; Sastre et al, 2004; Taupin et al, 2000), consistent with it being both a secreted protein as well as one that can be internalized into a cell via endocytosis (Ekstrom et al, 2008; Kolodziejczyk et al, 2010; Merz et al, 1997).…”