HEREDITARY CYSTATIN-C AMYLOID ANGIOPATHY - MONITORING THE PRESENCE OFTHE LEU-68-]GLN CYSTATIN-C VARIANT IN CEREBROSPINAL FLUIDS AND MONOCYTE CULTURES BY MS

Hereditary cystatin C amyloid angiopathy (HCCAA) is an autosomal domin ant condition in which the patients suffer at an early age from repeat ed cerebral haemorrhages. The development of HCCAA is directly linked to a Leu-68 --> Gln (L68Q) mutation in the cystatin C protein sequence . The concentration of cystatin C in cerebrospinal fluid (CSF) of HCCA A patients is markedly diminished and cultivated monocytes from affect ed individuals accumulate cystatin C. The goal of this work was to cha racterize cystatin C isolated from CSF and monocyte cultures originati ng from healthy persons and HCCAA patients with respect to the L68Q mu tation. Cystatin C was isolated by carboxymethylpapain affinity chroma tography. Proteins from CSF and monocyte cultures that bound specifica lly to the carboxymethylated papain column were resolved by reverse-ph ase HPLC chromatography and tryptic peptides were subsequently analyse d by matrix-assisted laser desorption ionization MS. No evidence for m utated cystatin C protein was found in CSF samples from healthy subjec ts or HCCAA patients, but approx. 60 % of the protein was found to be hydroxylated on Pro-3. No evidence was found for secretion of mutated cystatin C from HCCAA monocytes. However, we obtained evidence for the presence of mutated cystatin C in HCCAA monocytes. These results supp ort the conclusion that the mutated cystatin C is retained in associat ion with the monocytes and not secreted. An increased intracellular co ncentration would presumably promote the aggregation and denaturation of the mutated cystatin C, leading to the formation of amyloid fibrils and cell death.