Cystatin F is a cysteine peptidase inhibitor and a member of the type II cystatin family. It is synthesized as an 145 amino acid (AA) long inactive dimer. N-linked glycans alow it to transfer to endosomes/lysosomes, where it is activated. Monomerization and activation of cystatin F is facilitated by the proteolytic cleavege of the 15 AA at the N-terminal part. The proteolytic cleavage also alters the inhibitory profile of the monomeric form. Consequently, the N-terminal truncated cystatin F becomes a strong inhibitor of cathepsin C. The secreted inactive dimeric form of cystatin F can also be taken up by bystander cells and activated within them. Cystatin F is specifically expressed in immune cells, however, in pathological conditions its expression can be present in different cell types. In multiple sclerosis, during the phase of demyelination it is expressed in microglia. Elevated expression of the Cst 7 gene, which encodes the cystatin F, has also been shown in a mouse model of Alzheimerˊs disease. Microglia are macrophage-like cells of the central nervous system (CNS). The aim of our study was to determine the intracellular localization of cistatin F in human microglia, its secretion and uptake into the bystander cells. Main targets of cystatin F in microglia were determined by measuring the activity of cysteine proteases. For this purpose, h-TERT cells were transfected with plasmids containing dimeric (wild type), active N-terminally truncated or non-glycosylated form of cistatin F. The expression of cystatin F in microglia, its secretion from cells and uptake into bystander cells were determined by using western blot. Localization of individual forms of cystatin F within the cells were determined by using fluorescence confocal microscopy. Both dimeric (wild type) and active N-terminally truncated cystatin F were found to be internalized and transfered to endosomes/lysosomes of microglia. However, due to the absence of N-linked glycans, the non-glycosylated form of cystatin F was not taken up by recipient cells and did not enter the endo/lysosomal system. The absence of N-linked glycans thus inhibits the inhibition of cysteine cathepsins and the action of cystatin F in trans in human microglia. The expression of cystatin F in microglia reduces the activity of cathepsin L wereas its effects on cathepsin C activity were negligible.
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