Book of abstract 2008

Monomerisation of cystatin F: who’s in charge?
Tomaž Langerholc1, Boris Turk2, Janko Kos1 ,3
1Dept. of Biotechnology, Jožef Stefan Institute, Ljubljana, Slovenia; 2Dept. of Biochemistry, Molecular
and Structural Biology, Jožef Stefan Institute, Ljubljana, Slovenia; 3University of Ljubljana, Faculty of
Pharmacy, Ljubljana, Slovenia
Cystatin F is a cysteine protease inhibitor expressed mainly in cells important for the
immune system. Glycosylation, predominant lysosomal localisation and 6 cysteines instead
of four make cystatin F different from other members in the type II cystatin family. As a
monomer cystatin F is a potent inhibitor of C1 family of lysosomal cysteine proteases.
Inhibitory potential is abrogated in disulfide bonded dimers, which are predominantly
found in the cells. Cystatin F was proposed to regulate proteolytic activity in lysosome
– like organelles and hence the antigen presentation on MHC II molecules.
It is not known, whether active monomeric cystatin F is present in the lysosomes.
Monomeric cystatin F could be a transient form before dimerisation in the endoplasmic
reticulum (ER), or in the lysosomes after transportation of the monomers from the
ER. Alternatively, it may be created de novo by reduction of dimers in the reducing
lysosomal environment, or by assistance of GILT, the only known lysosomal reductase.
Monomerisation of cystatin F could also be performed by a specific protease, cleaving in
the N-terminal region after cys26. Cys26 is involved in the intermolecular disulfide bond
with cys44 from another molecule.
U937 promonocytic cells were subjected to ultracentrifugation experiment. We showed,
that monomeric form of cystatin F is present in the lysosomal fractions as well. The ratio
between the monomers and dimers changes in favour of the monomers upon activation
or differentiation of U937 cells. Disruption of the lysosomal pH effectively prevents or
delays this process, suggesting that acidic environment or an enzyme active in acidic pH
is involved in the process of monomerisation. Co-transfection of cystatin F and GILT
reductase into HEK293 cell line did not increase the ratio of the monomeric form,
suggesting that dimeric cystatin F is not an endogenous substrate for GILT. N-terminal
sequence of
l21
immunoprecipitated dimeric and monomeric cystatin F from transfected
HEK293 cells was as expected (GPSP), suggesting that a protease is not involved in
the generation of monomers. In contrast, monomers in U937 cells had a shorter N-
terminus. On the basis of cellular experiments we conclude, that a U937 cell line specific
protease acts downstream on removing the N-terminus of monomeric cystatin F, but it
is not necessary for the monomerisation process. We suggest, that lysosomal reductive
environment alone is responsible for the monomerisation of cystatin F, since we were
able to achieve monomerisation of recombinant dimeric cystatin F in buffers mimicking
lysosomal conditions (pH, redox potential).
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