Tài liệu Báo cáo khoa học: Inhibitory properties of cystatin F and its localization in U937

Inhibitory properties of cystatin F and its localization

in U937 promonocyte cells

Tomazˇ Langerholc1

, Valentina Zavasˇnik-Bergant1

, Boris Turk1

, Vito Turk1

, Magnus Abrahamson2

and Janko Kos3

1 Department of Biochemistry and Molecular Biology, Jozˇef Stefan Institute, Ljubljana, Slovenia

2 Department of Clinical Chemistry, Institute of Laboratory Medicine, University of Lund, Sweden

3 Faculty of Pharmacy, Department of Pharmaceutical Biology, University of Ljubljana, Slovenia

Human papain-like cathepsins were long believed to be

responsible for terminal protein degradation in the

lysosomes. This view changed dramatically when they

were found to be involved in a number of important

cellular processes, such as antigen presentation [1],

bone resorption [2], apoptosis [3] and protein process￾ing [4], as well as several pathologies such as cancer

progression [5], inflammation [6] and neurodegenera￾tion [7]. Their high proteolytic potential, which can be

very harmful, requires the activity of papain-like cath￾epsins to be strictly regulated. Their endogenous pro￾tein inhibitors act as one of the main means of

regulation [8]. The best characterized are the cystatins,

which comprise a superfamily of evolutionarily related

proteins, each consisting of at least one domain of

100–120 amino acid residues with conserved sequence

motifs [8–11]. Type I cystatins (the stefins), stefins A

and B, are cytosolic,
100 amino acid residue-long

proteins lacking disulfide bridges. Type II cystatins,

cystatins C, D, E⁄M, F, S, SA, SN are longer extra￾cellular proteins, consisting of
120 amino acid resi￾dues and containing two disulfide bridges. Type III

cystatins, the kininogens, are large multifunctional

plasma proteins, containing three type II cystatin-like

domains.

Cystatin F was discovered recently by three inde￾pendent groups. Two of them identified it by cDNA

cloning and named the new inhibitor leukocystatin [12]

Keywords

cathepsin; cysteine protease; inhibition;

cystatin; antigen presentation

Correspondence

T. Langerholc, Department of Biochemistry

and Molecular Biology, Jozˇef Stefan

Institute, Ljubljana, Slovenia

Fax: +386 14773984

Tel: +386 14773573

E-mail: [email protected]

(Received 9 November 2004, revised 31

January 2005, accepted 2 February 2005)

doi:10.1111/j.1742-4658.2005.04594.x

Cystatin F is a recently discovered type II cystatin expressed almost exclu￾sively in immune cells. It is present intracellularly in lysosome-like vesicles,

which suggests a potential role in regulating papain-like cathepsins involved

in antigen presentation. Therefore, interactions of cystatin F with several

of its potential targets, cathepsins F, K, V, S, H, X and C, were studied

in vitro. Cystatin F tightly inhibited cathepsins F, K and V with Ki values

ranging from 0.17 nm to 0.35 nm, whereas cathepsins S and H were inhib￾ited with 100-fold lower affinities (Ki
30 nm). The exopeptidases, cathep￾sins C and X were not inhibited by cystatin F. In order to investigate the

biological significance of the inhibition data, the intracellular localization

of cystatin F and its potential targets, cathepsins B, H, L, S, C and K,

were studied by confocal microscopy in U937 promonocyte cells. Although

vesicular staining was observed for all the enzymes, only cathepsins H and

X were found to be colocalized with the inhibitor. This suggests that cysta￾tin F in U937 cells may function as a regulatory inhibitor of proteolytic

activity of cathepsin H or, more likely, as a protection against cathepsins

misdirected to specific cystatin F containing endosomal ⁄ lysosomal vesicles.

The finding that cystatin F was not colocalized with cystatin C suggests

distinct functions for these two cysteine protease inhibitors in U937 cells.

Abbreviations

mAb, monoclonal antibody; pAb, polyclonal antibody; M6P, mannose-6-phosphate.

FEBS Journal 272 (2005) 1535–1545 ª 2005 FEBS 1535