Tài liệu Báo cáo Y học: Regulated expression and intracellular localization of cystatin F in human

Regulated expression and intracellular localization of cystatin F

in human U937 cells

Carl-Michael Nathanson1

, Johan Wasse´ lius2

, Hanna Wallin1 and Magnus Abrahamson1

1

Department of Clinical Chemistry, Institute of Laboratory Medicine, and 2

Department of Ophtalmology, University of Lund,

University Hospital, Lund, Sweden

Cystatin F is a cysteine peptidase inhibitor recently discov￾ered in haematopoietic cells by cDNA cloning. To further

investigate the expression,distribution and properties of the

native human inhibitor the promyeloid cell line U937 has

been studied. The cells expressed relatively large quantities of

cystatin F,which was found both secreted and intracellu￾larly. The intracellular levels were unusually high for a

secreted cystatin (
25% of the cystatin F in 2- or 4-day

culture medium). By contrast,U937 cells contained only

3–4% of the related inhibitor,cystatin C. Cystatin F purified

from lysates of U937 cells showed three major forms car￾rying two,one or no carbohydrate chains. Immunocyto￾chemistry demonstrated a marked cytoplasmic cystatin F

staining in a granular pattern. Double staining with a marker

for endoplasmic reticulum revealed no colocalization for

cystatin F. Analysis of the promoter region of the cystatin F

gene (CST7) showed that it,like that of the cystatin C gene

(CST3),is devoid of typical TATA- and CAAT-box ele￾ments. In contrast to the cystatin C promoter,it does not

contain multiple Sp1 binding sites,but has a unique site for

C/EBPa,possibly explaining the restricted expression of the

cystatin F gene. Cells stimulated with all-trans retinoic acid

to differentiate them towards a granulocytic pathway,

showed a strong (
18-fold) down-regulation of intracellu￾lar cystatin F and almost abolished secreted levels of the

inhibitor. Stimulation with tetradecanoyl phorbol acetate,

causing monocytic differentiation,also resulted in down￾regulation (two fold to threefold) of cystatin F expression,

whereas the cystatin C expression was essentially unaltered

in both experiments. The results suggest that cystatin F as an

intracellular cysteine peptidase inhibitor with readily regu￾lated expression,may be a candidate to control the cysteine

peptidase activity known to be essential for antigen presen￾tation in different blood cell lineages.

Keywords: cysteine protease; cysteine protease inhibitor;

cystatin F; cystatin C; expression pattern.

Mammalian papain-like (family C1) peptidases,such as

cathepsins B,H,L,S and K,can be regulated by natural

inhibitors belonging to the cystatin superfamily. These

inhibitors are of three major types [1]. Those of type 1,

cystatins A and B (also called stefins),are approximately

100 amino-acid long polypeptides lacking signal peptides

and disulfide bridges. Type 2 cystatins,synthesized with

signal peptides,are approximately 120 amino acids long and

contain at least two disulfide bridges. Type 3 cystatins,the

kininogens,are larger proteins containing three tandemly

repeated type 2-like cystatin domains. Type 2 cystatins

identified in higher animals include cystatins C,D,S,SA,

and SN [2],but also the recently reported cystatin E/M [3,4]

and cystatin F [5–7].

Human cystatin F is synthesized as a 145-amino-acid

residue preprotein,with the first 19 residues theoretically

constituting a signal peptide. Secretion of a 126-residue

mature protein has been verified for recombinant cystatin F

produced in insect cells [5]. The mature sequence shows 29–

34% identity when compared to the sequences of other

human type 2 cystatins. It has two possible N-glycosylation

motifs at positions 36–38 and 88–90 (cystatin C numbering).

Recombinant cystatin F expressed in Sf9 insect cells is

indeed glycosylated [5].

Compared to other cystatins,cystatin F is an unusually

specific inhibitor. Among the family C1 cysteine peptidases

studied,cystatin F binds papain and cathepsin L with high

affinity (Ki 0.1–1 nM),but does not inhibit cathepsin B

(Ki > 1000 nM) [5,6]. Cathepsin L is a peptidase involved in

the normal lysosomal turnover of proteins. However,more

specialized functions have also been attributed to the

enzyme. So has cathepsin L been shown to be involved in

the loading of MHC II complex at antigen presentation. In

cortical thymic epithelial cells from mice devoid of the

cathepsin L gene,CLIP 22 and CLIP 10,two intermediate

processing products of the invariant chain accumulate [8].

This indicates that the amount of cysteine peptidase activity

in the processing compartment for the invariant chain is

crucial for antigen presentation and,moreover,that this

activity may be regulated by a cystatin [9].

Besides the inhibitory site for family C1 peptidases,cyst￾atin F carries a second site for inhibition of the family C13

enzyme,mammalian legumain or asparginyl endopeptidase

Correspondence to M. Abrahamson,Department of Clinical

Chemistry,Institute of Laboratory Medicine,University Hospital,

S-221 85 Lund,Sweden. Fax: + 46 46 130064,Tel.: + 46 46 173445

E-mail: [email protected]

Abbreviations: ATRA,all-trans retinoic acid; TPA,tetradecanoyl

phorbol acetate; GAPDH,glyceraldehyde-3-phosphate

dehydrogenase.

Note: web page available at http://www.klinkem.lu.se/E/abrahamson

Note: nucleotide sequences are available in the DDBJ/EMBL/

GenBank databases under accession nos AJ51067,AJ51068,AJ51069

and AJ51070.

(Received 12 June 2002,revised 12 August 2002,

accepted 11 September 2002)

Eur. J. Biochem. 269,5502–5511 (2002)
FEBS 2002 doi:10.1046/j.1432-1033.2002.03252.x