A Cellulose-Binding Domain-Fused Recombinant Human T Cell Connective Tissue-Activating Peptide-III Manifests Heparanase Activity Meirav Rechter,* Ofer Lider,* ,1 Liora Cahalon,* Ehud Baharav,* Mara Dekel,† Daniel Seigel,† Israel Vlodavsky,‡ Helena Aingorn,‡ Irun R. Cohen,* and Oded Shoseyov† *Department of Immunology, The Weizmann Institute of Science, Rehovot 76100, Israel; †Kennedy Leigh Centre for Horticultural Research and the Otto Warburg Center for Agricultural Biotechnology, The Faculty of Agriculture, The Hebrew University of Jerusalem, Rehovot, Israel; and ‡Department of Oncology, Hadassah-Hebrew University Hospital, Jerusalem, Israel Received December 29, 1998 The chemokine connective tissue-activating peptide (CTAP)-III, which belongs to the leukocyte-derived growth factor family of mediators, was previously shown to be mitogenic for fibroblasts. However, it has recently been shown that CTAP-III, released from platelets, can act like a heparanase enzyme and de- grade heparan sulfate. This suggests that CTAP-III may also function as a proinflammatory mediator. We have successfully cloned CTAP-III from a gt11 cDNA library of PHA-activated human CD4  T cells and pro- duced recombinant CTAP-III as a fusion protein with a cellulose-binding domain moiety. This recombinant CTAP-III exhibited heparanase activity and released degradation products from metabolically labeled, nat- urally produced extracellular matrix. We have also developed polyclonal and monoclonal antibodies, and these antibodies against the recombinant CTAP-III de- tected the CTAP-III molecule in human T cells, poly- morphonuclear leukocytes, and placental extracts. Thus, our study provides tools to examine further im- mune cell behavior in inflamed sites rich with extra- cellular moieties and proinflammatory mediators. © 1999 Academic Press Chemokines are a recently characterized family of pro-inflammatory mediators that function biologically in both blood vessels and in extracellular matrix sites to promote immune cell-adhesion and migration (1-4). One member of the C-X-C chemokine sub-family, con- nective tissue-activating peptide (CTAP)-III, is mito- genic for connective tissue cells, stimulates glucose uptake, glycosaminoglycan synthesis, proliferation of fibroblasts and synovial cells (5,6), induces the release of histamine from basophils (7), and induces plasmin- ogen activator activity (8). Detection of CTAP-III in wound fluid (9), in the synovium in osteoarthritis, and in the sera of rheumatoid arthritis patients (10,11) suggests that CTAP-III may also be involved in inflam- mation. CTAP-III is a N'-truncated derivative of the leukocyte-derived growth factor (LDGF) molecule (12,13). Cleavage of LDGF (a 128-amino acid protein) at its Arg-Asn site generates CTAP-III, a 85-amino acid protein with a molecular weight of 9278 Da (13). CTAP-III has been identified in platelets (6), activated macrophages, neutrophils, and more recently, also in T lymphocytes (12), all of which are involved in inflam- matory reactions. In addition to the stimulatory activities of CTAP-III, heparanase-like heparan sulfate-degrading activity of platelet-derived CTAP-III has been recently reported (14). Specifically, a heparan sulfate-degrading mole- cule was purified from human platelets. N-terminal sequencing revealed that this enzymatic activity was due mainly to CTAP-III and neutrophil-activating pep- tide (NAP)-2, and partially to platelet basic protein (PBP), all of which are derivatives of the LDGF gene. This heparanase activity is also present in commer- cially prepared CTAP-III isolated from human plate- lets (14), but not in commercially prepared recombi- nant NAP-2. Heparan sulfate proteoglycans (HSPG) are associ- ated with cell surfaces and the extracellular matrix (ECM; 15) and bind growth factors and cytokines. Cleavage of the heparan sulfate moieties of HSPG in 1 To whom correspondence should be addressed at Department of Immunology, The Weizmann Institute of Science, Rehovot 76100, Is- rael. Fax: 792-8-9343657. E-mail: lclider@weizmann.weizmann.ac.il. Abbreviations used: CTAP-III, connective tissue activating pep- tide-III; LDGF, leukocyte derived growth factor; NAP-2, neutrophil activating peptide-2; HSPG, heparan sulfate proteoglycans; ECM, extracellular matrix; CBD, cellulose binding domain. Biochemical and Biophysical Research Communications 255, 657– 662 (1999) Article ID bbrc.1999.0181, available online at http://www.idealibrary.com on 657 0006-291X/99 $30.00 Copyright © 1999 by Academic Press All rights of reproduction in any form reserved.