CXC Chemokines Connective Tissue Activating Peptide-III and Neutrophil Activating Peptide-2 are Heparin/Heparan Sulfate-degrading Enzymes

Abstract

Heparan sulfate proteoglycans at cell surfaces or in extracellular matrices bind diverse molecules, including growth factors and cytokines, and it is believed that the activities of these molecules may be regulated by the metabolism of heparan sulfate. In this study, purification of a heparan sulfate-degrading enzyme from human platelets led to the discovery that the enzymatic activity residues in at least two members of the platelet basic protein (PBP) family known as connective tissue activating peptide-III (CTAP-III) and neutrophil activating peptide-2. PBP and its N-truncated derivatives, CTAP-III and neutrophil activating peptide-2, are CXC chemokines, a group of molecules involved in inflammation and wound healing. SDS-polyacrylamide gel electrophoresis analysis of the purified heparanase resulted in a single broad band at 8-10 kDa, the known molecular weight of PBP and its truncated derivatives. Gel filtration chromatography of heparanase resulted in peaks of activity corresponding to monomers, dimers, and tetramers; these higher order aggregates are known to form among the chemokines. N-terminal sequence analysis of the same preparation indicated that only PBP and truncated derivatives were present, and commercial CTAP-III from three suppliers had heparanase activity. Antisera produced in animals immunized with a C-terminal synthetic peptide of PBP inhibited heparanase activity by 95%, compared with activity of the purified enzyme in the presence of the preimmune sera. The synthetic peptide also inhibited heparanase by 95% at 250 microM, compared to the 33% inhibition of heparanase activity by two other peptides. The enzyme was determined to be an endoglucosaminidase, and it degraded both heparin and heparan sulfate with optimal activity at pH 5.8. Chromatofocusing of the purified heparanase resulted in two protein peaks: an inactive peak at pI7.3, and an active peak at pI 4.8-5.1. Sequence analysis showed that the two peaks contained identical protein, suggesting that a post-translational modification activates the enzyme.

Substrate Specificity of Heparanases from Human Hepatoma and Platelets

“…Similar endoglucuronidase activity was reported in subsequent similar studies on heparanases from different sources, such as human placenta (7), human platelets (23), mouse melanoma (24), and CHO cells (25). In contrast, Hoogewerf et al (26) attributed endo-␣-D-glucosaminidase properties to a heparanase purified from human platelets and further proposed, based on amino acid sequencing, that this enzyme was identical to connective tissue-activating peptide-III/neutrophil-activating peptide-2. These components and ␤-thromboglobulin are differently truncated forms of platelet basic protein, a member of the CXC-chemokine family.…”

“…Early studies of endoglycosidases from mouse mastocytoma (6) and from human platelets (9, 23) implicated ␤-D-glucuronidic linkages. More recently, however, it was proposed that a heparanase from platelets has endo-␣-D-glucosaminidase activity (26). The results of the present study, based on identification of fragments generated by heparanase cleavage of a well defined heparin octasaccharide, clearly point to glucuronidic linkages as the site of cleavage.…”

“…2), in accord with the postulated ␤-D-glucuronidase properties of the enzyme. By contrast, a platelet heparanase more recently isolated was claimed to be an ␣-D-glucosaminidase (26). The putative glucosaminidase activity was associated with purified connective tissue-activating peptide-III, which occurs in commercially available "␤-thromboglobulin" (a protein 4 amino acid residues shorter than connective tissue-activating peptide-III at the N terminus).…”

Heparanase and a Synthetic Peptide of Heparan Sulfate-interacting Protein Recognize Common Sites on Cell Surface and Extracellular Matrix Heparan Sulfate

“…Melanoma heparanase was found to produce 12-20 oligosaccharide fragments from bovine lung or kidney HS (3,27,38). In contrast, platelet heparitinase was reported to produce mainly di-and tetrasaccharides just as bacterial heparitinases (12,23,45,46). Heparanase was identified as an endo-␤-D-glucuronidase that cleaves ␤-D-glucuronosyl-N-acetylglucosaminyl linkages of the HS molecule (27).…”

Trans-activation of heparanase promoter by ETS transcription factors

“…The heparanase (Hpa) that degrades the HS side chains of HSPGs is found mainly in platelets, placental trophobasts, and leukocytes (Hoogewerf et al, 1995;Freeman and Parish, 1998). Therefore in tumor metastasis, the normal physiological functions of proteases and Hpa in embryogenesis, wound healing, tissue repair, and inflammation appear to be highjacked by tumor cells (Carmeliet and Jain, 2000).…”