CXC CHEMOKINES CONNECTIVE-TISSUE ACTIVATING PEPTIDE-III AND NEUTROPHIL-ACTIVATING PEPTIDE-2 ARE HEPARIN HEPARAN SULFATE-DEGRADING ENZYMES

Heparan sulfate proteoglycans at cell surfaces or in extracellular mat rices bind diverse molecules, including growth factors and cytokines, and it is believed that the activities of these molecules may be regul ated 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 resides 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 activat ing peptide-2, are CXC chemokines, a group of molecules involved in in flammation and wound healing. SDS-polyacrylamide gel electrophoresis a nalysis of the purified heparanase resulted in a single broad band at 8-10 kDa, the known molecular weight of PBP and its truncated derivati ves. Gel filtration chromatography of heparanase resulted in peaks of activity corresponding to monomers, dimers, and tetramers; these highe r order aggregates are known to form among the chemokines. N-terminaI 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 immun ized with a C-terminal synthetic peptide of PBP inhibited heparanase a ctivity by 95%, compared with activity of the purified enzyme in the p resence of the preimmune sera. The synthetic peptide also inhibited he paranase by 95% at 250 mu M, compared to the 33% inhibition of heparan ase 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 hep aranase resulted in two protein peaks: an inactive peak at pI 7.3, and an active peak at pI 4.8-5.1. Sequence analysis showed that the two p eaks contained identical protein, suggesting that a post-translational modification activates the enzyme.