FUNCTIONAL COMPARISON OF 2 HUMAN MONOCYTE CHEMOTACTIC PROTEIN-2 ISOFORMS, ROLE OF THE AMINO-TERMINAL PYROGLUTAMIC ACID AND PROCESSING BY CD26 DIPEPTIDYL PEPTIDASE-IV

Human Monocyte Chemotactic Protein (MCP)-2 has originally been isolate d from stimulated osteosarcoma cells as a chemokine coproduced with MC P-1 and MCP-3. Here, a 5'-end extended MCP-2 cDNA was cloned from a hu man testis cDNA library. It encoded a 76 residue MCP-2 protein, but di ffered from the reported bone marrow-derived MCP-2 cDNA sequence in co don 46, which coded for a Lys instead of a Gln. This MCP-2Lys(46) vari ant, caused by a single nucleotide polymorphism (SNP), was biologicall y compared with MCP-2GLn(46). The coding regions were subcloned into t he bacterial expression vector pHEN1, and after transformation of Esch erichia coli, the two MCP-2 protein variants were recovered from the p eriplasm. The recombinant proteins were purified to homogeneity by hep arin-Sepharose affinity chromatography and reversed-phase HPLC. Edman degradation revealed a Gin residue at the NH2 terminus instead of a pG lu. To evaluate the influence of the cyclization, this Gin was chemica lly converted into pGlu in both MCP-2 variants. The conversion was con firmed by electrospray mass spectrometry. rMCP-2Gln(46) and rMCP-2Lys( 46) and the NH2-terminal cyclic counterparts were tested on monocytic cells in calcium mobilization and chemotaxis assays. No significant di fference in biological activity was observed between the rMCP-2Gln46 a nd rMCP-2Lys(46) isoforms. However, for both MCP-2 variants the NH2-te rminal pyroglutamate was shown to be essential for chemotaxis, but not for calcium mobilization. NH2-terminal truncation of rMCP-2Lys46 by t he serine protease CD26/dipeptidyl peptidase IV (CD26/DPP IV) resulted in the cleavage of the NH2-terminal Gin-Pro dipeptide, whereas synthe tic MCP-2 with an amino-terminal pGlu remained unaffected. CD26/DPP IV -clipped rMCP-2Lys(46)(3-76) was almost completely inactive in both ch emotaxis and signaling assays. These observations indicate that the NH 2-terminal pGlu in MCP-2 is necessary for chemotactic activity but als o that it protects the protein against degradation by CD26/DPP IV.