Functional Comparison of Two Human Monocyte Chemotactic Protein-2 Isoforms, Role of the Amino-Terminal Pyroglutamic Acid and Processing by CD26/Dipeptidyl Peptidase IV

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• 作者：Els Van Coillie ; Paul Proost ; Ilse Van Aelst ; Sofie Struyf ; Machteld Polfliet ; Ingrid De Meester ; David J. Harvey ; Jo Van Damme ; and Ghislain Opdenakker
• 刊名：Biochemistry
• 出版年：1998
• 出版时间：September 8, 1998
• 年：1998
• 卷：37
• 期：36
• 页码：12672 - 12680
• 全文大小：250K
• 年卷期：v.37,no.36(September 8, 1998)
• ISSN：1520-4995

文摘

Human Monocyte Chemotactic Protein (MCP)-2 has originally been isolated from stimulatedosteosarcoma cells as a chemokine coproduced with MCP-1 and MCP-3. Here, a 5'-end extended MCP-2cDNA was cloned from a human testis cDNA library. It encoded a 76 residue MCP-2 protein, but differedfrom the reported bone marrow-derived MCP-2 cDNA sequence in codon 46, which coded for a Lysinstead of a Gln. This MCP-2Lys₄₆ variant, caused by a single nucleotide polymorphism (SNP), wasbiologically compared with MCP-2Gln₄₆. The coding regions were subcloned into the bacterial expressionvector pHEN1, and after transformation of Escherichia coli, the two MCP-2 protein variants were recoveredfrom the periplasm. The recombinant proteins were purified to homogeneity by heparin-Sepharose affinitychromatography and reversed-phase HPLC. Edman degradation revealed a Gln residue at the NH₂ terminusinstead of a pGlu. To evaluate the influence of the cyclization, this Gln was chemically converted intopGlu in both MCP-2 variants. The conversion was confirmed by electrospray mass spectrometry. rMCP-2Gln₄₆ and rMCP-2Lys₄₆ and the NH₂-terminal cyclic counterparts were tested on monocytic cells incalcium mobilization and chemotaxis assays. No significant difference in biological activity was observedbetween the rMCP-2Gln₄₆ and rMCP-2Lys₄₆ isoforms. However, for both MCP-2 variants the NH₂-terminal pyroglutamate was shown to be essential for chemotaxis, but not for calcium mobilization. NH₂-terminal truncation of rMCP-2Lys₄₆ by the serine protease CD26/dipeptidyl peptidase IV (CD26/DPPIV) resulted in the cleavage of the NH₂-terminal Gln-Pro dipeptide, whereas synthetic MCP-2 with anamino-terminal pGlu remained unaffected. CD26/DPP IV-clipped rMCP-2Lys₄₆(3-76) was almostcompletely inactive in both chemotaxis and signaling assays. These observations indicate that the NH₂-terminal pGlu in MCP-2 is necessary for chemotactic activity but also that it protects the protein againstdegradation by CD26/DPP IV.