Functional comparison of two 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 isolated from stimulated osteosarcoma cells as a chemokine coproduced with MCP-1 and MCP-3. Here, a 5'-end extended MCP-2 cDNA was cloned from a human testis cDNA library. It encoded a 76 residue MCP-2 protein, but differed from the reported bone marrow-derived MCP-2 cDNA sequence in codon 46, which coded for a Lys instead of a Gln. This MCP-2Lys46 variant, caused by a single nucleotide polymorphism (SNP), was biologically compared with MCP-2Gln46. The coding regions were subcloned into the bacterial expression vector pHEN1, and after transformation of Escherichia coli, the two MCP-2 protein variants were recovered from the periplasm. The recombinant proteins were purified to homogeneity by heparin-Sepharose affinity chromatography and reversed-phase HPLC. Edman degradation revealed a Gln residue at the NH2 terminus instead of a pGlu. To evaluate the influence of the cyclization, this Gln was chemically converted into pGlu 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 in calcium mobilization and chemotaxis assays. No significant difference in biological activity was observed between 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/DPP IV) resulted in the cleavage of the NH2-terminal Gln-Pro dipeptide, whereas synthetic MCP-2 with an amino-terminal pGlu remained unaffected. CD26/DPP IV-clipped rMCP-2Lys46(3-76) was almost completely 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 against degradation by CD26/DPP IV.
Van Coillie
E
E
Rega Institute for Medical Research, Laboratory of Molecular Immunology, University of Leuven, Belgium.
Proost
P
P
Van Aelst
I
I
Struyf
S
S
Polfliet
M
M
De Meester
I
I
Harvey
D J
DJ
Van Damme
J
J
Opdenakker
G
G
D000483:Alleles; D000595:Amino Acid Sequence; D001483:Base Sequence; D002118:Calcium / Q000378:metabolism; D054412:Chemokine CCL8; D002634:Chemotaxis, Leukocyte / Q000235:genetics; D003001:Cloning, Molecular; D018076:DNA, Complementary / Q000302:isolation & purification; D018819:Dipeptidyl Peptidase 4 / Q000378:metabolism*; D004926:Escherichia coli / Q000235:genetics; D005822:Genetic Vectors / Q000378:metabolism; D018698:Glutamic Acid / Q000235:genetics / Q000378:metabolism; D006801:Humans; D008239:Lysine / Q000235:genetics / Q000378:metabolism; D008297:Male; D008969:Molecular Sequence Data; D018945:Monocyte Chemoattractant Proteins / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism / Q000502:physiology*; D016366:Open Reading Frames; D020033:Protein Isoforms / Q000737:chemistry / Q000235:genetics / Q000378:metabolism / Q000502:physiology; D011499:Protein Processing, Post-Translational; D011761:Pyrrolidonecarboxylic Acid / Q000378:metabolism*; D019713:Receptors, CCR5 / Q000502:physiology; D011994:Recombinant Proteins / Q000096:biosynthesis / Q000302:isolation & purification / Q000378:metabolism; D017422:Sequence Analysis, DNA; D015398:Signal Transduction / Q000235:genetics; D013737:Testis / Q000737:chemistry; D014407:Tumor Cells, Cultured
C515453:CCL8 protein, human; D054412:Chemokine CCL8; D018076:DNA, Complementary; D018945:Monocyte Chemoattractant Proteins; D020033:Protein Isoforms; D019713:Receptors, CCR5; D011994:Recombinant Proteins; D018698:Glutamic Acid; D018819:Dipeptidyl Peptidase 4; D008239:Lysine; D002118:Calcium; D011761:Pyrrolidonecarboxylic Acid