Functional comparison of two human monocyte chemotactic protein-2 isoforms, role of the amino-terminal pyroglutamic acid and processing by CD26/dipeptidyl peptidase IV

Biochemistry. 1998 Sep 8;37(36):12672-80. doi: 10.1021/bi980497d.


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.

Publication types

  • Comparative Study
  • Research Support, Non-U.S. Gov't

MeSH terms

  • Alleles
  • Amino Acid Sequence
  • Base Sequence
  • Calcium / metabolism
  • Chemokine CCL8
  • Chemotaxis, Leukocyte / genetics
  • Cloning, Molecular
  • DNA, Complementary / isolation & purification
  • Dipeptidyl Peptidase 4 / metabolism*
  • Escherichia coli / genetics
  • Genetic Vectors / metabolism
  • Glutamic Acid / genetics
  • Glutamic Acid / metabolism
  • Humans
  • Lysine / genetics
  • Lysine / metabolism
  • Male
  • Molecular Sequence Data
  • Monocyte Chemoattractant Proteins / chemistry*
  • Monocyte Chemoattractant Proteins / genetics
  • Monocyte Chemoattractant Proteins / metabolism
  • Monocyte Chemoattractant Proteins / physiology*
  • Open Reading Frames
  • Protein Isoforms / chemistry
  • Protein Isoforms / genetics
  • Protein Isoforms / metabolism
  • Protein Isoforms / physiology
  • Protein Processing, Post-Translational*
  • Pyrrolidonecarboxylic Acid / metabolism*
  • Receptors, CCR5 / physiology
  • Recombinant Proteins / biosynthesis
  • Recombinant Proteins / isolation & purification
  • Recombinant Proteins / metabolism
  • Sequence Analysis, DNA
  • Signal Transduction / genetics
  • Testis / chemistry
  • Tumor Cells, Cultured


  • CCL8 protein, human
  • Chemokine CCL8
  • DNA, Complementary
  • Monocyte Chemoattractant Proteins
  • Protein Isoforms
  • Receptors, CCR5
  • Recombinant Proteins
  • Glutamic Acid
  • Dipeptidyl Peptidase 4
  • Lysine
  • Calcium
  • Pyrrolidonecarboxylic Acid

Associated data

  • GENBANK/Y16645