Functional comparison of two human monocyte chemotactic protein-2 isoforms, role of the amino-terminal pyroglutamic acid and processing by CD26/dipeptidyl peptidase IV. 1998

E Van Coillie, and P Proost, and I Van Aelst, and S Struyf, and M Polfliet, and I De Meester, and D J Harvey, and J Van Damme, and G Opdenakker
Rega Institute for Medical Research, Laboratory of Molecular Immunology, University of Leuven, Belgium.

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.

UI MeSH Term Description Entries
D008239 Lysine An essential amino acid. It is often added to animal feed. Enisyl,L-Lysine,Lysine Acetate,Lysine Hydrochloride,Acetate, Lysine,L Lysine
D008297 Male Males
D008969 Molecular Sequence Data Descriptions of specific amino acid, carbohydrate, or nucleotide sequences which have appeared in the published literature and/or are deposited in and maintained by databanks such as GENBANK, European Molecular Biology Laboratory (EMBL), National Biomedical Research Foundation (NBRF), or other sequence repositories. Sequence Data, Molecular,Molecular Sequencing Data,Data, Molecular Sequence,Data, Molecular Sequencing,Sequencing Data, Molecular
D011499 Protein Processing, Post-Translational Any of various enzymatically catalyzed post-translational modifications of PEPTIDES or PROTEINS in the cell of origin. These modifications include carboxylation; HYDROXYLATION; ACETYLATION; PHOSPHORYLATION; METHYLATION; GLYCOSYLATION; ubiquitination; oxidation; proteolysis; and crosslinking and result in changes in molecular weight and electrophoretic motility. Amino Acid Modification, Post-Translational,Post-Translational Modification,Post-Translational Protein Modification,Posttranslational Modification,Protein Modification, Post-Translational,Amino Acid Modification, Posttranslational,Post-Translational Amino Acid Modification,Post-Translational Modifications,Post-Translational Protein Processing,Posttranslational Amino Acid Modification,Posttranslational Modifications,Posttranslational Protein Processing,Protein Processing, Post Translational,Protein Processing, Posttranslational,Amino Acid Modification, Post Translational,Modification, Post-Translational,Modification, Post-Translational Protein,Modification, Posttranslational,Modifications, Post-Translational,Modifications, Post-Translational Protein,Modifications, Posttranslational,Post Translational Amino Acid Modification,Post Translational Modification,Post Translational Modifications,Post Translational Protein Modification,Post Translational Protein Processing,Post-Translational Protein Modifications,Processing, Post-Translational Protein,Processing, Posttranslational Protein,Protein Modification, Post Translational,Protein Modifications, Post-Translational
D011761 Pyrrolidonecarboxylic Acid A cyclized derivative of L-GLUTAMIC ACID. Elevated blood levels may be associated with problems of GLUTAMINE or GLUTATHIONE metabolism. 5-Oxoproline,Pidolic Acid,Pyroglutamic Acid,5-Ketoproline,5-Oxopyrrolidine-2-Carboxylic Acid,Magnesium Pidolate,Pyroglutamate,Pidolate, Magnesium
D011994 Recombinant Proteins Proteins prepared by recombinant DNA technology. Biosynthetic Protein,Biosynthetic Proteins,DNA Recombinant Proteins,Recombinant Protein,Proteins, Biosynthetic,Proteins, Recombinant DNA,DNA Proteins, Recombinant,Protein, Biosynthetic,Protein, Recombinant,Proteins, DNA Recombinant,Proteins, Recombinant,Recombinant DNA Proteins,Recombinant Proteins, DNA
D002118 Calcium A basic element found in nearly all tissues. It is a member of the alkaline earth family of metals with the atomic symbol Ca, atomic number 20, and atomic weight 40. Calcium is the most abundant mineral in the body and combines with phosphorus to form calcium phosphate in the bones and teeth. It is essential for the normal functioning of nerves and muscles and plays a role in blood coagulation (as factor IV) and in many enzymatic processes. Coagulation Factor IV,Factor IV,Blood Coagulation Factor IV,Calcium-40,Calcium 40,Factor IV, Coagulation
D002634 Chemotaxis, Leukocyte The movement of leukocytes in response to a chemical concentration gradient or to products formed in an immunologic reaction. Leukotaxis,Leukocyte Chemotaxis
D003001 Cloning, Molecular The insertion of recombinant DNA molecules from prokaryotic and/or eukaryotic sources into a replicating vehicle, such as a plasmid or virus vector, and the introduction of the resultant hybrid molecules into recipient cells without altering the viability of those cells. Molecular Cloning
D004926 Escherichia coli A species of gram-negative, facultatively anaerobic, rod-shaped bacteria (GRAM-NEGATIVE FACULTATIVELY ANAEROBIC RODS) commonly found in the lower part of the intestine of warm-blooded animals. It is usually nonpathogenic, but some strains are known to produce DIARRHEA and pyogenic infections. Pathogenic strains (virotypes) are classified by their specific pathogenic mechanisms such as toxins (ENTEROTOXIGENIC ESCHERICHIA COLI), etc. Alkalescens-Dispar Group,Bacillus coli,Bacterium coli,Bacterium coli commune,Diffusely Adherent Escherichia coli,E coli,EAggEC,Enteroaggregative Escherichia coli,Enterococcus coli,Diffusely Adherent E. coli,Enteroaggregative E. coli,Enteroinvasive E. coli,Enteroinvasive Escherichia coli

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