Takumasa Nishimura

Identification of Receptor-Binding Sites of Monocyte Chemotactic S19 Ribosomal Protein Dimer

The S19 ribosomal protein (RP S19) cross-linked homo-dimer attracts monocyte migration by binding to C5a receptor on monocytes (H Nishiura, Y Shibuya, T Yamamoto, Laboratory Investigation, 1998, 78:1615-1623). Using site-directed mutants of recombinant RP S19 and synthetic peptides mimicking RP S19 molecular regions, we currently identified the binding sites of the RP S19 dimer to the C5a receptor. The RP S19 dimer activated the receptor by a two-step binding mechanism as in the case of C5a. The first binding site was a basic cluster region containing a -Lys41-His42-Lys43- sequence. The second one was the -Leu131-Asp132-Arg133- moiety, localized 12 residues upstream from the COOH-terminal. The second binding triggered the chemotactic response. The first binding would have a role in achieving a high-binding affinity between the ligand and receptor. The first and second ligand-binding sites of C5a receptor seem to be shared by C5a and the RP S19 dimer, although overall homology between the amino acid sequences of these ligands is only 4%.

Switch Moiety in Agonist/Antagonist Dual Effect of S19 Ribosomal Protein Dimer on Leukocyte Chemotactic C5a Receptor

The cross-linked homodimer of S19 ribosomal protein (RP S19) induces monocyte predominant infiltration due to a dual effect on the C5a receptor in leukocyte chemotaxis, agonistic to monocytes and antagonistic to polymorphonuclear leukocytes (PMN) (H. Nishiura, Y. Shibuya, T. Yamamoto, Lab Invest 1998, 78:1615-1623). The agonistic ligand moiety was recently determined to be -Leu131-Asp132-Arg133- (Y. Shibuya et al, Am J Pathol 2001, 159:2293-2301). In this study we determined the moiety responsible for the antagonistic function. A C-terminal analogue peptide of RP S19, with 18 residues containing the agonistic ligand moiety as a part, reproduced the dual function in the leukocyte chemotaxis. A C5a analogue peptide attracted PMN as well as monocytes. When C-terminal 12 residues of RP S19 after the agonistic moiety, IAGQVAAANKKH, were connected to the C5a peptide, the chimeric peptide newly obtained the dual function, indicating that the C-terminal portion of RP S19 functions as a converter from the agonist to the antagonist. C-terminal truncation analyses indicated that the C-terminal His was not essential but the next Lys was necessary for the converter function. The homodimer of a mutant RP S19 that was truncated for the C-terminal 4 residues lost the monocyte selectivity in the leukocyte infiltration in vivo as in the case of the leukocyte chemotaxis in vitro. These results indicated that the conversion of the RP S19 dimer from agonist to antagonist of C5a receptor is attributed to the IAGQVAAANKK moiety between Ile134 and Lys144.