Inhibition of complement activation by recombinant Sh-CRIT-ed1 analogues.
Sh-CRIT-ed1 is a potent anti-complement peptide that inhibits the classical complement-activation pathway by interfering with the formation of the C3-convertase complex, C4b2a. C2 is an essential serum glycoprotein that provides the catalytic subunit of the C3 and C5 convertases of the classical pathways of complement activation. Because only in its C4-bound state is C2a capable of cleaving its physiological protein substrates C3 and C5, the interaction of Sh-CRIT-ed1 with C2 plays a decisive role of inhibition in the classical complement-activation process. However, the role of individual Sh-CRIT-ed1 amino acid residues in C2 binding is not fully understood. We constructed nine recombinant Sh-CRIT-ed1 (rSh1) analogues, substituted at conserved residues, and evaluated their anti-complement and C2-binding activities. Results from glutathione S-transferase (GST) pull-down and haemolytic assays suggested that residues 10K, 17E, 19K and 26Y are critical for the interaction of rSh1 with C2. We then constructed an improved anti-complement peptide by duplicating Sh-CRIT-ed1 C-terminal motifs (17H-26Y). This linear homodimer (rH17d) was more potent than rSh1 with respect to binding to C2 and anti-complement activity (the 50% inhibitory concentration value was approximately equal 1.2 micro m versus approximately equal 6.02 micro m for rSh1). Furthermore, rH17d showed higher anti-complement activity in vivo, providing additional evidence that this duplication is a more effective inhibitor of complement activation than rSh1. Taken together, these results identify four key residues in rSh1 and strongly suggest that rH17d is a potent inhibitor of complement activation that may have therapeutic applications.
Oh
Kwang-Seok
KS
Graduate School of Biotechnology, Korea University, Seoul, Korea, Kolon Central Research Park, Yongin-city, Kyunggi-do, Korea. hcsung@korea.ac.kr
Kweon
Mee-Hyang
MH
Rhee
Ki-Hyeong
KH
Ho Lee
Keyong
K
Sung
Ha-Chin
HC
D000595:Amino Acid Sequence; D000818:Animals; D000947:Antigens, Helminth / Q000235:genetics / Q000276:immunology*; D003175:Complement C2 / Q000276:immunology / Q000378:metabolism; D003171:Complement Pathway, Classical / Q000276:immunology*; D005577:Forssman Antigen / Q000276:immunology; D006168:Guinea Pigs; D015801:Helminth Proteins / Q000235:genetics / Q000276:immunology*; D006461:Hemolysis / Q000276:immunology; D008297:Male; D008969:Molecular Sequence Data; D011956:Receptors, Cell Surface / Q000235:genetics / Q000276:immunology*; D011994:Recombinant Proteins / Q000276:immunology; D016415:Sequence Alignment; D012769:Shock / Q000276:immunology; D013329:Structure-Activity Relationship
D000947:Antigens, Helminth; D003175:Complement C2; D015801:Helminth Proteins; D011956:Receptors, Cell Surface; D011994:Recombinant Proteins; C120046:TM3 protein, Schistosoma; D005577:Forssman Antigen