Ruiz S, Henschen-Edman A H, Tenner A J
Department of Molecular Biology and Biochemistry, University of California, Irvine 92717-3900, USA.
J Biol Chem. 1995 Dec 22;270(51):30627-34. doi: 10.1074/jbc.270.51.30627.
C1q, the recognition subunit of the classical complement pathway, interacts with specific cell surface molecules via its collagen-like region (C1q-CLR). This binding of C1q to neutrophils triggers the generation of toxic oxygen species. To identify the site on C1q that interacts with the neutrophil C1q receptor, C1q was isolated, digested with pepsin to produce C1q-CLR, and further cleaved with either trypsin or endoproteinase Lys-C. The resulting fragments were separated by gel filtration chromatography and analyzed functionally (activation of the respiratory burst in neutrophils) and structurally. Cleavage of C1q-CLR with endoproteinase Lys-C did not alter its ability to trigger neutrophil superoxide production. However, when C1q-CLR was incubated with trypsin under conditions permitting optimal cleavage, the ability of C1q-CLR to stimulate superoxide production in neutrophils was completely abrogated. Fractionation of the digests obtained with the two enzymes and identification by amino acid sequencing permitted localization of the receptor interaction site to a specific region of the C1q-CLR. Circular dichroism analyses demonstrated that cleavage by trypsin does not denature the remaining uncleaved collagen-like structure, suggesting that after trypsin treatment, the loss of activity was not due to a loss of secondary structure of the molecule. However, irreversible heat denaturation of C1q-CLR also abrogated all activity. Thus, a specific conformation conferred by the collagen triple helix constitutes the functional receptor interaction site. These data should direct the design of future specific therapeutic reagents to selectively modulate this response.
