LOCALIZATION OF THE SITE ON THE COMPLEMENT COMPONENT C1Q REQUIRED FORTHE STIMULATION OF NEUTROPHIL SUPEROXIDE PRODUCTION

C1q, the recognition subunit of the classical complement pathway, inte racts with specific cell surface molecules via its collagen like regio n (C1q-CLR). This binding of C1q to neutrophils triggers the generatio n of toxic oxygen species. To identify the site on C1q that interacts with the neutrophil C1q receptor, C1q was isolated, digested with peps in to produce C1q-CLR, and further cleaved with either trypsin or endo proteinase Lys-C. The resulting fragments were separated by gel filtra tion chromatography and analyzed functionally (activation of the respi ratory burst in neutrophils) and structurally. Cleavage of C1q-CLR wit h endoproteinase Lys-C did not alter its ability to trigger neutrophil superoxide production. However, when C1q-CLR was incubated with tryps in under conditions permitting optimal cleavage, the ability of C1q-CL R to stimulate superoxide production in neutrophils was completely abr ogated. 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. Circul ar dichroism analyses demonstrated that cleavage by trypsin does not d enature the remaining uncleaved collagen-like structure, suggesting th at after trypsin treatment, the loss of activity was not due to a loss of secondary structure of the molecule. However, irreversible heat de naturation of C1q-CLR also abrogated all activity. Thus, a specific co nformation conferred by the collagen triple helix constitutes the func tional receptor interaction site. These data should direct the design of future specific therapeutic reagents to selectively modulate this r esponse.