IDENTIFICATION OF THE CLEAVAGE SITE INVOLVED IN PRODUCTION OF PLASMA SOLUBLE FC-GAMMA RECEPTOR-TYPE-III (CD16)

CD16 (Fc gamma R type III) is a low-affinity IgG Fc receptor (R) that exists in two isoforms, a transmembrane Fc gamma RIIIa expressed by NK cells and monocytes, and a phosphatidylinositol-linked Fc gamma RIIIb expressed by neutrophils. A soluble form of CD16 (sCD16) circulates i n plasma. The cleavage site and the nature of the enzyme(s) involved i n production of sCD16 were investigated. Soluble CD16 was purified to apparent homogeneity from human serum by eight steps, including anion exchange and immunoaffinity chromatography. Serum sCD16 was sequenced at both ends, as well as a recombinant form of sCD16 used as control. N-terminal sequencing demonstrated that serum sCD16 originates from ne utrophil Fc gamma RIIIb and C-terminal sequencing suggested that the c leavage site is between Val 196 and Ser 197, close to the membrane anc hor. Addition of a hydroxamate-based inhibitor of Zn2+ metalloproteina ses (RU36156) led to a dramatic decrease of sCD16 production by phorbo l 12-myristate 13-acetate-activated neutrophils, whereas inhibitors of serine proteinases had no significant effect, showing the metalloprot einase dependence of this cleavage process.