Chymotryptic cleavage of lipoprotein lipase. Identification of cleavage sites and functional studies of the truncated molecule.

Treatment of bovine lipoprotein lipase (LPL) with chymotrypsin results in cleavage between residues Phe390-Ser391 and between Trp392-Ser393, indicating that this region is exposed in the native conformation of LPL. Two main fragments are generated, one large including the amino-terminus (chymotrypsin-truncated LPL = c-LPL) and one small, carboxy-terminal fragment. The small fragment is not stable, but is further degraded by the protease. Isolated c-LPL has full catalytic activity against tributyryl glycerol (tributyrin) and p-nitrophenyl butyrate, while the activity against emulsions of long-chain triacylglycerols and against liposomes is reduced and the activity against milk fat globules and chylomicrons is lost. Several properties of c-LPL were investigated. It was found that c-LPL interacts with apolipoprotein CII (apo CII) as efficiently as intact LPL. The truncated enzyme bound to liposomes and to emulsions of long-chain triacylglycerols as well as the intact enzyme did. In contrast, c-LPL did not bind to milk fat globules or to chylomicrons. The activity of c-LPL was more sensitive to inhibition by other lipid-binding proteins, e.g. apolipoprotein CIII (apo CIII), than was the intact enzyme. The affinity for heparin was as high with c-LPL as with intact LPL. Like intact LPL, c-LPL is dimeric in its active form, as evidenced by sucrose density gradient centrifugation. It is concluded that the reduced catalytic and lipid-binding properties of c-LPL compared with intact LPL are related to the properties of the substrate interface. It is speculated that the carboxy-terminal part of LPL contains a secondary lipid-binding site, which is important for activity against chylomicrons and related substrates.