Subdomain chimeras of hepatic lipase and lipoprotein lipase. Localization of heparin and cofactor binding.
To specify and localize carboxyl-terminal domain functions of human hepatic lipase (HL) and human lipoprotein lipase (LPL), two subdomain chimeras were created in which portions of the carboxyl-terminal domain were exchanged between the two lipases. The first chimera (HL-LPLC1) was composed of residues 1-344 of human HL, residues 331-388 of human LPL, and residues 415-476 of human HL. The second chimera (HL-LPLC2) consisted of just two segments, residues 1-414 of human HL and residues 389-448 of human LPL. These chimeric constructs effectively divided the HL C-terminal domain into halves, with corresponding LPL sequences either in the first or second portion of that domain. Both chimeras were lipolytically active and hydrolyzed triolein emulsions to a similar extent compared with native HL and LPL. Heparin-Sepharose chromatography demonstrated that HL-LPLC1 and HL-LPLC2 eluted at 0.80 and 1.3 M NaCl, respectively, elution positions that corresponded to native HL and LPL. Hence, substitution of LPL sequences into the HL carboxyl-terminal domain resulted in the production of functional lipases, but with distinct heparin binding properties. In addition, HL-LPLC2 trioleinase activity was responsive to apoC-II activation, although the -fold stimulation was less than that observed with native LPL. Moreover, an apoC-II fragment (residues 44-79) was specifically cross-linked to LPL and HL-LPLC2, but not to HL or HL-LPLC1. Finally, both chimeras hydrolyzed phospholipid with a specific activity similar to that of HL, which was unaffected by the presence of apoC-II. These findings indicated that in addition to a region found within the amino-terminal domain of LPL, apoC-II also interacted with the last half of the carboxyl-terminal domain (residues 389-448) to achieve maximal lipolytic activation. In addition, the relative heparin affinity of HL and LPL was determined by the final 60 carboxyl-terminal residues of each enzyme.
Hill
J S
JS
Lipid Research Laboratory, West Los Angeles Veterans Affairs Medical Center, Los Angeles, California 90073, USA.
Yang
D
D
Nikazy
J
J
Curtiss
L K
LK
Sparrow
J T
JT
Wong
H
H
D053304:Apolipoprotein C-II; D001056:Apolipoproteins C / Q000378:metabolism*; D001665:Binding Sites; D002846:Chromatography, Affinity; D003432:Cross-Linking Reagents; D004789:Enzyme Activation; D006493:Heparin / Q000378:metabolism*; D006801:Humans; D008049:Lipase / Q000235:genetics / Q000378:metabolism*; D008066:Lipolysis; D008071:Lipoprotein Lipase / Q000235:genetics / Q000378:metabolism*; D011993:Recombinant Fusion Proteins / Q000378:metabolism; D013329:Structure-Activity Relationship; D014304:Triolein / Q000378:metabolism
D053304:Apolipoprotein C-II; D001056:Apolipoproteins C; D003432:Cross-Linking Reagents; D011993:Recombinant Fusion Proteins; D014304:Triolein; D006493:Heparin; D008049:Lipase; D008071:Lipoprotein Lipase