Mutational analysis of human lipoprotein lipase by carboxy-terminal truncation.
We have previously reported a Trp382 (TGG)-->stop (TGA) mutation that causes familial lipoprotein lipase (LPL) deficiency. Expression study of the Trp382-->stop mutant showed that the truncated LPL was catalytically inactive with a marked reduction in the expressed mass. To investigate the minimal amino-terminal sequence of human LPL required for an active enzyme, a series of carboxy-terminal (C-terminal) truncated LPLs were expressed in vitro, and the enzyme activity, mass, and LPL mRNA levels were analyzed. The lipolytic activity showed a stepwise reduction between LPL-437 (Cys438-->stop; 68% of normal LPL activity in medium) and LPL-434 (Phe435-->stop; 3%). Without affecting LPL mRNA levels, LPL mass was reduced with the mutants not larger than LPL-437. In terms of specific activity, a significant difference was observed between LPL-436 (Lys437-->stop; 88% of that of normal LPL in medium) and LPL-435 (Val436-->stop; 22%), implying the importance of the role of Val436 in LPL action. Furthermore, our results unexpectedly showed that LPL-446 (Ser447-->stop), which is considered to be a common polymorphic form of LPL, exhibited higher activity than normal LPL (185% in medium). These results demonstrate that the C-terminal region of human LPL is closely associated with the expression of enzyme mass and catalytic activity.
Kozaki
K
K
Third Department of Internal Medicine, Faculty of Medicine, University of Tokyo, Japan.
Gotoda
T
T
Kawamura
M
M
Shimano
H
H
Yazaki
Y
Y
Ouchi
Y
Y
Orimo
H
H
Yamada
N
N
D000595:Amino Acid Sequence; D000818:Animals; D001483:Base Sequence; D015152:Blotting, Northern; D002460:Cell Line; D002522:Chlorocebus aethiops; D003241:Consanguinity; D005260:Female; D006801:Humans; D008072:Hyperlipoproteinemia Type I / Q000201:enzymology / Q000235:genetics*; D007223:Infant; D007668:Kidney; D008071:Lipoprotein Lipase / Q000737:chemistry / Q000235:genetics* / Q000378:metabolism; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D009154:Mutation; D012333:RNA, Messenger / Q000032:analysis; D014162:Transfection
D012333:RNA, Messenger; D008071:Lipoprotein Lipase