In vitro expression and site-specific mutagenesis of the cloned human lipoprotein lipase gene. Potential N-linked glycosylation site asparagine 43 is important for both enzyme activity and secretion.
Detailed structure-function information about human lipoprotein lipase (LPL) is unavailable because it is difficult to purify large amounts of the enzyme for study. To circumvent this problem, we constructed an in vitro LPL expression vector. Human LPL cDNA was cloned and inserted into the expression vector p91023(B). After transfection of COS M-6 cells with the human LPL cDNA construct, LPL enzyme activity was detected in cell extracts and culture medium. Purified human apolipoprotein C-II caused a 5-fold stimulation of the recombinant human LPL expressed in vitro. Using site-specific mutagenesis, Ala residues were substituted for Asn residues at two potential N-linked glycosylation sites (positions 43 and 359) and at a third unrelated Asn (position 257) in the LPL cDNA. RNA blot analysis demonstrated the presence of a single mRNA species in COS cells transfected with wild-type and mutant LPL expression vectors. Intracellular and secreted LPL activity was absent in the construct containing an Ala for Asn mutation at position 43, whereas the same substitutions at positions 257 and 359 did not appreciably affect activity. LPL activity was also absent in another construct containing a Gln for Asn mutation at position 43. Quantitation of LPL protein mass concomitant with measurement of enzyme activity showed that substitution of Ala or Gln for Asn at position 43 resulted in the production of an enzymatically inactive protein which accumulated intracellularly but was not secreted into the culture medium. Our report represents an initial documentation of the expression of cloned human LPL in vitro and of the importance of Asn-43 for both enzyme activity and secretion.
Semenkovich
C F
CF
Department of Cell Biology, Baylor College of Medicine, Houston, Texas.
Luo
C C
CC
Nakanishi
M K
MK
Chen
S H
SH
Smith
L C
LC
Chan
L
L
D000595:Amino Acid Sequence; D053304:Apolipoprotein C-II; D001056:Apolipoproteins C / Q000494:pharmacology; D001216:Asparagine; D002460:Cell Line; D003001:Cloning, Molecular; D004247:DNA / Q000235:genetics; D015870:Gene Expression; D005822:Genetic Vectors; D006031:Glycosylation; D006801:Humans; D008071:Lipoprotein Lipase / Q000235:genetics*; D008969:Molecular Sequence Data; D009154:Mutation; D009206:Myocardium / Q000201:enzymology; D009693:Nucleic Acid Hybridization; D015183:Restriction Mapping; D014162:Transfection
D053304:Apolipoprotein C-II; D001056:Apolipoproteins C; D001216:Asparagine; D004247:DNA; D008071:Lipoprotein Lipase