Structure-function relationships of lipoprotein lipase: mutation analysis and mutagenesis of the loop region.
The molecular models of two microbial lipases and human pancreatic lipase (PL) have suggested the existence of common structural motifs including a buried active site shielded by an amphipathic surface loop. In an effort to explore the role of residues comprising the loop of lipoprotein lipase (LPL), we have used site-directed mutagenesis to generate three new LPL variants. In variant LPLM1 we deleted 18 amino acids leaving a loop of only 4 residues which resulted in an LPL protein inactive against triolein substrates. In contrast, two other LPL variants with only partial deletions, involving the apical section of the loop [LPLM2 (-8 amino acids) and LPLM3 (-2 amino acids)] manifested normal lipolytic activity. These findings indicate a critical requirement for the maintenance of charge and periodicity in the proximal and distal segments of the LPL loop in normal catalytic function. This is further highlighted by the detection of a mutation in the proximal section of the loop in a patient with LPL deficiency at position 225 which results in a substitution of threonine for isoleucine. The intact catalytic activity of the partial deletion variants (LPLM2 and LPLM3) further suggests that the apical residues of the loop contribute minimally to the functional motifs of the active site. We support this postulate by showing that the conserved glycine in the apical turn section (G229) can be substituted by glutamine, lysine, proline, or threonine without significantly affecting catalytic activity.
Henderson
H E
HE
Department of Medical Genetics, University of British Columbia, Vancouver, Canada.
Ma
Y
Y
Liu
M S
MS
Clark-Lewis
I
I
Maeder
D L
DL
Kastelein
J J
JJ
Brunzell
J D
JD
Hayden
M R
MR
D001483:Base Sequence; D002478:Cells, Cultured; D003198:Computer Simulation; D004247:DNA / Q000032:analysis; D006801:Humans; D008071:Lipoprotein Lipase / Q000737:chemistry / Q000172:deficiency / Q000235:genetics* / Q000502:physiology; D008956:Models, Chemical; D008969:Molecular Sequence Data; D016296:Mutagenesis / Q000235:genetics; D009154:Mutation / Q000235:genetics*; D017354:Point Mutation; D013329:Structure-Activity Relationship; D014162:Transfection
D004247:DNA; D008071:Lipoprotein Lipase