Systematic mutagenesis of potential glycosylation sites of lysosomal acid lipase.
Lysosomal acid lipase (LAL; EC 3.1.1.13) is a key enzyme in the intracellular lipid metabolism. It hydrolyzes exogenous triglycerides and cholesterol esters taken up by various cell types. LAL has six potential N-glycosylation sites and one potential O-glycosylation site. Elimination of each of the six Asn-(X)-Ser/Thr sites by site-directed mutagenesis and expression in baculovirus-infected Spodoptera frugiperda cells resulted in two single-mutant enzymes without lipolytic activities (N134Q and N246Q) and four mutants with preserved activities. The two inactive mutants were not detectable on immunoblot analysis, indicating that they were not secreted. Six double mutants in all possible combinations except for the two inactive single mutants were produced and expressed. Double mutants in combination with the N9 glycosylation site showed reduced activities as compared to the other mutants or the wild-type enzyme. Kinetic data of LAL glycosylation mutants indicate that substrate affinity of N9Q was not changed, but k (cat) of N9 mutants was reduced distinctly compared to the wild-type enzyme. Peanut agglutinin lectin did not recognize LAL, demonstrating that the protein has no core1 structure (Galbeta 1-3 GalNAc) of O-glycosylation. These data indicate that at least two of the six N-glycosylation sites are used in native lipase. N134 and N246 were found to be essential for LAL activity. We conclude that glycosylation plays an important role in the formation of functional LAL.
Zschenker
Oliver
O
Department of Medicine, University Hospital Eppendorf, Martinistrasse 52, 20246 Hamburg, Germany. zschenker@uke.uni-hamburg.de
Bähr
Christina
C
Hess
Utz-Florian
UF
Ameis
Detlev
D
D000595:Amino Acid Sequence; D000818:Animals; D001216:Asparagine / Q000378:metabolism; D001483:Base Sequence; D004591:Electrophoresis, Polyacrylamide Gel; D015870:Gene Expression / Q000187:drug effects; D006031:Glycosylation; D008247:Lysosomes / Q000201:enzymology*; D016297:Mutagenesis, Site-Directed; D017924:Nucleopolyhedroviruses / Q000235:genetics; D043524:Peptide-N4-(N-acetyl-beta-glucosaminyl) Asparagine Amidase / Q000378:metabolism; D018411:Spodoptera / Q000378:metabolism; D002787:Sterol Esterase / Q000737:chemistry / Q000235:genetics* / Q000378:metabolism; D014415:Tunicamycin / Q000494:pharmacology
D014415:Tunicamycin; D001216:Asparagine; D002787:Sterol Esterase; D043524:Peptide-N4-(N-acetyl-beta-glucosaminyl) Asparagine Amidase