Effects of site-directed mutagenesis on the N-glycosylation sites of human lecithin:cholesterol acyltransferase.
There are four potential N-glycosylation site (Asn-X-Ser/Thr) in human lecithin:cholesterol acyltransferase (LCAT, residues 20, 84, 272, and 384). To study the role of the N-linked sugars, the codon for Asn at these positions was replaced with one for Thr (AAC to ACC). The wild-type and mutant LCAT cDNAs were used to transfect COS-6 cells from which RNA was isolated; cDNAs were synthesized by reverse transcription and subjected to the polymerase chain reaction, which showed that all transfectants synthesized LCAT-specific mRNA. No intracellular or secreted LCAT was detected with the Asn272-->Thr transfectants, indicating that this residue is essential for intracellular processing. All other single-point transfectants were secretion-competent. Although there was detectable LCAT protein inside the cells and in the media of the transfectant, Asn84-->Thr, its specific activity and secreted amount were only 26% and 58% of the wild type, respectively. This implies that Asn84 is critical for full activity but not for intracellular processing. The amount secreted, specific activity, and Vmax of LCAT (Asn20-->Thr) were similar to those of the wild-type LCAT. LCAT (Asn384-->Thr) differed from the wild-type LCAT only by a lower Km. These results suggest that glycosylation at residues 20 and 384 is not essential for intracellular processing, secretion, or activity.
Qu
S J
SJ
Department of Medicine, Baylor College of Medicine, Houston, Texas 77030.
Fan
H Z
HZ
Blanco-Vaca
F
F
Pownall
H J
HJ
D000595:Amino Acid Sequence; D001483:Base Sequence; D015153:Blotting, Western; D002478:Cells, Cultured; D004247:DNA; D005822:Genetic Vectors; D006031:Glycosylation; D006801:Humans; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D007862:Phosphatidylcholine-Sterol O-Acyltransferase / Q000235:genetics / Q000378:metabolism*; D016133:Polymerase Chain Reaction; D013379:Substrate Specificity; D014162:Transfection
D004247:DNA; D007862:Phosphatidylcholine-Sterol O-Acyltransferase