There are four potential N-glycosylation sites (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.
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