CHARACTERIZATION OF RECOMBINANT HUMAN PLASMA LECITHIN - CHOLESTEROL ACYLTRANSFERASE (LCAT) - N-LINKED CARBOHYDRATE STRUCTURES AND CATALYTICPROPERTIES

The major N-linked carbohydrate structures were determined for recombi nant human plasma lecithin:cholesterol acyltransferase (LCAT). The ana lysis of the structure of oligosaccharides by fast atom bombardment ma ss spectrometry (FAB-MS) and linkage analysis was preceded by reductio n and carboxymethylation of the intact glycoproteins and digestion wit h trypsin and proline specific endopeptidase. The N-glycans were subse quently released from the glycopeptides by PNGase F digestion and the oligosaccharides were separated using a C18 Sep-pak(R) cartridge. The data from the combination of FAB spectrometry and linkage analysis sho w that the N-linked glycans present on recombinant LCAT (rLCAT) were c omposed primarily of triantennary and tetraantennary structures with a nd without core fucosylation. A minor population of glycans (less than 5%) contained up to three repeats of N-acetyllactosamine in one or mo re antennae. The LCAT activities of both recombinant and circulating f orms of plasma LCAT were determined using low molecular weight and lip oprotein substrates. The catalytic behavior of these two enzyme forms were found to be very similar if not identical. These findings validat e the concept that the recombinant enzyme can serve as an appropriate model for structure/function studies of LCAT and provide the foundatio n for subsequent structural studies.