Wicker-Planquart_1999_Eur.J.Biochem_262_644

Human gastric lipase (HGL) is a highly glycosylated protein, as glycan chains account for about 15% of the molecular mass of the native HGL. Four potential N-glycosylation consensus sites (Asn15, 80, 252 and 308) can be identified from the HGL amino acid sequence. We studied the functional role of the individual N-linked oligosaccharide chains by removing one by one all the N-glycosylation sites, via Ala residue replacement by site-directed mutagenesis of Ser and Thr residues from the consensus sequences Asn-X-Ser/Thr. Mutagenized cDNA constructs were heterologously expressed in the baculovirus/insect cell system. Removal of oligosaccharides either at Asn15, 80 or 252 was found to have no significant influence on the enzymatic activity measured in vitro. However, the absence of glycosylation at Asn308, as well as a total deglycosylation, reduced the specific enzymatic activity of recombinant HGL (r-HGL), measured on short- and long-chain triglycerides, to about 50% of normal values. Furthermore, biosynthesis and secretion of r-HGL markedly dropped when all four potential glycosylation sites were mutated. The kinetics of the interfacial adsorption of r-HGL and the completely deglycosylated r-HGL (four-site mutant) were found to be identical when recording the changes with time of the surface pressure either at the air-water interface or in the presence of an egg phosphatidylcholine (PtdCho) monomolecular film spread at various initial surface pressures. This indicates that both recombinant HGLs are identical, as far as recognition of phospholipid film and adsorption on PtdCho are concerned. The N-glycosylation of HGL may contribute to the enzyme stability in the stomach, as under acidic conditions the degradation by pepsin of the unglycosylated r-HGL is increased.