Site-directed removal of N-glycosylation sites in human gastric lipase

Human gastric lipase (HGL) is a highly glycosylated protein, as glycan chai ns account for about 15% of the molecular mass of the native HGL. Four pote ntial N-glycosylation consensus sites (Asn15, 80, 352 and 308) can be ident ified from the HGL amino acid sequence. We studied the functional role of t he individual N-linked oligosaccharide chains by removing one by one all th e N-glycosylation sites, via Ala residue replacement by site-directed mutag enesis of Ser and Thr residues from the consensus sequences Asn-X-Ser/Thr. Mutagenized cDNA constructs were heterologously expressed in the baculoviru s/insect cell system. Removal of oligosaccharides either at Asn15, 80 or 25 2 was found to have no significant influence on the enzymatic activity meas ured in vitro. However, the absence of glycosylation at Asn308, as well as a total deglycosylation, reduced the specific enzymatic activity of recombi nant HGL (r-HGL), measured on short- and long-chain triglycerides, to about 50% of normal values. Furthermore, biosynthesis and secretion of r-HGL mar kedly dropped when all four potential glycosylation sites were mutated. The kinetics of the interfacial adsorption of r-HGL and the completely deglyco sylated 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 inter face or in the presence of an egg phosphatidylcholine (PtdCho) monomolecula r film spread at various initial surface pressures. This indicates that bot h recombinant HGLs are identical, as far as recognition of phospholipid fil m and adsorption on PtdCho are concerned. The N-glycosylation of HGL may co ntribute to the enzyme stability in the stomach, as under acidic conditions the degradation by pepsin of the unglycosylated r-HGL is increased.