Tryptic cleavage of gastric lipases: Location of the single disulfide bridge

• Published in: Biochimica et biophysica acta Vol. 1213; no. 3; pp. 319 - 324
• Main Authors: Aoubala, Mustapha, Bonicel, Jacques, Bénicourt, Claude, Verger, Robert, De Caro, Alain
• Format: Journal Article
• Language: English
• Published: Amsterdam Elsevier B.V 04.08.1994; Elsevier
• Subjects: Amino Acid Sequence; Analytical, structural and metabolic biochemistry; Animals; Binding Sites; Biological and medical sciences; Disulfide bridge; Disulfides - analysis; Domain structure; Enzymes and enzyme inhibitors; Fundamental and applied biological sciences. Psychology; Gastric lipase; Humans; Hydrolases; Immunoblotting; Lipase - chemistry; Lipase - metabolism; Molecular Sequence Data; Monoclonal antibody; Peptide Fragments - chemistry; Peptide Fragments - immunology; Rabbits; Stomach - enzymology; Trypsin - metabolism; Tryptic digestion; Gastric lipase; pAb; TPCK; SDS; Domain structure; RGL; Tryptic digestion; Monoclonal antibody; HGL; Disulfide bridge; mAb; Human; Stomach; Molecular processing; Serine endopeptidases; Enzyme; Rabbit; Triacylglycerol lipase; Esterases; Lagomorpha; Carboxylic ester hydrolases; Vertebrata; Trypsin; Mammalia; Immunogenicity; Peptide fragment; Disulfide bond; Hydrolases; Proteinases; Structural analysis
• ISSN: 0005-2760; 0006-3002; 1879-145X
• DOI: 10.1016/0005-2760(94)00058-1

Human (HGL) and rabbit (RGL) gastric lipases were cleaved by trypsin and the resulting peptides were characterized. Exposure of HGL to trypsin led to the production of three identified fragments (H1, H2 and H3) resulting from cleavage sites at Lys-4 and Arg-229. Fragments H2 (Lys-4-Arg-229) and H3 (Glu-230-Lys-379) were derived from fragment H1 (Lys-4-Lys-379). The single disulfide bridge (Cys-236-Cys-244) of the molecule is localized in fragment H3. Out of the three cysteine residues conserved in all known gastric lipases, the free sulfhydryl group (Cys-227) was localized in fragment H2. Immunoblots, carried out with the tryptic fragments of HGL and anti-HGL mAbs, revealed that five inhibitory mAbs immunoreacted selectively with the N-terminal fragment H2, whereas two other non inhibitory mAbs immunoreacted exclusively with the C-terminal fragment H3. Trypsin also cleaved RGL at two sites (Arg-55 and Arg-229) leading to four identifiable fragments (R1, R2, R3 and R4). One cleavage site (Arg-229) was found to be identical in both RGL and HGL. We propose that this latter site is localized between the two domains of native gastric lipases.