Closed and open conformations of the lid domain induce different patterns of human pancreatic lipase antigenicity and immunogenicity.
Epitope mapping was performed on human pancreatic lipase (HPL) using the SPOTscan method. A set of 146 short (12 amino acid residues) synthetic overlapping peptides covering the entire amino acid sequence of HPL were used to systematically assess the immunoreactivity of antisera raised in rabbits against native HPL, HPL without a lid (HPL(-lid)) and HPL covalently inhibited by diethyl p-nitrophenyl phosphate (DP-HPL). In the latter form of HPL, the lid domain controlling the access to the active site was assumed to exist in the open conformation. All the anti-lipase sera were tested in a direct ELISA, anti-HPL serum showing the greatest antibody titer. Although from the structural point of view, the differences between the various forms of HPL were restricted to the lid domain, differences in the antigenic properties of HPL were observed with the SPOTscan method, and the anti-DP-HPL antibodies showed the strongest reactivity. Most of the peptide stretches recognized included amino acid residues which are accessible at the surface of the lipase, except for those located near the active site. Two small peptides (T173-P180, V199-A207) were identified in the vicinity of the active site, their antipeptide antibodies were produced and their reactivity towards the various forms of HPL was tested in a double sandwich ELISA. No reactivity was observed under these conditions. Two antipeptide antibodies directed against two other selected peptides, P208-V221 (belonging to the beta9 loop) and I245-F258 (belonging to the lid domain) were prepared and found to react much more strongly with DP-HPL than with HPL or HPL(-lid) in a double sandwich ELISA. These antibodies should provide useful tools for monitoring the conformational changes taking place during the opening of the HPL lid domain.
Halimi
Hubert
H
Laboratoire d'Enzymologie Interfaciale et de Physiologie de la Lipolyse, UPR 9025, CNRS-Institut de Biologie Structurale et Microbiologie, 31, Chemin JosephBAiguier, 13402, Marseille Cedex 20, France.
De Caro
Josiane
J
Carrière
Frédéric
F
De Caro
Alain
A
D000818:Animals; D001665:Binding Sites / Q000276:immunology; D004797:Enzyme-Linked Immunosorbent Assay; D018604:Epitope Mapping; D000939:Epitopes / Q000737:chemistry* / Q000276:immunology; D006801:Humans; D008049:Lipase / Q000737:chemistry* / Q000276:immunology; D009842:Oligopeptides / Q000737:chemistry* / Q000276:immunology; D010179:Pancreas / Q000201:enzymology* / Q000276:immunology; D017433:Protein Structure, Secondary; D011817:Rabbits; D013329:Structure-Activity Relationship
D000939:Epitopes; D009842:Oligopeptides; D008049:Lipase