Human Pancreatic Lipase:  An Exposed Hydrophobic Loop from the C-terminal Domain May Contribute to Interfacial Binding

Epitope mapping was performed using four anti-HPL monoclonal antibodies (mAb's 81−23, 146−40, 315−25, and 320−24) directed against human pancreatic lipase (HPL). Three HPL mutants produced in insect cells were tested for this purpose: (i) N−HPL, which consists of only the N-terminal domain of HPL, (ii) HPL(-lid), in which a short loop consisting of 5 amino acid residues replaces the full-length 23-residue lid domain present in HPL, and (iii) N-GPLRP2/C−HPL chimera, a chimeric mutant consisting of the N-terminal domain of the guinea pig pancreatic lipase related protein 2 (GPLRP2) fused to the C-terminal domain of HPL. The C-terminal domain of HPL (C−HPL) was prepared in a pure form after performing chymotryptic digestion of HPL. The mAb 146−40 recognizes HPL, HPL(-lid), and N−HPL but not GPLRP2, N-GPLRP2/C−HPL chimera, or the C−HPL. The antibody mAb 146−40 therefore specifically recognizes the N-terminal domain of HPL, and the epitope recognized does not include the amphiphilic lid. On the other hand, mAb's 81−23, 315−25, and 320−24 react specifically to the C-terminal domain of HPL, since they recognize HPL, HPL(-lid), the N-GPLRP2/C−HPL chimera, and the C−HPL but not N−HPL or GPLRP2. It was further established that these three mAb's recognize the same conformational epitope, the structure of which is stabilized by the N-terminal domain in the presence of SDS at concentrations greater than its critical micellar concentration. This conformational epitope was found to be located in the vicinity of Met 397 and Arg 414. These two residues delineate a highly exposed peptide stretch extending from the HPL C-terminal domain, which includes a hydrophobic surface loop (β5‘). Kinetic studies on the HPL/mAb's complexes showed that the lipase activity was much lower in these complexes than in HPL. The results of the present study suggest for the first time that the β5‘ loop from the C-terminal domain may be involved in the interaction of HPL with a lipid/water interface.