Human Pancreatic Lipase: IMPORTANCE OF THE HINGE REGION BETWEEN THE TWO DOMAINS, AS REVEALED BY MONOCLONAL ANTIBODIES

Abstract

Several monoclonal antibodies (mAbs) were prepared against human pancreatic lipase (HPL). Two enzyme-linked immunosorbent assay (ELISA) procedures were set up for screening hybridomas producing specific antibodies. Four mAbs (81-23, 146-40, 315-25, and 320-24) of the IgG1 isotype were found to react with HPL in both simple sandwich and double sandwich ELISAs, while mAb 248-31, of the IgG2b isotype, reacted only with HPL in a double sandwich ELISA. The results of Western blot analysis carried out with native and SDS-denatured HPLs indicated that mAb 248-31 recognized only native HPL, while all the other mAbs recognized both forms of HPL. Since mAb 248-31 did not recognize SDS-denatured HPL, it was not possible to localize its epitope. To carry out epitope mapping along the primary sequence of HPL, four fragments (14, 26, 30, and 36 kDa) resulting from a limited chymotryptic cleavage of HPL were characterized by Western blotting as well as N-terminal amino acid sequence analysis. Of the above five anti-HPL mAbs, four (81-23, 248-31, 315-25, and 320-24) were found to inhibit the lipolytic activity of HPL (in both the presence and absence of bile salts and colipase), while mAb 146-40 had no inhibitory effects. The epitope recognized by mAb 146-40 was found to be located in the N-terminal domain (Lys1-Phe335). Combined immunoinactivation and epitope mapping studies showed that three inhibitory mAbs (81-23, 315-25, and 320-24) recognize overlapping epitopes from the hinge region between the N- and C-terminal domains of HPL, belonging to the 26-kDa fragment. In the presence of lipids, a significant decrease has been observed in the bending angle between the N- and C-terminal domains of the HPL tertiary structure (van Tilbeurgh, H., Egloff, M. P., Martinez, C., Rugani, N., Verger, R. and Cambillau, C. (1993) Nature 362, 814-820). From the present immunochemical data, we further propose that locking the hinge movement with mAbs may induce lipase immunoinactivation.