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Inactivation of papain by antithrombin due to autolytic digestion: a model of serpin inactivation of cysteine proteinases.

ABSTRACT: Cross-class inhibition of cysteine proteinases by serpins differs from serpin inhibition of serine proteinases primarily in that no stable serpin-cysteine proteinase complex can be demonstrated. This difference in reaction mechanism was elucidated by studies of the inactivation of the cysteine proteinases, papain and cathepsin L, by the serpin antithrombin. The two proteinases were inactivated with second-order rate constants of (1.6+/-0.1)x10(3) and (8.6+/-0. 4)x10(2) M-1.s-1 respectively. An antithrombin to papain inactivation stoichiometry of approximately 3 indicated extensive cleavage of the inhibitor concurrent with enzyme inactivation, a behaviour verified by SDS/PAGE. N-terminal sequence analyses showed cleavage predominantly at the P2-P1 bond, but also at the P2'-P3' bond of antithrombin. The papain band in SDS/PAGE progressively disappeared on reaction of the enzyme with increasing amounts of antithrombin, but no band representing a stable antithrombin-papain complex appeared. SDS/PAGE with 125I-labelled papain showed that the disappearance of papain was caused by cleavage of the enzyme into small fragments. These results suggest a mechanism in which papain attacks a peptide bond in the reactive-bond loop of antithrombin adjacent to that involved in serine proteinase inhibition. The reaction proceeds, similarly to that between serpins and serine proteinases, to form an inactive acyl-intermediate complex, although with the substrate pathway dominating in the papain reaction. In this complex, papain is highly susceptible to proteolysis and is degraded by still active papain, which greatly decreases the lifetime of the complex and results in liberation of fragmented, inactive enzyme. This model may have relevance also for the inactivation of physiologically or pathologically important cysteine proteinases by serpins.

SUBMITTER: Bjork I

PROVIDER: S-EPMC1219835 | biostudies-other | 1998 Nov

REPOSITORIES: biostudies-other

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Project description:BackgroundPlant-derived cysteine proteinases of the papain family (CPs) attack nematodes by digesting the cuticle, leading to rupture and death of the worm. The nematode cuticle is composed of collagens and cuticlins, but the specific molecular target(s) for the proteinases have yet to be identified.MethodsThis study followed the course of nematode cuticle disruption using immunohistochemistry, scanning electron microscopy and proteomics, using a free-living nematode, Caenorhabditis elegans and the murine GI nematode Heligmosomoides bakeri (H. polygyrus) as target organisms.ResultsImmunohistochemistry indicated that DPY-7 collagen is a target for CPs on the cuticle of C. elegans. The time course of loss of DPY-7 from the cuticle allowed us to use it to visualise the process of cuticle disruption. There was a marked difference in the time course of damage to the cuticles of the two species of nematode, with H. bakeri being more rapidly hydrolysed. In general, the CPs' mode of attack on the nematode cuticle was by degrading the structural proteins, leading to loss of integrity of the cuticle, and finally death of the nematode. Proteomic analysis failed conclusively to identify structural targets for CPs, but preliminary data suggested that COL-87 and CUT-19 may be important targets for the CPs, the digestion of which may contribute to cuticle disruption and death of the worm. Cuticle globin was also identified as a cuticular target. The presence of more than one target protein may slow the development of resistance against this new class of anthelmintic.ConclusionsScanning electron microscopy and immunohistochemistry allowed the process of disruption of the cuticle to be followed with time. Cuticle collagens and cuticlins are molecular targets for plant cysteine proteinases. However, the presence of tyrosine cross-links in nematode cuticle proteins seriously impeded protein identification by proteomic analyses. Multiple cuticle targets exist, probably making resistance to this new anthelmintic slow to develop.

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Inactivation of papain by antithrombin due to autolytic digestion: a model of serpin inactivation of cysteine proteinases.

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