Bernd Gerhartz

Inhibition of mammalian legumain by some cystatins is due to a novel second reactive site

We have investigated the inhibition of the recently identified family C13 cysteine peptidase, pig legumain, by human cystatin C. The cystatin was seen to inhibit enzyme activity by stoichiometric 1:1 binding in competition with substrate. TheK i value for the interaction was 0.20 nm, i.e. cystatin C had an affinity for legumain similar to that for the papain-like family C1 cysteine peptidase, cathepsin B. However, cystatin C variants with alterations in the N-terminal region and the “second hairpin loop” that rendered the cystatin inactive against cathepsin B, still inhibited legumain with K i values 0.2–0.3 nm. Complexes between cystatin C and papain inhibited legumain activity against benzoyl-Asn-NHPhNO2 as efficiently as did cystatin C alone. Conversely, cystatin C inhibited papain activity against benzoyl-Arg-NHPhNO2 whether or not the cystatin had been incubated with legumain, strongly indicating that the cystatin inhibited the two enzymes with non-overlapping sites. A ternary complex between legumain, cystatin C, and papain was demonstrated by gel filtration supported by immunoblotting. Screening of a panel of cystatin superfamily members showed that type 1 inhibitors (cystatins A and B) and low M r kininogen (type 3) did not inhibit pig legumain. Of human type 2 cystatins, cystatin D was non-inhibitory, whereas cystatin E/M and cystatin F displayed strong (K i 0.0016 nm) and relatively weak (K i 10 nm) affinity for legumain, respectively. Sequence alignments and molecular modeling led to the suggestion that a loop located on the opposite side to the papain-binding surface, between the α-helix and the first strand of the main β-pleated sheet of the cystatin structure, could be involved in legumain binding. This was corroborated by analysis of a cystatin C variant with substitution of the Asn39 residue in this loop (N39K-cystatin C); this variant showed a slight reduction in affinity for cathepsin B (K i 1.5 nm) but ≫5,000-fold lower affinity for legumain (K i ≫1,000 nm) than wild-type cystatin C.

Subcellular localization suggests novel functions for prolyl endopeptidase in protein secretion

For a long time, prolyl endopeptidase (PEP) was believed to inactivate neuropeptides in the extracellular space. However, reports on the intracellular activity of PEP suggest additional, as yet unidentified, physiological functions for this enzyme. Here, we demonstrate using biochemical methods of subcellular fractionation, immunocytochemical double‐labelling procedures and localization of PEP–enhanced green fluorescent protein fusion proteins that PEP is mainly localized to the perinuclear space, and is associated with the microtubulin cytoskeleton in human neuroblastoma and glioma cell lines. Disassembly of the microtubules by nocodazole treatment disrupts both the fibrillar tubulin and PEP labelling. Furthermore, in a two‐hybrid screen, PEP was identified as binding partner of tubulin. These findings indicate novel functions for PEP in axonal transport and/or protein secretion. Indeed, a metabolic labelling approach revealed that both PEP inhibition and PEP antisense mRNA expression result in enhanced peptide/protein secretion from human U‐343 glioma cells. Because disturbances in intracellular transport and protein secretion mechanisms are associated with a number of ageing‐associated neurodegenerative diseases, cell‐permeable PEP inhibitors may be useful for the application in a variety of related clinical conditions.

Inhibition of mammalian legumain by Michael acceptors and AzaAsn-halomethylketones

Abstract Legumain is a lysosomal cysteine peptidase specific for an asparagine residue in the P1-position. It has been classified as a member of clan CD peptidases due to predicted structural similarities to caspases and gingipains. So far, inhibition studies on legumain are limited by the use of endogenous inhibitors such as cystatin C. A series of Michael acceptor inhibitors based on the backbone CbzLAlaLAlaLAsn (Cbz= benzyloxycarbonyl) has been prepared and resulted in an irreversible inhibition of porcine legumain. Variation of the molecular size within the war head revealed the best inhibition for the compound containing the allyl ester (kobs/I=766 M 1s 1). To overcome cyclisation between the amide moiety of the Asn residue and the war head, several asparagine analogues have been synthesised. Integrated in halomethylketone inhibitors, azaasparagine is accepted by legumain in the P1-position. The most potent inhibitor of this series, CbzLAlaLAlaAzaAsnchloromethylketone, displays a kobs/I value of 139 000 M 1s 1. Other cysteine peptidases, such as papain and cathepsin B, are not inhibited by this compound at concentrations up to 100 M. The synthetic inhibitors described here represent useful tools for the investigation of the structural and physiological properties of this unique asparaginespecific peptidase.

Characterisation of Human Dipeptidyl Peptidase IV Expressed in Pichia pastoris. A Structural and Mechanistic Comparison between the Recombinant Human and the Purified Porcine Enzyme

Abstract Dipeptidyl peptidase IV/CD26 (DP IV) is a multifunctional serine protease cleaving off dipeptides from the N-terminus of peptides. The enzyme is expressed on the surface of epithelial and endothelial cells as a type II transmembrane protein. However, a soluble form of DPIV is also present in body fluids. Large scale expression of soluble human recombinant His(6)-37-766 DP IV, using the methylotrophic yeast Pichia pastoris, yielded 1.7 mg DP IV protein per litre of fermentation supernatant. The characterisation of recombinant DP IV confirmed proper folding and glycosylation similar to DP IV purified from porcine kidney. Kinetic comparison of both proteins using short synthetic substrates and inhibitors revealed similar characteristics. However, interaction analysis of both proteins with the gastrointestinal hormone GLP-17 -36 resulted in significantly different binding constants for the human and the porcine enzyme (Kd=153.0±17.0 M and Kd=33.4± 2.2 uM, respectively). In contrast, the enzyme adenosine deaminase binds stronger to human than to porcine DP IV (Kd=2.15±0.18 nM and Kd=7.38±0.54 nM, respectively). Even though the sequence of porcine DP IV, amplified by RT-PCR, revealed 88% identity between both enzymes, the species-specific variations between amino acids 328 to 341 are likely to be responsible for the differences in ADA-binding.

Physico-chemical properties of the N-terminally truncated L68Q cystatin C found in amyloid deposits of brain haemorrhage patients.

Abstract Cystatin C, a major extracellular cysteine proteinase inhibitor, is deposited as amyloid in brain haemorrhage patients with hereditary cystatin C amyloid angiopathy (HCCAA). A diseasecausing mutation on the genetic level results in the substitution Leu68-> Gln (L68Q) in cystatin C, which causes protein instability. Besides carrying the L68Q substitution, cystatin C in amyloid deposits isolated from patients is Nterminally truncated by 10 amino acids. To elucidate the role of the Nterminal truncation for protein stability and aggregation properties, (δ1-10,L68Q)cystatin C was produced in an Escherichia coli expression system and characterised. Unlike wildtype cystatin C, this variant rapidly dimerised under physiological conditions. Two unfolding intermediates of (δ1- 10,L68Q)cystatin C were identified, under the same pH and ionic strength conditions as required to form intermediates of fulllength L68Q cystatin C. No evidence was found that the Nterminal truncation per se alters protein stability and leads to higher forms of aggregation. Monomeric as well as dimeric L68Q cystatin C incubated with neutrophil elastase was truncated as in HCCAA patients amyloid. A protein variant with a thrombin cleavage site placed in front of residue Gly11 in L68Q cystatin C was constructed and used to confirm that the Nterminal segment is similarly accessible to proteinases in the monomeric and dimeric states of L68Q cystatin C. Thus, the Nterminal segment of L68Q cystatin C is exposed to proteolytic attack and does not seem to be involved in intramolecular contacts leading to dimerisation or higherorder aggregation. We conclude that the Nterminal truncation likely is an event secondary to amyloid formation, and of no relevance for the development of HCCAA.

Acylated Hydroxamates as Selective and Highly Potent Inhibitors of Dipeptidyl Peptidase I

The compounds 10–14 which are N-dipeptidyl derivatives of O-acyl hydroxamates proved to be potent, selective and irreversible inhibitors of DP I.