Structure of human dipeptidyl peptidase I (cathepsin C): exclusion domain added to an endopeptidase framework creates the machine for activation of granular serine proteases.

Dipeptidyl peptidase I (DPPI) or cathepsin C is the physiological activator of groups of serine proteases from immune and inflammatory cells vital for defense of an organism. The structure presented shows how an additional domain transforms the framework of a papain-like endopeptidase into a robust oligomeric protease-processing enzyme. The tetrahedral arrangement of the active sites exposed to solvent allows approach of proteins in their native state; the massive body of the exclusion domain fastened within the tetrahedral framework excludes approach of a polypeptide chain apart from its termini; and the carboxylic group of Asp1 positions the N-terminal amino group of the substrate. Based on a structural comparison and interactions within the active site cleft, it is suggested that the exclusion domain originates from a metallo-protease inhibitor. The location of missense mutations, characterized in people suffering from Haim-Munk and Papillon-Lefevre syndromes, suggests how they disrupt the fold and function of the enzyme.

Tetrameric dipeptidyl peptidase I directs substrate specificity by use of the residual pro-part domain.

The crystal structure of mature dipeptidyl peptidase I reveals insight into the unique tetrameric structure, substrate binding and activation of this atypical papain family peptidase. Each subunit is composed of three peptides. The heavy and light chains form the catalytic domain, which adopts the papain fold. The residual pro-part forms a beta-barrel with the carboxylate group of Asp1 pointing towards the substrate amino-terminus. The tetrameric structure appears to stabilize the association of the two domains and encloses a 12700 A3 spherical cavity. The tetramer contains six chloride ions, one buried in each S2 pocket and two at subunit interfaces.

Human recombinant pro-dipeptidyl peptidase I (cathepsin C) can be activated by cathepsins L and S but not by autocatalytic processing.

Human dipeptidyl peptidase I was expressed in the insect cell/baculovirus system and purified in its active (rhDPPI) and precursor (pro-rhDPPI) forms. RhDPPI was very similar to the purified enzyme (hDPPI) with respect to glycosylation, enzymatic processing, oligomeric structure, CD spectra, and catalytic activity. The precursor, which was a dimer, could be activated approximately 2000-fold with papain. Cathepsin L efficiently activated pro-rhDPPI in vitro at pH 4.5 (k(app) approximately 2 x 10(3) min(-)(1) M(-)(1)), and two cleavage pathways were characterized. The initial cleavage was within the pro region between the residual pro part and the activation peptide. Subsequently, the activation peptide was cleaved from the catalytic region, and the latter was cleaved into the heavy and light chains. Alternatively, the pro region was first separated from the catalytic region. Cathepsin S was a less efficient activating enzyme. Cathepsin B and rhDPPI did not activate pro-rhDPPI, and the proenzyme was incapable of autoactivation. Incubation of both pro-rhDPPI and rhDPPI with cathepsin D resulted in degradation. Cystatin C and stefins A and B inhibited rhDPPI with K(i) values in the nanomolar range (K(i) = 0.5-1.1 nM). The results suggest that cathepsin L could be an important activator of DPPI in vivo and that cathepsin D and possibly the cystatins may contribute to DPPI downregulation.

The residual pro-part of cathepsin C fulfills the criteria required for an intramolecular chaperone in folding and stabilizing the human proenzyme.

The 13.5 kDa N-terminal part of the propeptide remains associated with mature cathepsin C after proteolytic activation and excision of the activation peptide. This residual pro-part, isolated from the recombinant enzyme, folds spontaneously and rapidly to a stable, compact monomer with secondary structure and stable tertiary interactions. Folding and unfolding kinetics of the residual pro-part with intact disulfides are complex, and accumulation of transient intermediates is observed. The cleaved form of the pro-part isolated from natural human cathepsin C also folds, suggesting that the intact form comprises two folding domains. The linkages of the two disulfide bridges have been established as 30-118 and 54-136 for the native enzyme. The native disulfide bonds can be re-formed from the fully reduced and denatured state by oxidative refolding, resulting in a domain that is spectroscopically indistinguishable from the original refolded residual pro-part. Both disulfides are solvent-exposed and can be reduced in the absence of denaturant. The reduced form retains most or all of the native tertiary structure and is only approximately 2 kcal.mol(-1) less stable than the oxidized form. It folds fast relative to the rate of biosynthesis, to the same conformation as the oxidized form. Folding and disulfide formation are sequential. These results indicate that the proenzyme folds sequentially in vivo and that the residual pro-part constitutes a rapidly and independently folding domain that stabilizes the mature enzyme. It thus fulfills the criteria required of an intramolecular chaperone. It may also be involved in stabilizing the tetrameric structure of the mature enzyme.

Carica papaya glutamine cyclotransferase belongs to a novel plant enzyme subfamily: cloning and characterization of the recombinant enzyme.

A full-length cDNA encoding Carica papaya glutamine cyclotransferase was cloned by RT-PCR on the basis of results from amino acid sequencing of tryptic fragments of the native enzyme. The cDNA of 1036 nucleotides encodes a typical 22-residue signal peptide and a mature protein of 266 residues with a calculated molecular mass of 30,923 Da. Five plant ESTs encoding putative QCs highly homologous to PQC were identified and the numbers and locations of cysteines and N-glycosylation sites are conserved. The plant QC amino acid sequences are very different from the known mammalian QC sequences and no clear homology was observed. The PQC cDNA was expressed in Escherichia coli as either His-tagged PQC, with three different signal peptides and in fusions with thioredoxin, glutathione S-transferase, and (pre-) maltose-binding protein. In all cases, the expressed protein was either undetectable or insoluble. Expression in Pichia pastoris of PQC fused to the alpha-factor leader resulted in low levels of PQC activity. Extracellular expression of PQC in the insect cell/baculovirus system was successful and 15-50 mg/liter of active PQCs with three different secretion signals was expressed and purified. Further, PQC N-terminally fused to a combined secretion signal/His-tag peptide was correctly processed by the host signal peptidase and the His-tag could subsequently be removed with dipeptidyl peptidase I. The expressed products were characterized by activity assays, SDS-PAGE, N-terminal amino acid sequencing, MALDI-TOF mass spectroscopy, and peptide mass fingerprint analysis.

Active recombinant rat dipeptidyl aminopeptidase I (cathepsin C) produced using the baculovirus expression system.

An active form of rat dipeptidyl aminopeptidase I (DPPI, cathepsin C) was obtained by heterologous expression in insect cells. Baculoviruses carrying a cDNA sequence encoding the entire rat DPPI precursor was used to infect High Five cells in a serum-free medium. Recombinant DPPI (rDPPI) was secreted into the medium from which it was purified by a combination of ammonium sulfate fractionation, hydrophobic interaction chromatography (HIC), and ion-exchange chromatography. A polyhistidine-tagged form of the enzyme (HT-rDPPI) was purified from the medium by immobilized metal affinity chromatography (IMAC). In vivo activation of native rat DPPI involves at least three chain cleavages per subunit and the ability of the expression system to imitate this processing was investigated. Both rDPPI and HT-rDPPI were secreted into the medium as unprocessed and inactive proenzymes and gradually converted into their active forms in the medium. This process was not completed at the time of harvest but mature enzyme processed similarly to native rat and human DPPI could be obtained by incubating the eluates from the HIC and IMAC columns at pH 4.5 and 5 degrees C for 18-40 h. The yield of purified and matured enzyme was approximately 50 mg/liter, and it was shown that rDPPI and HT-rDPPI were active against both a dipeptide-p-nitroanilide substrate and human growth hormone N-terminally extended with an Ala-Glu dipeptide.

Heterologous expression of three plant serpins with distinct inhibitory specificities.

For the first time, inhibitory plant serpins, including WSZ1 from wheat, BSZ4, and the previously unknown protein BSZx from barley, have been expressed in Escherichia coli, and a procedure for fast purification of native plant serpins has been developed. BSZx, BSZ4, and WSZ1 were assayed for inhibitory activity against trypsin, chymotrypsin, and cathepsin G, and cleavage sites in the reactive center loop were identified by sequencing. BSZx proved to be a potent inhibitor with specific, overlapping reactive centers either at P1 Arg for trypsin or at P2 Leu for chymotrypsin. At 22 ;C, the apparent rate constant for chymotrypsin inhibition at P2 (ka = 9.4 x 10(5) M-1 s-1) was only four times lower than for trypsin at P1 (ka = 3.9 x 10(6) M-1 s-1), and the apparent inhibition stoichiometries were close to 1. Furthermore, our data suggest that cathepsin G was inhibited by BSZx (ka = 3.9 x 10(6) M-1 s-1) at both the P1 Arg and P2 Leu. These results indicate a unique adaptability of the reactive center loop of BSZx. WSZ1 inhibited chymotrypsin (ka = 1.1 x 10(5) M-1 s-1) and cathepsin G (ka = 7.6 x 10(3) M-1 s-1) at P1 Gln and not, as for BSZx, at the more favorable P2 Leu. BSZ4 inhibited cathepsin G (ka = 2.7 x 10(4) M-1 s-1) at P1 Met but was hydrolyzed by trypsin and chymotrypsin. The three plant serpins formed stable SDS-resistant complexes with the proteinases in accordance with the kinetic data.

Inhibition of coagulation factors by recombinant barley serpin BSZx.

Barley serpin BSZx is a potent inhibitor of trypsin and chymotrypsin at overlapping reactive sites (Dahl, S.W., Rasmussen, S.K. and Hejgaard, J. (1996) J. Biol. Chem., in press). We have now investigated the interactions of BSZx with a range of serine proteinases from human plasma, pancreas and leukocytes, a fungal trypsin and three subtilisins. Thrombin, plasma kallikrein, factor VIIa/tissue factor and factor Xa were inhibited by BSZx at heparin independent association rates (k(ass)) of 4.5 X 10(3)-1.3 x 10(5) M(-1) s(-1) at 22 degrees C. Only factor Xa turned a significant fraction of BSZx over as substrate. Complexes of these proteinase with BSZx resisted boiling in SDS, and amino acid sequencing showed that cleavage in the reactive center loop only occurred after P1 Arg. Activated protein C and leukocyte elastase were slowly inhibited by BSZx (k(ass)=1-2 x 10(2) M(-1) s(-1)) whereas factor XIIa, urokinase and tissue type plasminogen activator, plasmin and pancreas kallikrein and elastase were not or only weakly affected. The inhibition pattern with mammalian proteinases reveal a specificity of BSZx similar to that of antithrombin III. Trypsin from Fusarium was not inhibited while interaction with subtilisin Carlsberg and Novo was rapid but most BSZx was cleaved as a substrate. Identification of a monoclonal antibody specific for native BSZx indicate that complex formation and loop cleavage result in similar conformational changes.

A recombinant wheat serpin with inhibitory activity.

A full-length clone encoding the wheat (Triticum aestivum L.) serpin WSZ1 was isolated from a cDNA library based on mRNA from immature grain. The 398 amino acid sequence deduced from the cDNA was corroborated by sequencing CNBr peptides of WSZ1 purified from resting grain. WSZ1 belongs to the subfamily of protein Z-type serpins and the amino acid sequence is 70% identical with the barley serpins BSZ4 and BSZx and 27-33% identical with human serpins such as alpha 1-proteinase inhibitor, antithrombin III, and plasminogen activator inhibitor. The cDNA was subcloned in the pET3d expression vector, equipped with a histidine affinity tag at the N-terminus and expressed in Escherichia coli BL(21) DE3 pLysS. Recombinant WSZ1 from the soluble fraction was partially purified on Ni-NTA agarose and MonoQ columns and shown to form SDS-stable complexes with alpha-chymotrypsin. Southern blots and amino acid sequencing indicated that only few serpins are encoded by wheat, but at least three distinct genes are expressed in the grain. Cleavage experiments on a chymotrypsin column suggested a Gln-Gln reactive site bond not previously observed in inhibitory serpins.