Action of human pepsins 1,2,3 and 5 on the oxidized B-chain of insulin.

Human pepsins 1 and 2 attack the B-chain of oxidized insulin at pH 1.7 at the same bonds as does human pepsin 3. At pH 3.5, pepsins 1 and 2 attack insulin B-chain at essentially the same bonds as at pH 1.7, but more slowly. For all three enzymes, the first bond to be hydrolysed is Phe(25)-Tyr(26), followed simultaneously by Glu(13)-Ala(14), Leu(15)-Tyr(16) and Tyr(16)-Leu(17). Human pepsin 5, however, attacks Phe(24)-Phe(25) first of all, followed by Leu(15)-Tyr(16) and Tyr(16)-Leu(17). The results suggest that each pepsin has only one active site. Acid hydrolysis indicates that the sites of enzymic cleavage are not bonds with an inherent instability at low pH.     

Dissecting cardosin B trafficking pathways in heterologous systems

In cardoon pistils, while cardosin A is detected in the vacuoles of stigmatic papillae, cardosin B accumulates in the extracellular matrix of the transmitting tissue. Given cardosins’ high homology and yet different cellular localisation, cardosins represent a potentially useful model to understand and study the structural and functional plasticity of plant secretory pathways. The vacuolar targeting of cardosin A was replicated in heterologous species so the targeting of cardosin B was examined in these systems. Inducible expression in transgenic Arabidopsis and transient expression in tobacco epidermal cells were used in parallel to study cardosin B intracellular trafficking and localisation. Cardosin B was successfully expressed in both systems where it accumulated mainly in the vacuole but it was also detected in the cell wall. The glycosylation pattern of cardosin B in these systems was in accordance with that observed in cardoon high-mannose-type glycans, suggesting that either the glycans are inaccessible to the Golgi processing enzymes due to cardosin B conformation or the protein leaves the Golgi in an early step before Golgi-modifying enzymes are able to modify the glycans. Concerning cardosin B trafficking pathway, it is transported through the Golgi in a RAB-D2a-dependent route, and is delivered to the vacuole via the prevacuolar compartment in a RAB-F2b-dependent pathway. Since cardosin B is secreted in cardoon pistils, its localisation in the vacuoles in cardoon ovary and in heterologous systems, suggests that the differential targeting of cardosins A and B in cardoon pistils results principally from differences in the cells in which these two proteins are expressed.     

Purification and characterization of goat pepsinogens and pepsins

Three type-A and two type-C pepsinogens, namely, pepsinogens A-1, A-2, A-3, C-1, and C-2, were purified from adult goat abomasum. Their relative levels in abomasal mucosa were 27, 19, 14, 25, and 15%, respectively. Amino acid compositions were quite similar between isozymogens of respective types, but different between the two types especially in the Glx/Asx and Leu/Ile ratios. NH₂-terminal amino acid sequences of pepsinogens A-3 and C-2 were SFFKIPLVKKKSLRQNLIEN- and LVKIPLKKFKSIRETM-, respectively. Pepsins A and C showed maximal hemoglobin-digestive activity at around pH 2 and 3, respectively, and specific activities of pepsins C were higher than those of pepsins A. Two subtypes of pepsin A were obvious, namely pepsin A-2/3 which maintains its activity in the weakly acidic pH region over pH 3 and pepsin A-1, which does not. Hydrolysis of oxidized insulin B chain by goat pepsins A occurred primarily at Ala14-Leu15 and Leu15-Tyr16 bonds.     

Functional and structural characterization of synthetic cardosin B-derived rennet

The potential of using a synthetic cardosin-based rennet in cheese manufacturing was recently demonstrated with the development and optimization of production of a recombinant form of cardosin B in Kluyveromyces lactis. With the goal of providing a more detailed characterization of this rennet, we herein evaluate the impact of the plant-specific insert (PSI) on cardosin B secretion in this yeast, and provide a thorough analysis of the specificity requirements as well as the biochemical and structural properties of the isolated recombinant protease. We demonstrate that the PSI domain can be substituted by different linker sequences without substantially affecting protein secretion and milk clotting activity. However, the presence of small portions of the PSI results in dramatic reductions of secretion yields in this heterologous system. Kinetic characterization and specificity profiling results clearly suggest that synthetic cardosin B displays lower catalytic efficiency and is more sequence selective than native cardosin B. Elucidation of the structure of synthetic cardosin B confirms the canonical fold of an aspartic protease with the presence of two high mannose-type, N-linked glycan structures; however, there are some differences in the conformation of the flap region when compared to cardosin A. These subtle variations in catalytic properties and the more stringent substrate specificity of synthetic cardosin B help to explain the observed suitability of this rennet for cheese production.     

Multiplicity of aspartic proteinases from Cynara cardunculus L.

Aspartic proteinases (AP) play major roles in physiologic and pathologic scenarios in a wide range of organisms from vertebrates to plants or viruses. The present work deals with the purification and characterisation of four new APs from the cardoon Cynara cardunculus L., bringing the number of APs that have been isolated, purified and biochemically characterised from this organism to nine. This is, to our knowledge, one of the highest number of APs purified from a single organism, consistent with a specific and important biological function of these protein within C. cardunculus. These enzymes, cardosins E, F, G and H, are dimeric, glycosylated, pepstatin-sensitive APs, active at acidic pH, with a maximum activity around pH 4.3. Their primary structures were partially determined by N- and C-terminal sequence analysis, peptide mass fingerprint analysis on a MALDI-TOF/TOF instrument and by LC–MS/MS analysis on a Q-TRAP instrument. All four enzymes are present on C. cardunculus L. pistils, along with cyprosins and cardosins A and B. Their micro-heterogeneity was detected by 2D-electrophoresis and mass spectrometry. The enzymes resemble cardosin A more than they resemble cardosin B or cyprosin, with cardosin E and cardosin G being more active than cardosin A, towards the synthetic peptide KPAEFF(NO₂)AL. The specificity of these enzymes was investigated and it is shown that cardosin E, although closely related to cardosin A, exhibits different specificity. Electronic supplementary material  The online version of this article (doi:) contains supplementary material, which is available to authorized users.     

Purification of an acid proteinase from Aspergillus saitoi and determination of peptide bond specificity.

The specificity and mode of action of an acid proteinase (EC 3.4.23.6) from Aspergillus saitoi were investigated with oxidized B-chain of insulin, angiotensin II and bradykinin. Further purification of acid proteinase was performed with N,O-dibenzyloxycarbonyl-tyrosine hexamethylene-diamino-Sepharose 4B affinity chromatography and isoelectric focusing. The purified enzyme was free of any other proteolytic activity demonstrated in Asp. saitoi. Acid proteinase from Asp. saitoi hydrolyzed primarily two peptide bonds in the oxidized B-chain of insulin, the Leu(15)-Tyr(16) bond and the Phe(24)-Phe(25) bond. Additional cleavages of the bonds His(10)-Leu(11), Ala(14)-Leu(15) and Tyr(16)-Leu(17) were also noted. Primary splitting sites at Leu(15)-Tyr(16) and Phe(24-)-Phe(25) with acid proteinase from Asp. saitoi were identical with those reported in the work of cathepsin D (EC 3.4.23.5) from human erythrocyte. Hydrolysis of angiotensin II was observed at the Tyr(4)-Ile(5) bond. In conclusion, peptide bonds which have a hydrophobic amino acid such as phenylalanine, tyrosine, leucine and isoleucine in the P'1 position (as defined by Berger and Schechter, [29]) are preferentially cleaved by the trypsinogenactivating acid proteinase from Asp. saitoi.     

Exogenous adenosine 3': 5'-monophosphate can release yeast from catabolite repression.

A new simple fast and reproducible purification procedure for the proteinase from rat liver mitochondria has been worked out. The specificity of cleavage of peptide bonds in glucagon, oxidized A and B chains of insulin and yeast proteinase B inhibitor by the proteinase of the inner mitochondrial membrane has been studied. The proteinase hydrolyzed three peptide bonds in glucagon, Tyr (13) - Leu (14), Trp (25) - Leu (26) and Phe (22) - Val (23) (minor cleavage site); none in the insulin A chain; one in the B chain of insulin, Tyr (16) - Leu (17); and three in the yeast proteinase B inhibitor, Phe (4) - Ile (5), Phe (20) - Leu (21) and Tyr (41) - Thr (42) (minor cleavage site).Thus, the mitochondrial proteinase cleaves peptide bonds at the carboxyl site of an aromatic amino acid and the amino site of a leucine, isoleucine, threonine or valine. The comparison with chymotrypsin A shows that the mitochondrial proteinase cleaves peptide bonds in a more restricted manner.     

Characterization and expression analysis of the aspartic protease gene family of Cynara cardunculus L

Cardosin A and cardosin B are two aspartic proteases mainly found in the pistils of cardoon Cynara cardunculus L., whose flowers are traditionally used in several Mediterranean countries in the manufacture of ewe's cheese. We have been characterizing cardosins at the biochemical, structural and molecular levels. In this study, we show that the cardoon aspartic proteases are encoded by a multigene family. The genes for cardosin A and cardosin B, as well as those for two new cardoon aspartic proteases, designated cardosin C and cardosin D, were characterized, and their expression in C. cardunculus L. was analyzed by RT-PCR. Together with cardosins, a partial clone of the cyprosin B gene was isolated, revealing that cardosin and cyprosin genes coexist in the genome of the same plant. As a first approach to understanding what dictates the flower-specific pattern of cardosin genes, the respective gene 5' regulatory sequences were fused with the reporter beta-glucuronidase and introduced into Arabidopsis thaliana. A subsequent deletion analysis of the promoter region of the cardosin A gene allowed the identification of a region of approximately 500 bp essential for gene expression in transgenic flowers. Additionally, the relevance of the leader intron of the cardosin A and B genes for gene expression was evaluated. Our data showed that the leader intron is essential for cardosin B gene expression in A. thaliana. In silico analysis revealed the presence of potential regulatory motifs that lay within the aforementioned regions and therefore might be important in the regulation of cardosin expression.     

Occurrence of cathepsin D isozymes with different specificities in monkey skeletal muscle

Cathepsin D was highly purified from the skeletal muscle of Japanese monkey (Macaca fuscata fuscata) by a procedure including affinity chromatography on concanavalin A-Sepharose and pepstatin-Sepharose, and then resolved into ten isozymes (A through J) by isoelectric focusing. When examined for specificity toward oxidized insulin B chain, isozyme A was highly specific and cleaved exclusively the Leu15-Tyr16 bond, whereas isozyme F was less specific, cleaving the Leu15-Tyr16 and Glu13-Ala14 bonds, with slower cleavages at several other bonds. These results demonstrate for the first time the occurrence of isozymes with different specificities among cathepsin D isozymes obtained from a single source.     

Metallo-proteinase from the seedlings of kale (Brassica oleracea L. var. sabellica):

Metallo-proteinase from 8-d-old seedlings of kale was isolated. The enzyme was extracted with 1% NaCl, concentrated by ammonium sulfate and finally purified by high-performance liquid chromatography. The isolated enzyme had a molecular weight of 22.4 kDa and showed a maximum activity at pH 9.0 using casein as a substrate. Proteolytic activity of proteinase was inhibited by chelators. The inhibition by ethylenediaminetetraacetate (EDTA) was abolished by some divalent metals ions, especially by Zn²⁺. The enzyme showed activity against the synthetic peptides Suc-Ala-Ala-Pro-Leu-pNA and Suc-Ala-Ala-Pro-Phe-pNA, and hydrolized the following peptide bonds in the oxidized insulin B-chain: Leu6-Cya7, Leu15-Tyr16, Leu17—Val18 and Phe25-Tyr26.Abbreviations EDTA ethylenediaminotetraacetic acid - HPLC high-performance liquid chromatography - NEM N-ethylmaleimide - PCMB p-mecuribenzoic acid - PMSF phenylmethylsulfonyl fluoride This work was supported by the University Science Programme, Ministry of National Education, and Polish Academy of Science, Warsaw, Poland.     

Hydrolysis of beta-casein by gastric proteases. I. Comparison of proteolytic action of bovine chymosin and pepsin A

Hydrolysis of beta A2-casein by bovine chymosin and pepsin A was performed in order to compare the hydrolysis of the two enzymes on this protein. Different conditions have been tested: pH 5.5 for 116h and pH 3.5 for 7 h [E/S = 1/100 (w/w)] for chymosin. pH 3.0 for 24 h [E/S = 1/1000 (w/w)] for pepsin A. Under these conditions 17 peptides were obtained after the action of chymosin and 23 after the action of pepsin A. They corresponded respectively to the cleavage of 14 and 15 peptide bonds for chymosin and pepsin A. However, six of the peptide bonds were only hydrolyzed by chymosin and seven other bonds only by pepsin A. Our results showed a preferential splitting at the Leu-X, Ser-X, and Trp-X bonds for chymosin and Leu-X, Met-X, and Thr-X, for pepsin A. Some of the identified peptides contained sequences with possible physiological roles.     

Cardosin A endocytosis mediated by integrin leads to lysosome leakage and apoptosis of epithelial cells

Cardosin A is an aspartic protease present in large amount in the pistils of cardoon flowers. This protease is known to contain an -Arg-Gly-Asp- (RGD) motif located on the molecular surface. In this study, we found that isolated recombinant cardosin A attached to human epithelial cells A549, mediated by the binding of its RGD motif to cell surface integrins. The cell bound cardosin A was internalized to endosomes and lysosomes and triggered the permeability of lysosomal membrane leading to apoptosis of the epithelial cells. These events are identical to those observed for three RGD-containing aspartic proteases, Saps 4-6, secreted by Candida albicans. Such a process, which has been called the Trojan Horse mechanism, is believed to benefit the invasion of C. albican into the epithelium of the host. The location of the RGD motifs of cardosin A and Saps 4-6 are on the opposite ends of the homologous three-dimensional structures, suggesting that the Trojan Horse mechanism is insensitive to the RGD position. Current finding also suggests that cardosin A may have a defensive function against the ingestion of cardoon flowers by human, insects, and other herbivores.     

Cardosins in postembryonic development of cardoon: towards an elucidation of the biological function of plant aspartic proteinases

Summary. Following on from previous work, the temporal and spatial accumulation of the aspartic proteinases (EC 3.4.23) cardosin A and cardosin B during postembryonic seed development of cardoon (Cynara cardunculus) was studied. mRNA and protein analyses of both cardosins suggested that the proteins accumulate during seed maturation, and that cardosin A is later synthesised de novo at the time of radicle emergence. Immunocytochemistry revealed that the precursor form of cardosin A accumulates in protein bodies and cell walls. This localisation in seeds is different from that previously described for cardoon flowers, suggesting a tissue-dependent targeting of the protein. It is known that procardosins are active and may have a role in proteolysis and processing of storage proteins. However, the presence of procardosin A in seeds could be related to the proposed role of the plant-specific insert in membrane lipid conversion during water uptake and solute leakage in actively growing tissues. This is in accordance with the recently proposed bifunctional role of aspartic proteinase precursor molecules that possess a membrane-destabilising domain in addition to a protease domain. Mature cardosin B, but not its mRNA, was detected in the first hours after seed imbibition and disappeared at the time of radicle emergence. This extracellular aspartic protease has already been implicated in cell wall loosening and remodelling, and its role in seed germination could be related to loosening tissue constraints for radicle protusion. The described pattern of cardosin A and B expression suggests a finely tuned developmental regulation and prompts an analysis of their possible roles in the physiology of postembryonic development. Correspondence: C. S. Pereira, Institute for Molecular and Cell Biology, Rua do Campo Alegre 823, 4150-180 Porto, Portugal.     

Continuous coagulation of milk using immobilized enzymes in a fluidized-bed reactor

Milk-clotting enzymes such as pepsin, chymosin, chymotrypsin, and M. miehei proteases were immobilized on porous, alkylamine glass and incorporated into a fluidized-bed continuous coagulation scheme. Only pepsin and calf rennet retained sufficient activity towards skim milk to warrant further studies. Comparison of kinetic data with fixed-bed reactors revealed the overall superior performance of fluidized beds; higher clotting activities were possible while avoiding plugging problems and high pressure drops common to fixed-bed reactors. Film diffusion and catalyst back-mixing appear to be significant factors in the overall kinetics. All enzymes lost activity on exposure to skim milk. The inactivation rates were lower at high substrate pH and insignificantly affected by reactor temperature. Nitrogen and sialic acid accumulation on the porous glass paralleled the loss in activity in the initial stages. Attempts to regenerate the immobilized enzymes were partially successful.     

Direct Effect of Sodium Nitrite on Extracted Histones

Irpex lacteus milk-clotting enzyme hydrolyzed the Phe(105)-Met(106) bond of κ-casein, causing the precipitation of para-κ-casein along with other casein fractions in the presence of calcium ions, with a mechanism similar to other milk-clotting enzymes. Furhtermore, Irpex enzyme hydrolyzed at the positions Leu(79)-Ser(80) and Tyr(30)-Val(31) of para-κ-casein.

Degradation patterns of β-casein by Irpex and Mucor miehei enzymes were almost the same by polyacrylamide gel electrophoresis, but Endothia parasitica enzyme showed a different degradation pattern. Under the conditions employed, β-casein appeared to be scarcely hydrolyzed by chymosin.

Comparing the specificity of Irpex enzyme on β-casein with that of chymosin, the common cleaving points were Leu(165)-Ser(166), Ala(189)-Phe(190), and Tyr(192)-Glu(193). The difference in the specificity between the enzymes was exhibited in the cleavage at the Leu(139)-Leu(140) bond by chymosin and of the Ser(142)-Trp(143) bond by Irpex enzyme. Although the cleaving points of β-casein by both enzymes resembled each other, each enzyme exhibited different degradation patterns of β-casein because of thier different order of cleavage.     

News & Notes: Specificity of a Neutral Zn-Dependent Proteinase from Thermoactinomyces sacchari Toward the Oxidized Insulin B Chain

The proteolytic specificity of the neutral Zn-dependent proteinase from Thermoactinomyces sacchari was determined by analysis of the peptides obtained after incubation with the oxidized insulin B chain as a substrate. The enzyme is an endopeptidase with broad specificity. In total, 12 peptide bonds in the B chain of insulin were hydrolyzed. The major requirement is that a hydrophobic residue such as Leu, Val, or Phe should participate with the α-amino group in the bond to be cleaved. However, hydrolysis of bonds at the N-terminal side of His, Thr, and Gly was also observed. The peptide bond Leu 15–Tyr 16 in the oxidized insulin B chain, which is the major cleavage site for the alkaline microbial proteinases, is resistant to the attacks of the enzyme from Thermoactinomyces sacchari and other neutral proteinases. The proteolytic activity of the Zn-dependent proteinase from T. sacchari is different from those of other metalloendopeptidases from microorganisms. Received: 10 November 1999 / Accepted: 15 December 1999     

Characterization of meprin, a membrane-bound metalloendopeptidase from mouse kidney.

Meprin is an intrinsic protein of the brush border, a specialized plasma membrane, of the mouse kidney. It is a metalloendopeptidase that contains 1 mol of zinc and 3 mol of calcium per mol of the 85,000-Mr subunit. The enzyme is isolated, and active, as a tetramer. The behaviour of the enzyme on SDS/polyacrylamide gels in the presence and absence of beta-mercaptoethanol indicates that the subunits are of the same Mr (approx. 85,000) and held together by intersubunit S--S bridges. Eight S-carboxymethyl-L-cysteine residues were detected after reduction of the enzyme with beta-mercaptoethanol and carboxymethylation with iodoacetate. The enzyme is a glycoprotein and contains approx. 18% carbohydrate. Most of the carbohydrate is removed by endoglycosidase F, indicating that the sugar residues are N-linked. The isoelectric point of the enzyme is between pH 4 and 5, and the purified protein yields a pattern of evenly spaced bands in this range on isoelectric focusing. The peptide-bond specificity of the enzyme has been determined by using the oxidized B-chain of insulin as substrate. In all, 15 peptide degradation products were separated by h.p.l.c. and analysed for their amino acid content and N-terminal amino acid residue. The prevalent peptide-bond cleavages were between Gly20 and Glu21, Phe24 and Phe25 and between Phe25 and Tyr26. Other sites of cleavage were Leu6-Cysteic acid7, Ala14-Leu15, His10-Leu11, Leu17-Val18, Gly8-Ser9, Leu15-Tyr16, His5-Leu6. These results indicate that meprin has a preference for peptide bonds that are flanked by hydrophobic or neutral amino acid residues, but hydrolysis is not limited to these bonds. The ability of meprin to hydrolyse peptide bonds between small neutral and negatively charged amino acid residues distinguishes it from several other metalloendopeptidases.     

The specificity of bovine spleen cathepsin S. A comparison with rat liver cathepsins L and B.

The peptide-bond-specificity of bovine spleen cathepsin S in the cleavage of the oxidized insulin B-chain and peptide methylcoumarylamide substrates was investigated and the results are compared with those obtained with rat liver cathepsins L and B. Major cleavage sites in the oxidized insulin B-chain generated by cathepsin S are the bonds Glu13-Ala14, Leu17-Val18 and Phe23-Tyr26; minor cleavage sites are the bonds Asn3-Gln4, Ser9-His10 and Leu15-Tyr16. The bond-specificity of this proteinase is in part similar to the specificities of cathepsin L and cathepsin N. Larger differences are discernible in the reaction with synthetic peptide substrates. Cathepsin S prefers smaller neutral amino acid residues in the subsites S2 and S3, whereas cathepsin L efficiently hydrolyses substrates with bulky hydrophobic residues in the P2 and P3 positions. The results obtained from inhibitor studies differ somewhat from those based on substrates. Z-Phe-Ala-CH2F (where Z- represents benzyloxycarbonyl-) is a very potent time-dependent inhibitor for cathepsin S, and inhibits this proteinase 30 times more efficiently than it does cathepsin L and about 300 times better than it does cathepsin B. By contrast, the peptidylmethanes Z-Val-Phe-CH3 and Z-Phe-Lys(Z)-CH3 inhibit competitively both cathepsin S and cathepsin L in the micromolar range.     

Selective cleavage of pepsin by molybdenum metallopeptidase

In this study, the cleavage of protein by molybdenum cluster is reported for the first time. The protein target used is porcine pepsin. The data presented in this study show that pepsin is cleaved to at least three fragments with molecular weights of ～23, ～19 and ～16 kDa when the mixture of the protein and ammonium heptamolybdate tetrahydrate ((NH(4))(6)Mo(7)O(24)·4H(2)O) was incubated at 37°C for 24h. No self cleavage of pepsin occurs at 37 °C, 24h indicating that the reaction is mediated by the metal ions. N-terminal sequencing of the peptide fragments indicated three cleavage sites of pepsin between Leu 112-Tyr 113, Leu 166-Leu 167 and Leu 178-Asn 179. The cleavage reaction occurs after incubation of the mixture of pepsin and (NH(4))(6)Mo(7)O(24)·4H(2)O) only for 2h. However, the specificity of the cleavage decreases when incubation time is longer than 48 h. The mechanism for cleavage of pepsin is expected to be hydrolytic chemistry of the amide bonds in the protein backbone.