Potato cysteine proteinase inhibitor gene family: molecular cloning,characterisation and immunocytochemical localisation studies

Potato cysteine proteinase inhibitors (PCPIs) represent a distinct group of proteins as they show no homology to any other known cysteine proteinase inhibitor superfamilies, but they all belong to the Kunitz-type soybean trypsin inhibitor family. cDNA clones for five PCPIs have been isolated and sequenced. Amino acid substitutions occurring in the limited regions forming loops on the surface of these proteins suggest a further classification of PCPIs into three subgroups. Accumulation of PCPI was observed in vacuoles of stems after treatment with jasmonic acid (JA) using immunocytochemical localisation, implying that these inhibitors are part of a potato defence mechanism against insects and pathogens. Genomic DNA analysis show that PCPIs form a multigene family and suggest that their genes do not possess any introns.     

Characterization of a latent cysteine proteinase from ascitic fluid as a high molecular weight form of cathepsin B

The latent cysteine proteinase present in ascitic fluid of patients with neoplasia and released from ascites cells in culture has been partially purified and the enzyme after pepsin activation was shown to be immunologically related to the lysosomal proteinase, cathepsin B. The latent form was characterized as a single chain of M_r 40 000 as determined by SDS-polyacrylamide gel electrophoresis under reducing conditions followed by Western blotting and immune staining with an antiserum to human cathepsin B. Using the same techniques the enzyme after pepsin activation gave a single band of M_r 33 000. Analysis by isoelectric focusing showed that the latent enzyme before and after pepsin treatment is composed of several acidic isoenzymes. These findings suggest that this latent proteinase represents a precursor form of cathepsin B which is released extracellularly rather than being processed and directed to the lysosome.     

Purification and characterization of trypsin-like enzyme from sea urchin eggs: substrate specificity and physiological role

A trypsin-like enzyme has been purified to homogeneity from eggs of the sea urchin, Strongylocentrotus intermedius. The purified enzyme efficiently hydrolyzed Z-Phe-Arg-4- methylcoumaryl -7-amide (MCA) and Pro-Phe-Arg-MCA among 12 peptidyl-Arg (or Lys)- MCAs . The substrate specificity of the enzyme was closely similar to that of the enzyme activity in the egg cortical granule exudate. Among various peptidyl-argininal (Arg-H) derivatives, Z-Phe-Arg-H and Z-Phe-Leu-Arg-H showed the strongest inhibition against both the activity of the purified enzyme and the elevation of vitelline coat. Thus, the trypsin-like enzyme of sea urchin possesses a narrow substrate specificity and participates at least in the elevation of vitelline coat during fertilization.     

Sperm proteasomes are responsible for the acrosome reaction and sperm penetration of the vitelline envelope during fertilization of the sea urchin Pseudocentrotus depressus

The roles of sperm proteasomes in fertilization were investigated in the sea urchin Pseudocentrotus depressus. Two proteasome inhibitors, MG-132 and MG-115, inhibited fertilization at 100 microM, whereas chymostatin and leupeptin showed no inhibition. Among three proteasome substrates, Z-Leu-Leu-Glu-MCA showed the strongest inhibition toward fertilization. MG-132 inhibited the egg-jelly-induced, but not ionomycin-induced, acrosome reaction. In addition, MG-132, but not E-64-d, inhibited fertilization of dejellied eggs by acrosome-reacted sperm. MG-132 showed no significant inhibition toward the binding of reacted sperm to the vitelline layer. Proteasomes were detected by Western blotting in the acrosomal contents, which are partially released upon exocytosis. We also found that the inhibition pattern of the caspase-like activity of the proteasome in the acrosomal contents by chymostatin and proteasome inhibitors coincided well with their inhibitory abilities toward fertilization. Furthermore, the vitelline layer of unfertilized eggs appears to be ubiquitinated as revealed by immunocytochemistry and Western blotting. Extracellular ATP, required for the degradation of ubiquitinated proteins by the proteasome, was also necessary for fertilization. These results indicate that the sperm proteasome plays a key role not only in the acrosome reaction but also in sperm penetration through the vitelline envelope, most probably as a lysin, during sea urchin fertilization.     

Digestive proteolysis in the Colorado potato beetle,Leptinotarsa decemlineata: Activity-based profiling and imaging of a multipeptidase network

The Colorado potato beetle (CPB), Leptinotarsa decemlineata, is a major pest of potato plants, and its digestive system is a promising target for development of pest control strategies. This work focuses on functional proteomic analysis of the digestive proteolytic enzymes expressed in the CPB gut. We identified a set of peptidases using imaging with specific activity-based probes and activity profiling with selective substrates and inhibitors. The secreted luminal peptidases were classified as: (i) endopeptidases of cathepsin D, cathepsin L, and trypsin types and (ii) exopeptidases with aminopeptidase (cathepsin H), carboxypeptidase (serine carboxypeptidase, prolyl carboxypeptidase), and carboxydipeptidase (cathepsin B) activities. The proteolytic arsenal also includes non-luminal peptidases with prolyl oligopeptidase and metalloaminopeptidase activities. Our results indicate that the CPB gut employs a multienzyme network of peptidases with complementary specificities to efficiently degrade ingested proteins. This proteolytic system functions in both CPB larvae and adults and is controlled mainly by cysteine and aspartic peptidases and supported by serine and metallopeptidases. The component enzymes identified here are potential targets for inhibitors with tailored specificities that could be engineered into potato plants to confer resistance to CPB.