Diane Savaria

Lepidopteran-specific crystal toxins from Bacillus thuringiensis form cation- and anion-selective channels in planar lipid bilayers

SummaryPrevious studies in our laboratory have shown that CryIC, a lepidopteran-specific toxin from Bacillus thuringiensis, triggers calcium and chloride channel activity in SF-9 cells (Spodoptera frugiperda, fall armyworm). Chloride currents were also observed in SF-9 membrane patches upon addition of CryIC toxin to the cytoplasmic side of the membrane. In the present study the ability of activated CryIC toxin to form channels was investigated in a receptor-free, artificial phospholipid membrane system. We demonstrate that this toxin can partition in planar lipid bilayers and form ion-selective channels with a large range of conductances. These channels display complex activity patterns, often possess subconducting states and are selective to either anions or cations. These properties appeared to be pH dependent. At pH 9.5, cation-selective channels of 100 to 200 pS were most frequently observed. Among the channels recorded at pH 6.0, a 25–35 pS anion-selective channel was often seen at pH 6.0, with permeation and kinetic properties similar to those of the channels previously observed in cultured lepidopteran cells under comparable pH environment and for the same CryIC toxin doses. We conclude that insertion of CryIC toxin in SF-9 cell native membranes and in artificial planar phospholipid bilayers may result from an identical lipid-protein interaction mechanism.

α1-Antitrypsin Portland Inhibits Processing of Precursors Mediated by Proprotein Convertases Primarily within the Constitutive Secretory Pathway

We studied the extent of cellular inhibitory activity of α1-antitrypsin Portland (α1-PDX), a potent inhibitor of proprotein convertases of the subtilisin/kexin type. We compared the inhibitory effects of α1-PDX on the intracellular processing of two model precursors (pro-7B2 and POMC) mediated by six of the seven known mammalian convertases, namely furin, PC1, PC2, PACE4, PC5-A, PC5-B, and PC7. The substrates selected were pro7B2, a precursor cleaved within the trans-Golgi network (TGN), and pro-opiomelanocortin, which is processed in the TGN and secretory granules. Biosynthetic analyses were performed using either vaccinia virus expression in BSC40, GH4C1, and AtT20 cells, or stable transfectants of α1-PDX in AtT20 cells. Results revealed that α1-PDX inhibits processing of these precursors primarily within the constitutive secretory pathway and that α1-PDX is cleaved into a shorter form by some convertases. Evidence is presented demonstrating that in contrast to the full-length α1-PDX (64 kDa), the cleaved (56 kDa) secreted product does not significantly inhibit furin activity in vitro. Cellular expression of α1-PDX results in modified contents of mature secretory granules with increased levels of partially processed products. Biosynthetic and immunocytochemical analyses of AtT20/α1-PDX cells demonstrated that α1-PDX is primarily localized within the TGN, and that a small proportion enters secretory granules where it is mostly stored as the cleaved product.

7B2 Is a Specific Intracellular Binding Protein of the Prohormone Convertase PC2

Abstract: Biosynthetic pulse‐chase analyses have previously demonstrated that the prohormone convertase PC2 is first synthesized as a precursor pro‐PC2 and that zymogen activation to PC2 occurs following the slow exit of pro‐PC2 from the endoplasmic reticulum (ER) and its concentration within the trans‐Golgi network (TGN). The endocrine and neural protein 7B2 is first synthesized as a nonglycosylated precursor (pro‐7B2), which is cleaved within the TGN by a furin‐like ubiquitous convertase at the RRKRR155S site to generate 7B2. In this report, we demonstrate that within the ER, pro‐7B2 binds pro‐PC2 but not any of the other convertases furin, PC1, PACE4, or PC5. This specific binding is Ca2+ dependent and does not require an N‐glycosylated pro‐PC2. Mutagenesis of the RRKRRS sequence demonstrated that the intact hexapeptide is critical for this binding, because the latter was abolished by mutations of the RR152 and greatly diminished by mutations of either the R151 or S156 residues of pro‐7B2. Once the complex is formed in the ER, it is then transported to the TGN where furin or a furin‐like convertase cleaves both precursors, even when present as a complex. We also provide evidence that following zymogen cleavage, 7B2 remains bound to PC2, suggesting the presence of at least one other Ca2+‐dependent binding site within the 7B2 sequence. Coexpression of 7B2 and PC2, although resulting in an elevation of the level of pro‐PC2, did not eliminate the processing of pro‐PC2 to PC2. Accordingly, cellular coexpression of 7B2 together with PC2 and proopiomelanocortin only marginally diminished the ability of PC2 to cleave proopiomelanocortin into β‐endorphin in constitutive cells and had no effect in regulated cells. These results suggest that in vivo pro‐7B2 is a specific PC2‐binding protein that only transiently inhibits the processing of pro‐PC2 until it reaches the TGN.

In vitro characterization of the novel proprotein convertase PC7.

Biochemical and enzymatic characterization of the novel proprotein convertase rat PC7 (rPC7) was carried out using vaccinia virus recombinants overexpressed in mammalian BSC40 cells. Pro-PC7 is synthesized as a glycosylated zymogen (101 kDa) and processed into mature rPC7 (89 kDa) in the endoplasmic reticulum. No endogenously produced soluble forms of this membrane-anchored protein were detected. A deletion mutant (65 kDa), truncated well beyond the expected C-terminal boundary of the P-domain, produced soluble rPC7 in the culture medium. Enzymatic activity assays of rPC7 using fluorogenic peptidyl substrates indicated that the pH optimum, Ca2+ dependence, and cleavage specificity of this enzyme are largely similar to those of furin. However, with some substrates, cleavage specificity more closely resembled that of yeast kexin, suggesting differential processing of proprotein substrates by this novel convertase. We examined the rPC7- and human furin-mediated cleavage of synthetic peptides containing the processing sites of three proteins known to colocalize in situ with rPC7. Whereas both enzymes correctly processed the pro-parathyroid hormone tridecapeptide and the pro-PC4 heptadecapeptide, neither enzyme cleaved a pro-epidermal growth factor hexadecapeptide. Thus, this study establishes that rPC7 is an enzymatically functional subtilisin/kexin-like serine proteinase with a cleavage specificity resembling that of hfurin. In addition, we have demonstrated that rPC7 can correctly process peptide precursors that contain the processing sites of at least two potential physiological substrates.

Proteolytic Processing of Chromogranin B and Secretogranin II by Prohormone Convertases

Abstract: Two experimental approaches were used to study the processing of chromogranin B and secretogranin II by prohormone convertases. In GH3 cells various prohormone convertases were overexpressed together with the substrate chromogranin B by use of a vaccinia virus infection system. PC1 appeared to be by far the most active enzyme and converted chromogranin B to several smaller molecules, including the peptide PE‐11. In brain this peptide is cleaved physiologically from chromogranin B. Some processing of chromogranin B and formation of free PE‐11 were also observed with PC2 and PACE4. Furin produced larger fragments, whereas PC5‐A and PC5‐B had negligible effects. As a second model, PC12 cells were stably transfected with PC1 or PC2 to investigate the processing of endogenous chromogranins. Both enzymes effectively cleaved chromogranin B and secretogranin II, liberating the peptides PE‐11 and secretoneurin, respectively. However, in transfection experiments the ability to generate the free peptides was more pronounced with PC2 than with PC1. The extent of proprotein processing achieved by prohormone convertases apparently differed depending on the experimental system applied. This suggests that in vivo mechanisms to support and fine‐tune the activity of the processing enzymes exist, which might be overlooked by using only one methodological approach.

Comparative functional role of PC7 and furin in the processing of the HIV envelope glycoprotein gp160

© 1997 Federation of European Biochemical Societies.

Residues unique to the pro-hormone convertase PC2 modulate its autoactivation, binding to 7B2 and enzymatic activity.

The prohormone convertase PC2 is one of the major subtilisin/kexin‐like enzymes responsible for the formation of small bioactive peptides in neural and endocrine cells. This convertase is unique among the members of the subtilisin/kexin‐like mammalian serine proteinase family in that it undergoes zymogen processing of its inactive precursor proPC2 late along the secretory pathway and requires the help of a PC2‐specific binding protein known as 7B2. We hypothesized that some of these unique properties of PC2 are dictated by the presence of PC2‐specific amino acids, which in the six other known mammalian convertases are otherwise conserved but distinct. Accordingly, six sites were identified within the catalytic segment of PC2. Herein we report on the site‐directed mutagenesis of Tyr194 and of the oxyanion hole Asp309 and the consequences of such mutations on the cellular expression and enzyme activity of PC2. The data show that the Y194D mutation markedly increases the ex vivo ability of PC2 to process proopiomelanocortin (POMC) into β‐endorphin in cells devoid of 7B2, e.g. BSC40 cells. In these cells, expression of native PC2 does not result in the secretion of measurable in vitro activity against a pentapeptide fluorogenic substrate. In contrast, secreted Y194D‐PC2 exhibited significant enzymatic activity, even in the absence of 7B2. Based on co‐immunoprecipitations and Western blots, binding assays indicate that Tyr194 participates in the interaction of PC2 with 7B2, and that the oxyanion hole Asp309 is critical for the binding of proPC2 with pro7B2.

Calcium-activated potassium channels in the UCR-SE-la lepidopteran cell line from the beet armyworm (Spodoptera exigua)

Abstract Ion channels in the UCR-SE-1a cell line from the beet armyworm ( Spodoptera exigua ) were studied using the patch-clamp technique in the cell-attached, the inside-out and the outside-out configurations. Contrarily to SF-9 cells from the fall armyworm ( Spodoptera frugiperda ) which did not display ion channel activity, UCR-SE-1a cells possess 120-pS (in the cell-attached configuration), voltage-dependent potassium channels that are activated in the inside-out configuration by calcium exposure of the cytoplasmic side of the membrane. In the cell-attached configuration, similar channel activation takes place in the presence of the calcium ionophore A23187. The channels are blocked by tetraethylammonium and cobalt ions but are insensitive to the scorpion venom charybdotoxin. They do not appear to belong to any of the well-characterized calcium-activated potassium channels found in mammalian cells. Identical channel activity was observed when UCR-SE-1a cells were exposed to Bacillus thuringiensis CryIC toxin, suggesting that intracellular calcium rises as a result of toxin interaction with the cells. This effect was directly demonstrated by fluorescence videomicroscopy experiments showing that like SF-9 cells, UCR-SE-1a cells loaded with the calcium probe Fura 2 respond to CryIC toxin exposure by an intracellular calcium surge.

Structure-function studies on the biosynthesis and bioactivity of the precursor convertase PC2 and the formation of the PC2/7B2 complex

Site directed mutagenesis of the prohormone convertase PC2 was used to define the effect of certain residues on the zymogen activation of proPC2 and on its binding to the neuroendocrine protein 7B2. These included the oxyanion hole Asp309 (D309N), the N‐terminal Glu25 (E25Q and E25K) of proPC2 and the Asp519 (D519E) of the RGD motif within the P‐domain of PC2. Heterologous vaccinia virus expression of the wild type and mutant PC2s in endocrine pituitary cells such as AtT20 and GH3 cells demonstrated that the most dramatic effect was observed with the D309N mutant which no longer bound pro7B2 and which exhibited a significant reduction in its capacity to produce β‐endorphin from pro‐opiomelanocortin (POMC).