Tetrabasic Site Research Articles

Acquisition of a novel eleven amino acid insertion directly N-terminal to a tetrabasic cleavage site confers intracellular cleavage of an H7N7 influenza virus hemagglutinin

A critical feature of highly pathogenic avian influenza viruses (H5N1 and H7N7) is the efficient intracellular cleavage of the hemagglutinin (HA) protein. H7N7 viruses also exist in equine species, and a unique feature of the equine H7N7 HA is the presence of an eleven amino acid insertion directly N-terminal to a tetrabasic cleavage site. Here, we show that three histidine residues within the unique insertion of the equine H7N7 HA are essential for intracellular cleavage. An asparagine residue within the insertion-derived glycosylation site was also found to be essential for intracellular cleavage. The presence of the histidine residues also appear to be involved in triggering fusion, since mutation of the histidine residues resulted in a destabilizing effect. Importantly, the addition of a tetrabasic site and the eleven amino acid insertion conferred efficient intracellular cleavage to the HA of an H7N3 low pathogenicity avian influenza virus. Our studies show that acquisition of the eleven amino acid insertion offers an alternative mechanism for intracellular cleavage of influenza HA.

Endoproteolysis at Tetrabasic Amino Acid Sites in Procalcitonin Gene-Related Peptide by Pituitary Cell Lines

Rosenblatt, M. I. and I. M. Dickerson. Endoproteolysis at tetrabasic amino acid sites in procalcitonin gene-related peptide by pituitary cell lines. Peptides 18(4) 567–576, 1997.—The specificity of neuroendocrine prohormone convertases for tetrabasic amino acid sites was investigated. Mutations were introduced into the tetrabasic cleavage site of the procalcitonin gene-related peptide (proCGRP) cDNA and these mutated cDNA's were expressed in AtT-20 cells which predominantly express the endoprotease prohormone convertase-1 (PC1/3), and in GH3 cells which predominantly express prohormone convertase-2 (PC2). Mutations were introduced into the proCGRP cDNA which converted the wild-type ArgArgArgArg site to LysLysArgArg and ArgArgLysLys, and the proCGRP variants were stably transfected into AtT-20 and GH3 cells. ProCGRP containing each of the LysLysArgArg permutations were efficiently cleaved in both AtT-20 and GH3 cells. Cleavage of LysLysArgArg in exogenous proCGRP, but not in endogenous POMC, suggests that the specificity of cleavage at tetrabasic sites is not defined solely by the endoproteases expressed by the cell or by the amino acid sequence at the cleavage site, but is also dependent on the structure of the propeptide.

Post-translational processing of the alternative neuropeptide precursor encoded by the FMRFamide gene in the pulmonate snail Lymnaea stagnalis.

The neuropeptide gene encoding FMRFamide-like peptides in the pulmonate mollusc Lymnaea is subject to alternative splicing that generates cell-specific expression of distinct sets of peptides in the CNS. In this paper, we analyse the post-translational processing of the alternative protein precursor encoded by the exon I, III-V transcript (type 2 transcript). We raised anti-peptide antisera specific to distinct segments of the precursor in order to address the pattern of endoproteolytic cleavages, specifically around the tetrabasic site RRKR. We first showed that not all peptides predicted by the precursor structure are generated as final steady-state products. We then identified a novel peptide by biochemical purification, amino acid sequencing and mass spectrometry- the 35 amino acid SDPFFRFGKQQVATDDSGELDDEILSRVSDDDKNI, which we termed the acidic peptide, previously not predicted on the basis of the precursor structure. This novel peptide, abundant in the snail brain (0.7 pmol per central nervous system), includes the N-terminal sequence SDPFFRF, which was previously considered to be a variant of the known heptapeptide SDPFLRFamide, also encoded within the same protein precursor. We showed by in situ hybridization and immunocytochemistry that the acidic peptide is produced in all cells that transcribe type 2 FMRFamide mRNA. We mapped the expression of this novel peptide in the CNS and localized it mainly in three identifiable neuronal clusters - the E, F and B groups of cells - and some additional neurons, all situated in three of the eleven central ganglia. Immunoreactive neurons included the single identifiable visceral white interneuron (VWI or VD4), a key cell of the cardiorespiratory network.

Specificity and Kinetic Studies on the Cleavage of Various Prohormone Mono- and Paired-basic Residue Sites by Yeast Aspartic Protease 3

The specificity and relative efficiency of cleavage of mono- and paired-basic residue processing sites by YAP3p was determined in vitro for a number of prohormone substrates: human ACTH1 39, bovine proinsulin, porcine cholecystokinin 33, cholecystokinin (CCK) 13-33, dynorphin A(1-11), dynorphin B(1-13), and amidorphin. YAP3p generated ACTH1-15 from ACTH1-39. It cleaved proinsulin at the paired-basic residue sites of the B-C junction as well as the C-A junction. Leu-enkephalin-Arg and Leu-enkephalin-Arg-Arg were generated from dynorphin A and dynorphin B, respectively. YAP3p generated Met-enkephalin-Lys-Lys from amidorphin showing that cleavage by this enzyme can occur at a lone pair of Lys residues. CCK33 was cleaved at Lys23 and Arg9, each containing an upstream Arg residue at the P6 and P5 position, respectively. Km values were between 10(-4) and 10(-5) M for the various substrates, with the highest affinity exhibited for the tetrabasic site of ACTH1-39 (1.8 x 10(-5) M). The tetrabasic residue site of ACTH1-39 was cleaved with the highest relative efficiency (kcat/Km = 3.1 x 10(6) m-1 s-1), while that of the monobasic site of CCK13-33 and the paired-basic site of proinsulin B-C junction, were cleaved less efficiently at 4.2 x 10(4) m-1 s-1 and 1.6 x 10(4) m-1 s-1, respectively.

In Vitro Processing of Anthrax Toxin Protective Antigen by Recombinant PC1(SPC3) and Bovine Intermediate Lobe Secretory Vesicle Membranes

Protective antigen (PA), an 83-kDa protein produced by Bacillus anthracis, requires proteolytic activation at a tetrabasic site (RKKR167) before it can combine with either edema factor or lethal factor on the cell surface. The complex is then endocytosed and the target cell intoxicated, Previous work has demonstrated that furin, a ubiquitously distributed, subtilisin-like protease, can perform this cleavage. In this study, another member of the furin family, PC1 (SPC3), was tested as a putative processing enzyme for PA, Recombinant PC1, partially purified from the medium of stably transfected L-cells, cleaved PA to a 63-kDa fragment (PA63) and a 20-kDa fragment (PA20). Amino-terminal sequence analysis of the 63 kDa product demonstrated that cleavage occurred between Arg167 and Ser168. The pH optimum for in vitro PA cleavage was 6.0 and the enzymatic activity was calcium-dependent. Medium from untransfected L-cells did not cleave PA. Site-directed mutagenesis of the tetrabasic cleavage site revealed that PC1 preferred to cleave sequences containing basic residues at positions −1 and −4 relative to the wild-type cleavage site, demonstrating that PC1 can cleave substrates at a monobasic residue site in vitro. Substrates having basic residues at the −1 and −2 positions were cleaved with approximately twofold less efficiency than wild-type PA. Mutants of PA containing basic residues in positions −1 and either −2 or −4 of the cleavage site were predicted to be substrates for PC1 and were more toxic to L-cells expressing PC1 than to untransfected L-cells. These results demonstrate that PA is cleaved by PC1 in vivo. Membranes from bovine intermediate lobe secretory vesicles which contain both prohormone convertases, PC1 and PC2, also cleaved PA to PA63 with a pH optimum of 5.5. Immunodepletion studies using antisera against PC1 and PC2 showed that these are the enzymes primarily responsible for the cleavage of PA in the membrane preparation. Thus, both recombinant PC1 and a membrane preparation containing endogenous PC1 can activate PA.

Expression of mutant ELH prohormones in AtT-20 cells: the relationship between prohormone processing and sorting.

Posttranslational processing of many proteins is essential to the synthesis of fully functional molecules. The ELH (egg-laying hormone) prohormone is cleaved by endoproteases in a specific order at a variety of basic residue processing sites to produce mature peptides. The prohormone is first cleaved at a unique tetrabasic site liberating two intermediates (amino and carboxy) which are sorted to different classes of dense core vesicles in the bag cell neurons of Aplysia. When expressed in AtT-20 cells, the ELH prohormone is also first cleaved at the tetrabasic site. The amino-terminal intermediate is then sorted to the constitutive pathway, and a portion of the carboxy-terminal intermediate is sorted to the regulated pathway. Here, we use mutant constructs of the ELH prohormone expressed in AtT-20 cells to examine the relationship between prohormone processing and consequent sorting. Prohormone which has a dibasic site in place of the tetrabasic site is processed and sorted similarly to wild type. Furthermore, mutant prohormone which lacks the tetrabasic site is processed at an alternative site comprising three basic residues. In these mutant prohormones, mature ELH is still produced and stored in dense core vesicles while amino-terminal products are constitutively secreted. However, deletion of the tetrabasic and tribasic sites results in the rerouting of the amino-terminal intermediate products from the constitutive pathway to the regulated secretory pathway. Thus, in the ELH prohormone, the location of the proteolytic processing events within the secretory pathway and the order of cleavages regulate the sorting of peptide products.

Molluscan Subtilisin-Related Endoproteases Involved in the Activation of Prohormones and Proproteins in the Secretory Pathway

Neuropeptides and pcptide hormones are synthesized as part of larger precursor proteins that are processed post-translationally by subtilisin-related prohormone convertases (PCs), frequently at multiple basic sites, to generate biologically active peptides. The neuroendocrine bag cells and exocrine atrial gland cells of Aplysia californica are model peptidergic cells that provide a unique opportunity to examine the processing and differential sorting of a family of egg-laying hormone (ELH)-related polyprotein precursors. Bag cell neurons synthesize and initially cleave the ELH prohormone at a unique tetrabasic site to generate NH2-terminal and COOH-terminal intermediates that are sorted to distinct vesicle classes in the trans-Golgi. The processing intermediates subsequently undergo an ordered series of cleavage steps to generate multiple products, including ELH. The atrial gland synthesizes and post-translationally processes large quantities of four ELH-related precursors to liberate multiple products, including ELH-related peptides. Four members of a family of Aplysia subtilisin-related endoproteases that are expressed in bag cell neurons or atrial gland cells have been cloned, and two of these enzymes are capable of correctly cleaving the ELH prohormone. The predicted primary structures of the preproendoproteases suggest that they undergo protcolytic cleavage at the consensus sequence Arg-Xaa-(Lys/Arg)-Arg prior to enzyme activation.