Identification of the RT-RH/IN cleavage site of HTLV-I

Human T-cell leukemia virus type 1 (HTLV-1) is a type C human retrovirus and is the causative agent of adult T-cell leukemia and other diseases. The enzymatic and structural proteins of HTLV-I are synthesized as part of a Gag-Pro-Pol precursor polyprotein, and the mature proteins are released by proteolytic processing catalyzed by HTLV-I protease. The locations of most of the proteolytic cleavage sites are known, however, the site that creates the N-terminus of HTLV-1 integrase has not been previously identified. A 15 residue peptide corresponding to junction of the C-terminus of RNaseH and N-terminus of integrase (DALLITPVLQLSPAF-OH) was incubated with HTLV-1 protease. Analysis of the cleavage products by LC-MS revealed fragments Ac-DALLITPVLQL-OH and H(2)N-SPAF-OH were produced, indicating cleavage between the leucine and serine. This is the first physical identification of the N-terminal amino acid sequence of the integrase of HTLV-1.     

Cleavage of cartilage proteoglycan between G1 and G2 domains by stromelysins

Normal and pathological turnover of proteoglycans in articular cartilage involves its cleavage close to the N-terminal G1 domain responsible for aggregation. A fragment containing G1 and G2 N-terminal domains of pig cartilage proteoglycans was therefore used as a substrate to investigate its degradation by the metalloproteinase stromelysin and related recombinant stromelysin enzymes. The stromelysins produced an apparent single cleavage yielding a G1 fragment of 56 kDa and a G2 fragment of 110 kDa. Rabbit bone stromelysin was much more active against the G1-G2 fragment and against proteoglycan aggregates than recombinant human stromelysin-1 and stromelysin-2. All metalloproteinase preparations were active against proteoglycan and the G1-G2 fragment at acid (pH 5.5) and neutral pH (7.4). N-terminal sequencing of the G2 fragment derived from the action of recombinant human stromelysin-1 revealed that cleavage between G1 and G2 occurred at the N-terminal end of the interglobular domain, close to the last cysteine in G1. The specific cleavage site was between an asparagine and a pair of phenylalanine residues, where the asparagine corresponds to residue 341 in human and rat mature core protein sequence.     

Proteolytic processing of the Ada protein that repairs DNA O6-methylguanine residues in E. coli

In extracts of E. coli treated with an adapting regime of MNNG, the induced 39kd Ada protein having O6-MeG-DNA methyltransferase activity is processed to a 19kd active domain corresponding to the C-terminal half of the intact protein. This proteolytic processing has been followed on Western immunoblots using antisera raised against the 19kd fragment. Initial processing at 25 degrees C or 37 degrees C mainly generates a fragment of mol. wt. 24kd which then undergoes a slower second cleavage to generate the 19kd active domain. Preceding this second cleavage site is a sequence of amino acids Thr- -Gly-Met-Thr- -Lys that also occurs at another site in the N-terminal half of the 39kd methyltransferase. It is proposed that this sequence is a recognition site for proteolytic activity. On the basis of cleavage of the Ada protein at either one or both of these sites, fragments may be generated of mol. wt. 24kd and 19kd containing the active site for O6-methylguanine and O4-methylthymine repair, and 15kd and 20kd, containing the active site for methylphosphotriester repair. These observations explain previous reports by others on the existence in cell extracts of multiple methyltransferase activities of different sizes recognizing O-methyl lesions in DNA. The cellular protease involved is resistant to a wide range of protease inhibitors.     

Specific cleavage sites of Nef proteins from human immunodeficiency virus types 1 and 2 for the viral proteases.

Human immunodeficiency virus type 2 (HIV-2) Nef is proteolytically cleaved by the HIV-2-encoded protease. The proteolysis is not influenced by the absence or presence of the N-terminal myristoylation. The main cleavage site is located between residues 39 and 40, suggesting a protease recognition sequence, GGEY-SQFQ. As observed previously for Nef protein from HIV-1, a large, stable core domain with an apparent molecular mass of 30 kDa is produced by the proteolytic activity. Cleavage of Nef from HIV-1 in two domains by its own protease or the protease from HIV-2 is also independent of Nef myristoylation. However, processing of HIV-1 Nef by the HIV-2 protease is less selective than that by the HIV-1 protease: the obtained core fragment is heterogeneous at its N terminus and has an additional cleavage site between amino acids 99 and 100. Preliminary experiments suggest that the full-length Nef of HIV-2 and the core domain are part of the HIV-2 particles, analogous to the situation reported recently for HIV-1.     

Maturation of the activities of recombinant mite allergens Der p 1 and Der f 1, and its implication in the blockade of proteolytic activity

Recombinant pro-Der p 1 expressed in yeast Pichia pastoris was convertible into the prosequence-removed mature Der p 1 with full activities of cysteine protease and IgE-binding with or without N-glycosylation of the mature sequence as well as pro-Der f 1. The active recombinant variants will be the basis for various future studies. The major N-terminus of pro-Der p 1 with low proteolytic activity was the putative signal-cleavage site, while that of pro-Der f 1 contained not only the equivalent site but also 21 residues downstream, and pro-Der f 1 retained significant activity. Contribution of the N-terminal region of the Der p 1 prosequence including an N-glycosylation motif on effective inhibition of proteolytic activity of pro-Der p 1 was suggested.     

Caspase-mediated parkin cleavage in apoptotic cell death

The parkin protein is important for the survival of the neurons that degenerate in Parkinson's disease as demonstrated by disease-causing lesions in the parkin gene. The Chinese hamster ovary and the SH-SY5Y cell line stably expressing recombinant human parkin combined with epitope-specific parkin antibodies were used to investigate the proteolytic processing of human parkin during apoptosis by immunoblotting. Parkin is cleaved during apoptosis induced by okadaic acid, staurosporine, and camptothecin, thereby generating a 38-kDa C-terminal fragment and a 12-kDa N-terminal fragment. The cleavage was not significantly affected by the disease-causing mutations K161N, G328E, T415N, and G430D and the polymorphism R366W. Parkin and its 38-kDa proteolytic fragment is preferentially associated with vesicles, thereby indicating that cleavage is a membrane-associated event. The proteolysis is sensitive to inhibitors of caspases. The cleavage site was mapped by site-directed mutagenesis of potential aspartic residues and revealed that mutation of Asp-126 alone abrogated the parkin cleavage. The tetrapeptide aldehyde LHTD-CHO, representing the amino acid sequence N-terminal to the putative cleavage site was an efficient inhibitor of parkin cleavage. This suggests that parkin function is compromised in neuropathological states associated with an increased caspase activation, thereby further adding to the cellular stress.     

Secretion of egg envelope protein ZPC after C-terminal proteolytic processing in quail granulosa cells.

In avian species, an egg envelope homologous to the mammalian zona pellucida is called the perivitelline membrane. We have previously reported that one of its components, a glycoprotein homologous to mammalian ZPC, is synthesized in the granulosa cells of the quail ovary. In the present study, we investigated the proteolytic cleavage of the newly synthesized ZPC and the secretion of ZPC from the granulosa cells. Western blot analysis of the cell lysates demonstrated that the 43-kDa protein is the precursor of mature ZPC (proZPC), and is converted to the 35-kDa protein before secretion. The accumulation of proZPC in the presence of brefeldin A, and conversion of proZPC to ZPC in the presence of monensin, indicate the possibility that the proteolytic processing of ZPC occurs in the Golgi apparatus. An analysis of amino-acid sequence identified that the C terminus of mature ZPC protein is Phe360, and the N-terminal amino-acid sequence of the proZPC-derived fragment was determined as Asp363. These results suggest that newly synthesized ZPC is cleaved at the consensus furin cleavage site, and the resulting two basic residues at the C terminus are subsequently trimmed off to generate mature ZPC prior to secretion.     

Synthetic HIV-2 protease cleaves the GAG precursor of HIV-1 with the same specificity as HIV-1 protease

A 99-amino acid protein having the deduced sequence of the protease from human immunodeficiency virus type 2 (HIV-2) was synthesized by the solid phase method and tested for specificity. The folded peptide catalyzes specific processing of a recombinant 43-kDa GAG precursor protein (F-16) of HIV-1. Although the protease of HIV-2 shares only 48% amino acid identity with that of HIV-1, the HIV-2 enzyme exhibits the same specificity toward the HIV-1 GAG precursor. Fragments of 34, 32, 24, 10, and 9 kDa were generated from F-16 GAG incubated with the protease. N-terminal amino acid sequence analysis of proteolytic fragments indicate that cleavage sites recognized by HIV-2 protease are identical to those of HIV-1 protease. The verified cleavage sites in F-16 GAG appear to be processed independently, as indicated by the formation of the intermediate fragments P32 and P34 in nearly equal ratios. The site nearest the amino terminus is quite conserved between the two viral GAG proteins (...VSQNY-PIVQN...in HIV-1,...KGGNY-PVQHV...in HIV-2). In contrast, the putative second site (...IPFAA-AQQKG...) of HIV-2 GAG shares minimal sequence identity with site 2 of HIV-1 GAG (...SATIM-MQRGN...). These sequence variations in the substrates suggest higher order structural features that may influence recognition by the proteases. Pepstatin A inhibits HIV-2 protease, whereas 1,10-phenanthroline and phenylmethylsulfonylfluoride do not; these results are in agreement with the finding that proteases of HIV and other retroviruses are aspartyl proteases.     

C-terminal residues of mature human T-lymphotropic virus type 1 protease are critical for dimerization and catalytic activity

HTLV-1 [HTLV (human T-cell lymphotrophic virus) type 1] is associated with a number of human diseases. HTLV-1 protease is essential for virus replication, and similarly to HIV-1 protease, it is a potential target for chemotherapy. The primary sequence of HTLV-1 protease is substantially longer compared with that of HIV-1 protease, and the role of the ten C-terminal residues is controversial. We have expressed C-terminally-truncated forms of HTLV-1 protease with and without N-terminal His tags. Removal of five of the C-terminal residues caused a 4-40-fold decrease in specificity constants, whereas the removal of an additional five C-terminal residues rendered the protease completely inactive. The addition of the N-terminal His tag dramatically decreased the activity of HTLV-1 protease forms. Pull-down experiments carried out with His-tagged forms, gel-filtration experiments and dimerization assays provided the first unequivocal experimental results for the role of the C-terminal residues in dimerization of the enzyme. There is a hydrophobic tunnel on the surface of HTLV-1 protease close to the C-terminal ends that is absent in the HIV-1 protease. This hydrophobic tunnel can accommodate the extra C-terminal residues of HTLV-1 protease, which was predicted to stabilize the dimer of the full-length enzyme and provides an alternative target site for protease inhibition.     

Molecular cloning of pertussis toxin genes.

We have cloned a 4.5 kb EcoRI/BamHI DNA fragment from Bordetella pertussis which contains at least two genes responsible for expression of pertussis toxin. The S4 subunit of the toxin was isolated by high pressure liquid chromatography and the NH2-terminal amino acid sequence determined. Using a mixed synthetic oligonucleotide probe designed by reverse translation of a portion of the protein sequence, a cloned DNA fragment was identified which contains the coding information for at least the S4 structural subunit of the toxin. Sequence analyses indicate that the mature protein is derived by proteolytic cleavage of a precursor molecule. Southern blot analyses of Tn5-induced B. pertussis toxin-deficient mutants show that the Tn5 DNA is inserted 1.3 kb downstream from the S4 subunit gene. This second gene could code for another subunit required for assembly of the mature toxin or a non-structural transport protein, possibly in the same polycistronic operon. The molecular cloning of pertussis toxin genes provides the basis for development of a safer recombinant "new generation" vaccine for whooping cough.     

Botulinum neurotoxin type A: sequence of amino acids at the N-terminus and around the nicking site

Clostridium botulinum synthesizes the type A botulinum neurotoxin (NT) as a approximately 150 kDa single chain protein. Post-translational proteolytic processing yields a approximately 150 kDa dichain protein composed of a approximately 50 kDa light and approximately 100 kDa heavy chain, which has higher toxicity. Trypsin's action mimics the endogenous proteolytic processing. The proteolytic cleavages could occur at 4 sites. We have examined 2 such sites and defined the peptide sequences before and after proteolytic processing. The N-terminal residues of the newly synthesized approximately 150 kDa single chain NT, Pro-Phe-Val-Asn-Lys-, remain intact at the N-terminus of the approximately 50 kDa light chain generated either in the clostridial culture or in vitro with trypsin or with a protease purified from the homologous bacterial culture. The clostridial protease cleaves the single chain NT in vitro, at 1/3 the distance from its N-terminus, on the amino side of Gly of the sequence -Gly-Tyr-Asn-Lys-Ala-Leu-Asn-Asp-Leu- before cleaving the bond Lys-Ala at a slower rate. The data indicate that the dichain NT is formed in the bacterial culture in at least 2 steps. Cleavage at X-Gly produces a approximately 100 kDa heavy chain-like fragment which is then truncated; cleavage 4 residues downstream at Lys-Ala, and excision of the tetrapeptide Gly-Tyr-Asn-Lys, generates the mature heavy chain with Ala as its N-terminal residue. The approximately 100 kDa heavy chain generated in vitro, by nicking the single chain NT with trypsin, also has Ala-Leu-Asn- as the N-terminal residues.