Cleavage Point Research Articles

Use of Edman degradation sequence analysis and matrix-assisted laser desorption/ionization mass spectrometry in designing substrates for matrix metalloproteinases

The matrix metalloproteinase (MMP) family has been implicated in the process of a variety of diseases such as arthritis, atherosclerosis, and tumor cell metastasis. We have been designing single-stranded peptides (SSPs) and triple-helical peptides (THPs) as potential discriminatory MMP substrates. Edman degradation sequence and matrix-assisted laser desorption/ionization mass spectrometric (MALDI-MS) analyses of proteolytic activity have been utilized to aid in further substrate design. THP models of the α1(I)772–786 sequence from type I collagen were synthesized to examine the triple-helical substrate specificity of MMP family members. Sequence and MALDI-MS analyses were used in conjunction with a fluorometric assay to determine the exact point of cleavage by each MMP. MMP-1 (interstitial collagenase) cleaved the substrates at a single Gly–Ile bond, analogous to the cleavage site in type I collagen. MMP-2 ( M r 72 000 type IV collagenase; gelatinase A) was found to cleave the substrates at two sites, a Gly–Ile bond and a Gly–Gln bond. MMP-3 (stromelysin 1) was found to cleave only one of the substrates after reaction for 48 h. Ultimately, sequence and MALDI-MS analyses allowed us to detect an additional cleavage site for MMP-2 in comparison to MMP-1, while MMP-3 was found to cleave a substrate after an extended time period. The second cleavage site would cause the kinetic parameters for MMP-2 to be overestimated by the fluorometric assay. Further design variations for these substrates need to consider the presence of more stable triple-helical conformation (to eliminate MMP-3 binding) and the removal of Gly–Gln bonds that may be susceptible to MMP-2.

Methods for the Prediction of Cleavage Sites of the Polypeptide in Certain Proteins

Many proteins critical to the existence of parasites from infectious agents that affect man, including malaria, leishmania, and trypansoma, are attached to the surface membrane by glycosyl-phosphatidyl inositol (GPI) anchors. Creation of GPI-anchored proteins involves elimination of a bond between adjacent amino acids in a newly formed, or nascent, polypeptide and attachment of a GPI anchor at this site. Because interfering with this process by altering the location of attachment is a potential infection-control strategy in humans, knowledge of the cleavage site in the nascent polypeptide is important. Determination of the cleavage point directly through biochemical analysis is both labor and resource intensive. We develop both likelihood-based and Bayesian approaches for use in the identification of the site of cleavage of GPI-anchored proteins. These methods are demonstrated using four examples where extensive biochemical analyses already have identified the bond in the newly formed polypeptide where a GPI anchor is attached. In contrast to sole reliance on biochemical analyses, use of the proposed methods has great potential for savings of both time and money.

The CafA Protein Required for the 5′-Maturation of 16 S rRNA Is a 5′-End-dependent Ribonuclease That Has Context-dependent Broad Sequence Specificity

The CafA protein, which was initially described as having a role in either Escherichia coli cell division or chromosomal segregation, has recently been shown to be required for the maturation of the 5'-end of 16 S rRNA. The sequence of CafA is similar to that of the N-terminal ribonucleolytic half of RNase E, an essential E. coli enzyme that has a central role in the processing of rRNA and the decay of mRNA and RNAI, the antisense regulator of ColE1-type plasmids. We show here that a highly purified preparation of CafA is sufficient in vitro for RNA cutting. We detected CafA cleavage of RNAI and a structured region from the 5'-untranslated region of ompA mRNA within segments cleavable by RNaseE, but not CafA cleavage of 9 S RNA at its "a" RNase E site. The latter is consistent with the finding that the generation of 5 S rRNA from its 9 S precursor can be blocked by inactivation of RNase E in cells that are wild type for CafA. Interestingly, however, a decanucleotide corresponding in sequence to the a site of 9 S RNA was cut efficiently indicating that cleavage by CafA is regulated by the context of sites within structured RNAs. Consistent with this notion is our finding that although 23 S rRNA is stable in vivo, a segment from this RNA is cut efficient by CafA at multiple sites in vitro. We also show that, like RNase E cleavage, the efficiency of cleavage by CafA is dependent on the presence of a monophosphate group on the 5'-end of the RNA. This finding raises the possibility that the context dependence of cleavage by CafA may be due at least in part to the separation of a cleavable sequence from the 5'-end of an RNA. Comparison of the sites surrounding points of CafA cleavage suggests that this enzyme has broad sequence specificity. Together with the knowledge that CafA can cut RNAI and ompA mRNA in vitro within segments whose cleavage in vivo initiates the decay of these RNAs, this finding suggests that CafA may contribute at some point during the decay of many RNAs in E. coli.

Preparation of oligomeric beta-glycosides from cellulose and hemicellulosic polysaccharides via the glycosyl transferase activity of a trichoderma reesei cellulase.

Oligoglycosyl (allyl, 2,3-dihydroxypropyl, ethyl, 2-hydroxyethyl, and methyl) beta-glycosides were generated by endo -transglycosylation reactions catalyzed by commercially available Trichoderma reesei cellulase. A polymeric donor substrate (xyloglucan or cellulose) was incubated with the enzyme in an aqueous solution containing 20% of the acceptor alcohol (allyl alcohol, glycerol, ethanol, ethylene glycol, and methanol, respectively). The products of these reactions included oligomeric alkyl beta-glycosides and reducing oligosaccharides. The high yield of alkyl beta-glycosides may be explained by the resistance of the xyloglucan beta-glycosides to cellulase-mediated hydrolysis. The resistance of the oligoxyloglucan beta-glycosides to endo glucanase catalyzed hydrolysis supports the hypothesis that productive binding of the glycan substrate depends on its interaction with enzyme subsites on both sides of the cleavage point, leading to distortion of the ring geometry of the residue whose glycosidic bond is cleaved. Oligoxyloglucan beta-glycosides were purified by a combination of gel-permeation and reversed-phase HPLC and were structurally characterized by MS and NMR spectroscopy. These results demonstrate that novel oligosaccharide beta-glycosides can be efficiently produced by enzyme-catalyzed fragmentation/transglycosylation reactions starting with a polysaccharide donor substrate. This class of reactions may represent a convenient source of beta-glycosides to be used as synthons for the rapid synthesis of complex glycans.

Inhibition of Hepatitis C Virus NS2/3 Processing by NS4A Peptides: IMPLICATIONS FOR CONTROL OF VIRAL PROCESSING

The NS2/3 protease of hepatitis C virus is responsible for a single cleavage in the viral polyprotein between the nonstructural proteins NS2 and NS3. The minimal protein region necessary to catalyze this cleavage includes most of NS2 and the N-terminal one-third of NS3. Autocleavage reactions using NS2/3 protein translated in vitro are used here to investigate the inhibitory potential of peptides likely to affect the reaction. Peptides representing the cleaved sequence have no effect upon reaction rates, and the reaction rate is insensitive to dilution. Both results are consistent with prior suggestions that the NS2/3 cleavage is an intramolecular reaction. Surprisingly, peptides containing the 12-amino acid region of NS4A responsible for binding to NS3 inhibit the NS2/3 reaction with K(i) values as low as 3 microM. Unrelated peptide sequences of similar composition are not inhibitory, and neither are peptides containing incomplete segments of the NS4A region that binds to NS3. Inhibition of NS2/3 by NS4A peptides can be rationalized from the organizing effect of NS4A on the N terminus of NS3 (the NS2/3 cleavage point) as suggested by the known three-dimensional structure of the NS3 protease domain (Yan, Y., Li, Y., Munshi, S., Sardana, V., Cole, J. L., Sardana, M., Steinkuhler, C., Tomei, L., De Francesco, R., Kuo, L. C., and Chen, Z. (1998) Protein Sci. 7, 837-847). These findings may imply a sequential order to proteolytic maturation events in hepatitis C virus.

Transcriptional control of development, protein synthesis, and heat-induced heat shock protein 70 synthesis in 2-cell bovine embryos.

Experiments were performed to evaluate the role of transcription in early development of bovine embryos. Two transcription inhibitors-5, 6-dichloro-1-beta-D-ribofuranosylbenzimidazole (DRB) and actinomycin D-were used to test whether 1) the inhibitors alter the rate of early embryonic development and protein synthesis, 2) heat shock increases the steady-state amounts of mRNA for the inducible form of heat shock protein 70 (HSP70) in embryos, and 3) this latter effect is blocked by transcription inhibitors. Addition of either DRB or actinomycin D to culture medium beginning 8 h postinsemination (hpi) reduced the proportion of oocytes that had undergone cleavage by 32-34 hpi. Both transcription inhibitors also reduced the proportion of cleaved embryos that reached the 4-cell stage by 32-34 hpi. Incorporation of (35)S-labeled amino acids into de novo synthesized protein by bovine 2-cell embryos was lower for embryos cultured with DRB. Using reverse transcription-polymerase chain reaction, HSP70 mRNA in 2- and 4-cell embryos was increased by exposure to 42 degrees C. Both inhibitors reduced amounts of HSP70 mRNA at 42 degrees C. Results indicate that bovine embryos can undergo transcription in response to heat shock as early as the 2-cell stage. Moreover, the observations that transcription inhibitors reduce rates of cleavage and early development point out the importance of transcription for development from the earliest period of embryonic life.

Cytosine Arabinoside Lesions Are Position-specific Topoisomerase II Poisons and Stimulate DNA Cleavage Mediated by the Human Type II Enzymes

Cytosine arabinoside (araC) is an important drug used for the treatment of human leukemias. In order to exert its cytotoxic effects, araC must be incorporated into chromosomal DNA. Although specific DNA lesions that involve base loss or modification stimulate nucleic acid cleavage mediated by type II topoisomerases, the effects of deoxyribose sugar ring modification on enzyme activity have not been examined. Therefore, the effects of incorporated araC residues on the DNA cleavage/religation equilibrium of human topoisomerase IIalpha and beta were characterized. AraC lesions were position-specific topoisomerase II poisons and stimulated DNA scission mediated by both human type II enzymes. However, the positional specificity of araC residues differed from that previously reported for other cleavage-enhancing DNA lesions. Finally, additive or synergistic increases in DNA cleavage were observed in the presence of araC lesions and etoposide. These findings broaden the range of DNA lesions known to alter topoisomerase II function and raise the possibility that this enzyme may mediate some of the cellular effects of araC.

Effect of flap modifications on human FEN1 cleavage.

The flap endonuclease, FEN1, plays a critical role in DNA replication and repair. Human FEN1 exhibits both a 5' to 3' exonucleolytic and a structure-specific endonucleolytic activity. On primer-template substrates containing an unannealed 5'-tail, or flap structure, FEN1 employs a unique mechanism to cleave at the point of annealing, releasing the 5'-tail intact. FEN1 appears to track along the full length of the flap from the 5'-end to the point of cleavage. Substrates containing structural modifications to the flap have been used to explore the mechanism of tracking. To determine whether the nuclease must recognize a succession of nucleotides on the flap, chemical linkers were used to replace an interior nucleotide. The nuclease could readily traverse this site. The footprint of the nuclease at the time of cleavage does not extend beyond 25 nucleotides on the flap. Eleven-nucleotide branches attached to the flap beyond the footprinted region do not prevent cleavage. Single- or double-thymine dimers also allow cleavage. cis-Platinum adducts outside the protected region are moderately inhibitory. Platinum-modified branch structures are completely inert to cleavage. These results show that some flap modifications can prevent or inhibit tracking, but the tracking mechanism tolerates a variety of flap modifications. FEN1 has a flexible loop structure through which the flap has been proposed to thread. However, efficient cleavage of branched structures is inconsistent with threading the flap through a hole in the protein.

Steroid-induced conformational changes of rat glucocorticoid receptor cause altered trypsin cleavage of the putative helix 6 in the ligand binding domain

Steroid-induced changes in receptor protein conformation constitute a logical means of translating the variations in steroid structures into the observed array of whole cell biological activities. One conformational change in the rat glucocorticoid receptor (GR) can be readily discerned by following the ability of trypsin digestion to afford a 16-kDa fragment. This fragment is seen after proteolysis of steroid-free receptors but disappears in digests of either glucocorticoid- or antiglucocorticoid-bound receptors. The location of this cleavage site has now been located unambiguously as R651, in helix 6 of the ligand binding domain, by a combination of point mutagenesis, arginine specific protease digestion, and radiochemical sequencing. This 16-kDa species, corresponding to amino acids 652–795, was non-covalently associated with another, ∼17-kDa species that was determined to be amino acids 518–651 after a comparison of co-immunoprecipitated fragments from wild type and two chimeric receptors. These assignments revise our earlier report of amino acids 537–673 being the 16-kDa fragment and suggest that sequences of the entire ligand binding domain are required for high affinity and specificity binding. This was supported by the observation that trypsin digestion of the steroid-free R651A mutant GR gave rise to the 30-kDa meroreceptor (amino acids 518–795), which displayed wild type affinity. This 30-kDa species is thus the smallest non-associated fragment of GR possessing wild type steroid binding affinity. This suggests that other GR regions do not influence steroid binding affinity. The above results are reminiscent of those observed for the estrogen receptor. However, unlike the estrogen receptor or the more closely related progesterone receptor, the precise proteolytic cleavage points of both the steroid-free and -bound GR fall within regions that are predicted, on the basis of X-ray crystal structures of related receptors, to be α-helical and resistant to proteolysis. Thus, the tertiary structure of the GR ligand binding domain may be distinctly different from that of estrogen and progesterone receptors.

Influenza Virus Inhibits Cleavage of the HSP70 Pre-mRNAs at the Polyadenylation Site

Influenza virus infection is known to shut off the expression of host genes. To study the mechanism, we examined the effects of influenza A/Udorn/72 virus infection on the heat induction of a major heat shock protein, HSP70, in Madin–Darby canine kidney cells. The induction of HSP70 protein synthesis was progressively suppressed with postinfection time when heat shock was applied. Northern hybridization analysis revealed the appearance of longer, heterogeneous HSP70 transcripts in the range of 2.7 to 30 kb with a concomitant decrease in the amount of the mature 2.7-kb mRNAs in the nucleus of the infected cells. Such longer β-actin transcripts were also observed but with much less intensity. The longer HSP70 transcripts contained the downstream sequence of the polyadenylation site, as demonstrated by RNase protection with an antisense RNA probe containing the sequence through the polyadenylation sites. This clearly proved that influenza virus infection inhibits the polyadenylation-site cleavage of the pre-mRNAs by the host cleavage and polyadenylation machinery. One temperature-sensitive mutant virus carrying a temperature-sensitive mutation on the NS1gene failed to inhibit the cleavage at the nonpermissive temperature, indicating that the NS1protein is involved in the inhibition of the pre-mRNA cleavage. This is the first report of the down-regulation of cellular mRNA maturation at the point of polyadenylation-site cleavage by virus infection and identifies a new mechanism by which the influenza virus shuts off host gene expression.

Active site specificity of plasmepsin II.

Members of the aspartic proteinase family of enzymes have very similar three-dimensional structures and catalytic mechanisms. Each, however, has unique substrate specificity. These distinctions arise from variations in amino acid residues that line the active site subsites and interact with the side chains of the amino acids of the peptides that bind to the active site. To understand the unique binding preferences of plasmepsin II, an enzyme of the aspartic proteinase class from the malaria parasite, Plasmodium falciparum, chromogenic octapeptides having systematic substitutions at various positions in the sequence were analyzed. This enabled the design of new, improved substrates for this enzyme (Lys-Pro-Ile-Leu-Phe*Nph-Ala/Glu-Leu-Lys, where * indicates the cleavage point). Additionally, the crystal structure of plasmepsin II was analyzed to explain the binding characteristics. Specific amino acids (Met13, Ser77, and Ile287) that were suspected of contributing to active site binding and specificity were chosen for site-directed mutagenesis experiments. The Met13Glu and Ile287Glu single mutants and the Met13Glu/Ile287Glu double mutant gain the ability to cleave substrates containing Lys residues.

Sequence analysis of the medium (M) segment of Cache Valley virus, with comparison to other Bunyaviridae.

The complete sequence of the medium (M) segment of Cache Valley virus (CVV), a human neuropathogen, has been determined using a series of overlapping cDNA clones. The viral complementary-sense RNA is comprised of 4463 nucleotides which encodes a polyprotein precursor of 1435 amino acids, starting at AUG at bases 49-51 to a UGA stop codon at bases 4351-4353. This polyprotein-encoding sequence is arranged as G2-NSm-G1. The base composition of the segment is 34.9% A, 17.0% C, 19.4% G and 28.7% U. Comparison of the nucleotide sequence to the prototype Bunyamwera virus sequence shows an identity of 63%, indicating several differences exist within the individual coding regions, most notably within the NSm and G1 coding regions. Based on two presumed cleavage points within the precursor, the G2 glycoprotein, encoded from nt 94-951, is 286 amino acids long, and has two sites of potential glycosylation. NSm, encoded from nt 952-1476, is 175 amino acids, while the largest glycoprotein, G1, encoded from nt 1477-4350, consists of 958 amino acids, and has five potential glycosylation sites, two of which appear to be unique to CVV. The subsequent study of these glycosylation sites and potential differences between the sequence of this prototype CVV strain and other geographic isolates may suggest the means for improving detection of human infections as well as mapping differences in neurovirulence, neuroinvasiveness and other aspects of pathogenicity.

Effect of oxidation of beta-amyloid precursor protein on its beta-secretase cleavage. A model study with synthetic peptides and candidate beta-secretases.

As the amyloidogenic processing of beta-amyloid precursor protein (betaAPP) proceeds under conditions of oxidative stress, the methionine-596 residue at the beta-secretase cleavage point is likely in an oxidized state. In the present work, possible consequences of the oxidation of Met-596 for the generation of the N-terminus of amyloid beta protein were modeled using synthetic peptide substrates, matching 587-606 sequence fragment of betaAPP and containing either intact methionine or methionine sulfoxide. Patterns and rates for the cleavage of these substrates by purified mast cell chymase, cathepsin G, cathepsin D, matrix metalloproteinase-3 and neutrophil elastase, were compared. Only the three first proteases, all previously suggested as candidate beta-secretases, preferentially cleaved the "intact" substrate after Met-596. For chymase and cathepsin G, the specificity of this cleavage increased upon a shift from optimal alkaline pH to acidic pH, which is also more compatible with the plausible intracellular localization of amyloidogenic betaAPP processing. The substitution of methionine sulfoxide for methionine in the substrate slowed down the cleavage rate for all the enzymes tested, by a factor of 6-15. This was associated with shifts of cleavage preferences to points of minor importance for the "intact" peptide, suggesting a specific resistance of the peptide bond after MetSO-596 against proteolysis.

Mutations in domain IIIα of the mu transposase: evidence suggesting an active site component which interacts with the Mu-Host junction

A series of point mutations was constructed in domain IIIα of the Mu A protein. The mutant transposases were purified and assayed for their ability to promote various aspects of the in vitro Mu DNA strand transfer reaction. All mutants with discernable phenotypes were inhibited in stable synapsis (Type 0 or Type 1 complex formation). In contrast, these mutant proteins were capable of LER formation (a transient early reaction intermediate in which the Mu left and right ends have been synapsed with the enhancer), at levels comparable to wild-type transposase. These proteins therefore comprise a novel class of transposase mutants, which are specifically inhibited in stable transpososome assembly. The defect in these proteins was also uniformly suppressed by either Mn2+, or the Mu B protein in the presence of ATP and target DNA. Striking phenotypic similarities were recognized between the domain IIIα transposase mutant characteristics noted above, and those for substrate mutants carrying a terminal base-pair substitution at the point of cleavage on the donor molecule. This phenotypic congruence suggests that the alterations in either protein or DNA are exerting an effect on the same step of the reaction i.e., engagement of the terminal nucleotide by the active site. We suggest that domain IIIα of the transposase comprises the substrate binding pocket of the active site which interacts with the Mu-host junction.

Chemical Cleavage of Heteroduplex DNA to Identify Mutations

Mutation screening by the chemical-cleavage method is based on the fact that mismatched cytosine (C) and thymidine (T) are more reactive with the compounds hydroxylamine and osmium tetroxide, respectively, than are Watson and Crick-paired cytosine and thymidine bases. In this protocol, an excess of unlabeled target DNA is hybridized with labeled wild-type DNA probe and heteroduplexes are formed. One aliquot is treated with hydroxylamine, which reacts with mismatched C bases. Another aliquot is treated with osmium tetroxide, which reacts with mismatched T bases. The reactions are mixed with piperidine; the strands are then cleaved at the sites where hydroxylamine and osmium tetroxide react. Cleaved fragments are then electrophoresed and sized on polyacrylamide gels, identifying the point of cleavage (and hence the position of the mutation). Then only a small portion of the mutant gene needs to be sequenced to define a single change between two DNA sequences.

A nuclease released from Colletotrichum coccodes is not a defence gene elicitor in pea tissue

An endo-DNase with single strand nicking activity from the bean pathogen, F. solani f. sp. phaseoli has been previously reported to induce the expression of defence genes in peas. Culture filtrates of the potato pathogen, Colletotrichum coccodes , also contain a single strand nicking endo-DNase (CCN). This nuclease has a molecular weight of ∼ 27 kDa, an isoelectric point of pH 7·8, optimal catalytic activity at pH 5–6 and is stable at temperatures below 50 °C. CCN activity increases are observed with addition of cation cofactors, favouring Mn 2+ > Ca 2+ > Ca 2+ > Na + > Zn 2+ . CCN is inhibited by aurintricarboxylic acid, a nuclease inhibitor. The point of CCN cleavage on the DNA molecule is between the 5′-phosphate and the 3′-hydroxyl. CCN also possesses RNase activity. Although the C. coccodes culture filtrate can induce the expression of disease resistance response genes and phytoalexin production in pea tisue, this inductive activity does not co-purify with the CCN DNase activity through the Sephacel G-75 purification step, and thus CCN applied singly does not serve as a signal for initiating the host defence reponses. Because CCN degrades both DNA and RNA, it is more probably a nutrient scavenging enzyme.

Some properties and action mode of [formula omitted] lyase from Enterobacter cloacae M-1

An intracellular alginate lyase was purified from Enterobacter cloacae M-1 by successive fractionation on Q Sepharose FF, SP Sepharose FF, and Sephacryl S-200 HR. The purified enzyme gave a single band on SDS-PAGE and isoelectric focusing. The enzyme easily degraded polyguluronate and produced unsaturated oligoguluronic acids with a wide range of dp. The major end product of the enzyme reaction on polyguluronate was unsaturated triuronic acid. The pattern of oligoguluronic acids (dp 2–9) generated with the enzyme was investigated by fluorophore-assisted carbohydrate electrophoresis. The enzyme was not capable of degrading oligoguluronic acids having a dp < 4. The degradation rate of heptaguluronic acid by this enzyme remarkably increased, compared with that of hexaguluronic acid, and heptaguluronic acid had a single preferential point of cleavage by this enzyme. On the basis of the cleavage pattern of oligoguluronic acids, the number of subsites was estimated to be seven for this enzyme. The catalytic site of the enzyme is located between the second and the third subsites from the non-reducing end.

Formation and cleavage of a DNA network during in vitro bacteriophage T7 DNA packaging: light microscopy of DNA metabolism.

To understand in vivo DNA metabolism, in vitro systems are developed that perform DNA metabolism, while maintaining in vivo (physiological) character. To determine the state of DNA during in vitro physiological metabolism, the present study develops procedures of fluorescence light microscopy for observation of stained DNA molecules during in vitro physiological metabolism in a crude extract of bacteriophage T7-infected cells. The extract inhibits illumination-induced breakage of DNA. The following DNA metabolism remains active for 2-3 min during microscopy: exonuclease-dependent end-to-end joining (concatemerization) of T7 DNA and subsequent cleavage of concatemers. When the T7 gene 3-encoded DNA debranching endonuclease is absent during in vitro T7 DNA concatemerization, DNA progressively partitions to form a continuous, mostly immobile (i.e., no detected Brownian motion) fibrous network that encloses the DNA-depleted solution; presumably because of reduced branching, a less extensive network forms when the gene 3-encoded debranching endonuclease is present. Most strands of the network consist of multiple DNA segments. After a time interval of 5-10 min, the DNA network undergoes cleavage that depends on the presence of both ATP, capsids, and the DNA packaging accessory proteins encoded by genes 18 and 19; multiple cleavages eventually disrupt the continuity of the DNA network. The dependence of the observed cleavage on these factors is explained by the hypothesis that this cleavage is the first of two cleavages known to occur during the packaging of T7 DNA concatemers both in vivo and in vitro. The first cleavage is also known to initiate entry of DNA into a T7 capsid. The cleavage observed here is usually preceded by an approximately 10 s burst of oscillatory motion of the DNA network near the point of eventual cleavage. If the in vivo presence of a similar concatemer-containing DNA network is assumed, requirement for DNA packaging-associated release of DNA from this network is a possible explanation for the evolution of a T7 DNA packaging pathway that is initiated by cleavage of a concatemer.

Isolation of a cDNA encoding Fasciola hepatica cathepsin L2 and functional expression in Saccharomyces cerevisiae

Cathepsin L2 is a major cysteine proteinase secreted by adult Fasciola hepatica. The enzyme differs from other reported cathepsin Ls in that it can cleave peptide substrates that contain proline in the P 2 position. A cDNA was isolated from an expression library by immunoscreening with antiserum prepared against purified native cathepsin L2. This cDNA was sequenced and shown to encode a complete preprocathepsin L proteinase. Functionally active recombinant cathepsin L proteinase was expressed and secreted by Saccharomyces cerevisiae transformed with the cDNA. The recombinant enzyme was purified from large-scale fermentation broths using ultrafiltration and gel filtration chromatography on Sephacryl S200 HR columns. NH 2-terminal amino acid sequencing showed that the cleavage point for activation of the recombinant pro-enzyme is identical to that of the F. hepatica-produced cathepsin L2. The mature active recombinant proteinase behaved similarly to the native enzyme when analysed by SDS-PAGE, immunoblotting and zymography and also cleaved peptides containing proline in the P 2 position. Finally, the recombinant cathepsin L2 cleaved fibrinogen to form a fibin clot, a property we described for F. hepatica cathepsin L2.

Generation of hemoglobin peptides in the acidic digestive vacuole of Plasmodium falciparum implicates peptide transport in amino acid production.

Intraerythrocytic malaria parasites avidly consume hemoglobin as a source of amino acids for incorporation into parasite proteins. An acidic organelle, the digestive vacuole, is the site of hemoglobin proteolysis. Early events in hemoglobin catabolism have been well studied. Two aspartic proteases, plasmepsins I and II, and a cysteine protease, falcipain, cleave hemoglobin into peptides. While it has been presumed that hemoglobin peptide fragments are degraded to individual amino acids by exopeptidase activity in the digestive vacuole, this hypothesis lacks experimental support. Incubation of human hemoglobin with P. falciparum digestive vacuole lysate generated a series of discrete peptide fragments with cleavage sites an average of 8.4 amino acids apart. No free amino acids could be detected and there was no evidence of peptide heterogeneity due to exopeptidase trimming. These sites correspond to points of cleavage previously established for plasmepsin I, plasmepsin II, and falcipain as well as some novel sites that suggest the existence of an additional endoproteinase. By colorimetric assay, P. falciparum has abundant aminopeptidase activity but this activity is not found in the digestive vacuoles and the parasite lacks detectable carboxypeptidase activity altogether. These data support a model for hemoglobin catabolism wherein small peptides are formed from cleavage of hemoglobin by the enzymes of the digestive vacuole and then are transported through the membrane of the digestive vacuole to the cytoplasm. There, exopeptidase activity converts the peptides to individual amino acids for parasite growth and maturation.