Appraisal of green fluorescent protein as a model substrate for seryl-histidine dipeptide cleaving agent

Summary We have investigated the action of seryl-histidine dipeptide (SH) on Green Fluorescent Protein (GFPxm) by SDS-PAGE and fluorescence techniques. It was found that 1 mmol SH showed a 75 units protease K (PK)-like cleavage effect at 50°C, pH 6.5–7.5. Compared with the results of SH cleavage on BSA, SDS-PAGE experiments showed no obvious fragments resulting from SH cleavage of GFPxm. SH quenched the GFPxm fluorescence greatly prior to cleavage. When the SH concentration reached 150 mM, the fluorescence quenching phenomena stopped. Compared cleavage. When the SH concentration reached 150 mM, the fluorescence quenching phenomena stopped. Compared with seryl-histidine dipeptide, the individual amino acids Ser and His, and these two amino acids mixed together, showed no effects on the spectral characteristics of GFPxm nor cleavage of GFPxm. Further study of the SH protein cleavage mechanism might provide insight for protease mechanisms and biomacromolecule evolution.     

Dipeptide seryl-histidine and related oligopeptides cleave DNA, protein, and a carboxyl ester

The amino acids histidine (His) and serine (Ser), or amino acids similar to Ser, function together as key catalytic amino acids in the active sites of such diverse enzymes as the serine- and thiol-proteases, lipases, and esterases. Ser and His are also conserved in the intein–extein junctions of the phylogenetically widespread self-splicing proteins and at the N- and C-termini of the homing endonucleases spliced from them. Here we show that the dipeptide seryl-histidine (Ser-His) and related oligopeptides can themselves cleave DNA, protein, and the ester p-nitrophenyl acetate (p-NPA) over wide ranges of pH and temperature. Denaturing polyacrylamide gel electrophoresis (PAGE) of 5′-end labeled DNA samples incubated with Ser-His reveals a pattern of two bands per nucleotide position, consistent with the generation of both 3′-hydroxyl and 3′-phosphate DNA cleavage fragments, as would be expected of phosphodiester hydrolysis by Ser-His. To the best of our knowledge, Ser-His is the shortest peptide ever reported to show cleavage activity with multiple categories of natural substrates. The amenability of the dipeptide to variation through addition of amino acid residues, either internally or to the C-terminus while retaining its multiple cleavage activities, combined with its reactivity over wide ranges of pH and temperature, demonstrates the evolutionary capacity of the Ser/His dyad and evokes many questions about possible roles it may have played in molecular evolution and its potential role as a core for selection of oligopeptides with enhanced cleavage activities and target specificity.     

Cleavage of BSA by a dipeptide seryl-histidine

A dipeptide seryl-histidine (Ser-His) was found to have theprotein cleavage activity. BSA was cleaved into smear at aroundpH 5.0–6.0, with a half-life around 15 hr at 60 °C.Phosphate could accelerate the reaction. This is a brand newprotein cleavage system. Since Ser and His are well-knowncatalytic residues at the active sites of many serine proteases,this results might provide clues to the possible roles of shortoligopeptides in the origins of modern enzymes.     

3C-like protease of rabbit hemorrhagic disease virus: identification of cleavage sites in the ORF1 polyprotein and analysis of cleavage specificity.

Rabbit hemorrhagic disease virus, a positive-stranded RNA virus of the family Caliciviridae, encodes a trypsin-like cysteine protease as part of a large polyprotein. Upon expression in Escherichia coli, the protease releases itself from larger precursors by proteolytic cleavages at its N and C termini. Both cleavage sites were determined by N-terminal sequence analysis of the cleavage products. Cleavage at the N terminus of the protease occurred with high efficiency at an EG dipeptide at positions 1108 and 1109. Cleavage at the C terminus of the protease occurred with low efficiency at an ET dipeptide at positions 1251 and 1252. To study the cleavage specificity of the protease, amino acid substitutions were introduced at the P2, P1, and P1' positions at the cleavage site at the N-terminal boundary of the protease. This analysis showed that the amino acid at the P1 position is the most important determinant for substrate recognition. Only glutamic acid, glutamine, and aspartic acid were tolerated at this position. At the P1' position, glycine, serine, and alanine were the preferred substrates of the protease, but a number of amino acids with larger side chains were also tolerated. Substitutions at the P2 position had only little effect on the cleavage efficiency. Cell-free expression of the C-terminal half of the ORF1 polyprotein showed that the protease catalyzes cleavage at the junction of the RNA polymerase and the capsid protein. An EG dipeptide at positions 1767 and 1768 was identified as the putative cleavage site. Our data show that rabbit hemorrhagic disease virus encodes a trypsin-like cysteine protease that is similar to 3C proteases with regard to function and specificity but is more similar to 2A proteases with regard to size.     

The study on the interaction between seryl-histidine dipeptide and proteins by circular dichroism and molecular modeling

The selective cleavage of proteins is very important in key biological processes. Chemical (nonenzymatic) reagents such as cyanogen bromide and transition metal complexes are used extensively with great defects. In this paper, the binding of seryl-histidine dipeptide (abbreviated as SH) with bovine serum albumin (BSA) and lysozyme were investigated by the circular dichroism spectroscopy (CD) at 298K, molecular docking studies and quantum chemical calculations based on the previous results of polyacrylamide gel electrophoresis (PAGE). From the studies of CD, it showed that SH interacted strongly with BSA and lysozyme. The change percentages of the secondary structures of BSA and lysozyme were calculated. The contents of the beta-sheets decreased remarkably. It indicated that the interactions between SH and proteins could break the hydrogen bonds of beta-sheets selectively. The docking studies between SH and BSA showed that the position of the oxygen atom of the hydroxyl group of SH (O(12)) was in favor of a nucleophilic attack on carbon atom of the amide bond of a beta-sheet (C(34)) because the distance between O(12) and C(34) was 3.37A. Natural charges, natural atomic hybrid percentages and square sums of HOMO coefficients calculated by the NBO and population analysis at HF/6-31G* supported the suggested mechanism. And so SH may be an interesting agent for the therapeutic use.     

Substrate specificity of recombinant dengue 2 virus NS2B-NS3 protease: influence of natural and unnatural basic amino acids on hydrolysis of synthetic fluorescent substrates

A recombinant dengue 2 virus NS2B-NS3 protease (NS means non-structural virus protein) was compared with human furin for the capacity to process short peptide substrates corresponding to seven native substrate cleavage sites in the dengue viral polyprotein. Using fluorescence resonance energy transfer peptides to measure kinetics, the processing of these substrates was found to be selective for the Dengue protease. Substrates containing two or three basic amino acids (Arg or Lys) in tandem were found to be the best, with Abz-AKRRSQ-EDDnp being the most efficiently cleaved. The hydrolysis of dipeptide substrates Bz-X-Arg-MCA where X is a non-natural basic amino acid were also kinetically examined, the best substrates containing aliphatic basic amino acids. Our results indicated that proteolytic processing by dengue NS3 protease, tethered to its activating NS2B co-factor, was strongly inhibited by Ca2+ and kosmotropic salts of the Hofmeister's series, and significantly influenced by substrate modifications between S4 and S6'. Incorporation of basic non-natural amino acids in short peptide substrates had significant but differential effects on Km and k(cat), suggesting that further dissection of their influences on substrate affinity might enable the development of effective dengue protease inhibitors.     

Cleavage of BSA by a dipeptide seryl-histidine

Summary A dipeptide seryl-histidine (Ser-His) was found to have the protein cleavage activity. BSA was cleaved into smear at around pH 5.0–6.0, with a half-life around 15 hr at 60 °C. Phosphate could accelerate the reaction. This is a brand new protein cleavage system. Since Ser and His are well-known catalytic residues at the active sites of many serine proteases, this results might provide clues to the possible roles of short oligopeptides in the origins of modern enzymes.     

Ser-His catalyses the formation of peptides and PNAs

The dipeptide seryl-histidine (Ser-His) catalyses the condensation of esters of amino acids, peptide fragments, and peptide nucleic acid (PNA) building blocks, bringing to the formation of peptide bonds. Di-, tri- or tetra-peptides can be formed with yields that vary from 0.5% to 60% depending on the nature of the substrate and on the conditions. Other simpler peptides as Gly-Gly, or Gly-Gly-Gly are also effective, although less efficiently. We discuss the results from the viewpoint of primitive chemistry and the origin of long macromolecules by stepwise fragment condensations.     

Variants of Green Fluorescent Protein GFPxm

As research progresses, fluorescent proteins useful for optical marking will evolve toward brighter, monomeric forms that are more diverse in color. We previously reported a new fluorescent protein from Aequorea macrodactyla, GFPxm, that exhibited many characteristics similar to wild-type green fluorescent protein (GFP). However, the application of GFPxm was limited because GFPxm expressed and produced fluorescence only at low temperatures. To improve the fluorescent properties of GFPxm, 12 variants were produced by site-directed mutagenesis and DNA shuffling. Seven of these mutants could produce strong fluorescence when expressed at 37°C. The relative fluorescence intensities of mutants GFPxm16, GFPxm18, and GFPxm19 were higher than that of EGFP (enhanced GFP) when the expression temperature was between 25 and 37°C, and mutants GFPxm16 and GFPxm163 could maintain a high fluorescence intensity even when expressed at 42°C. Meanwhile, at least 4 mutants could be successfully expressed in mammalian cell lines. The fluorescence spectra of 6 of the 12 mutants had a progressive red shift. The longest excitation-emission maximum was at 514/525 nm. In addition, 3 of the 12 mutants had two excitation peaks including an UV-excitation peak, while another mutant had only one UV-excitation peak.     

A novel location for dipeptidyl aminopeptidase processing sites in the alkaline extracellular protease of Yarrowia lipolytica

A stretch of 10 consecutive dipeptides with the sequence -X-Ala- or -X-Pro-, possible cleavage sites for dipeptidyl aminopeptidase (DPAPase) activity, are located in the prepro-region of the alkaline extracellular protease (AEP) beginning at Leu14. Evidence for DPAPase processing of this dipeptide stretch was obtained by characterizing the polypeptide secreted by a strain carrying a xpr6 mutation. The secreted polypeptide reacted with antibodies specific for AEP and was essentially identical to the 52-kilodalton intracellular AEP precursor based on mobility during sodium dodecyl sulfate-polyacrylamide gel electrophoresis, content of N-linked carbohydrate, and peptide mapping. Amino-terminal amino acid sequencing of this secreted precursor revealed that it consisted of at least three major polypeptides. One began at the end of the stretch of dipeptides, and two of the others began two and four amino acids upstream. These results confirm that DPAPase activity is involved in the formation of the 52-kilodalton AEP precursor. In other reported cases of DPAPase processing, the dipeptides are located directly upstream of the mature polypeptide. For AEP, the dipeptide stretch is located over 120 amino acids upstream from the N terminus of mature AEP. The novel location of the dipeptide stretch may provide a mechanism for preventing premature activation of AEP in the secretory pathway.     

Linkers for improved cleavage of fusion proteins with an engineered alpha-lytic protease.

Addition of an N-terminal fusion partner can greatly aid the expression and purification of a recombinant protein in Escherichia coli. We investigated two genetically engineered proteases designed to remove the fusion partner after the protein of interest has been expressed. Recombinant human insulin-like growth factor-II (hIGF-II) has been produced from E. coli-derived fusion proteins using a novel enzymatic cleavage system that uses a mutant of alpha-lytic protease. Initially, two potential fusion protein linkers were designed, Pro-Ala-Pro-His (PAPH) and Pro-Ala-Pro-Met (PAPM), and were tested as substrates in the form of synthetic dodecapeptides. Using mass spectrometry and reverse-phase HPLC, the position of cleavage was confirmed and the kinetics of synthetic peptide cleavage were examined. Use of the linkers in hIGF-II fusion proteins produced in E. coli was then evaluated. The fusion proteins constructed consist of the first 11 amino acids of porcine growth hormone linked N-terminally to hIGF-II by six amino acids that include the dipeptide Val-Asn followed by a variable tetrapeptide protease cleavage motif. Mass spectrometry and N-terminal sequencing confirmed that proteolytic cleavage of the fusion proteins had occurred at the predicted sites. Using the fusion proteins as substrates, the cleavage of the rationally designed motifs by the alpha-lytic protease mutant was compared. The fusion protein containing the motif PAPM had a k(cat)/K(M) ratio indicating a 1.6-fold preference over the PAPH fusion protein for cleavage by this enzyme. Furthermore, when hIGF-II fusion proteins containing the designed cleavable linkers were processed with the engineered alpha-lytic protease, they gave greatly improved yields of native hIGF-II compared to an analogous fusion protein cleaved by H64A subtilisin. Comparison of the peptide and protein cleavage studies shows that the efficient proteolysis of the cleavage motifs is an inherent property of the designed sequences and is not determined by secondary or tertiary structure in the fusion proteins.     

Evidence for pH-Dependent Protease Activity in the Adeno-Associated Virus Capsid

Incubation of highly purified adeno-associated virus (AAV) capsids in vitro at pH 5.5 induced significant autocleavage of capsid proteins at several amino acid positions. No autocleavage was seen at pH 7.5. Examination of other AAV serotypes showed at least two different pH-induced cleavage patterns, suggesting that different serotypes have evolved alternative protease cleavage sites. In contrast, incubation of AAV serotypes with an external protease substrate showed that purified AAV capsid preparations have robust protease activity at neutral pH but not at pH 5.5, opposite to what is seen with capsid protein autocleavage. Several lines of evidence suggested that protease activity is inherent in AAV capsids and is not due to contaminating proteins. Control virus preparations showed no protease activity on external substrates, and filtrates of AAV virus preparations also showed no protease activity contaminating the capsids. Further, N-terminal Edman sequencing identified unique autocleavage sites in AAV1 and AAV9, and mutagenesis of amino acids adjacent to these sites eliminated cleavage. Finally, mutation of an amino acid in AAV2 (E563A) that is in a conserved pH-sensitive structural region eliminated protease activity on an external substrate but did not seem to affect autocleavage. Taken together, our data suggested that AAV capsids have one or more protease active sites that are sensitive to pH induction. Further, it appears that acidic pHs comparable to those seen in late endosomes induce a structural change in the capsid that induces autolytic protease activity. The pH-dependent protease activity may have a role in viral infection.     

Sequence-specific destabilization of azurin by tetramethylguanidinium-dipeptide ionic liquids

The thermal unfolding of the copper redox protein azurin was studied in the presence of four different dipeptide-based ionic liquids (ILs) utilizing tetramethylguanidinium as the cation. The four dipeptides have different sequences including the amino acids Ser and Asp: TMG-AspAsp, TMG-SerSer, TMG-SerAsp, and TMG-AspSer. Thermal unfolding curves generated from temperature-dependent fluorescence spectroscopy experiments showed that TMG-AspAsp and TMG-SerSer have minor destabilizing effects on the protein while TMG-AspSer and TMG-SerAsp strongly destabilize azurin. Red-shifted fluorescence signatures in the 25 °C correlate with the observed protein destabilization in the solutions with TMG-AspSer and TMG-SerAsp. These signals could correspond to interactions between the Asp residue in the dipeptide and the azurin Trp residue in the unfolded state. These results, supported by appropriate control experiments, suggest that dipeptide sequence-specific interactions lead to selective protein destabilization and motivate further studies of TMG-dipeptide ILs.     

Effect of Vibrio cholerae non-O1 protease on lysozyme, lactoferrin and secretory immunoglobulin A

Abstract The effect of Vibrio cholerae non-O1 protease on host defense proteins (lysozyme, secretory immunoglobullin A and lactoferrin) was studied in relation to its virulence mechanism. The proteins treated with the protease were analysed by SDS-PAGE. There was no influence of the protease on lysozyme. The protease cleaved lactoferrin into two fragments of 50 kDa and 34 kDa. N-terminal amino acid sequencing of these fragments revealed that the cleavage site was near the hinge region, between serine 420 and serine 421. This cleavage could affect the transition from open to closed configuration which is involved in iron binding and release. The anti-bacterial activity of lactoferrin was not affected by protease treatment. Secretory immunoglobulin A yielded a 42-kDa protein as the cleavage product. The susceptibility of secretory immunoglobulin A to V. cholerae non-O1 protease suggests a mechanism by which bacteria might evade the effect of this immunoglobulin.     

Peptide substrate specificity of the membrane-bound metalloprotease of Leishmania

The promastigote surface protease (PSP) of Leishmania is a neutral membrane-bound zinc enzyme. The protease has no exopeptidase activity and does not cleave a large selection of substrates with chromogenic and fluorogenic leaving groups at the P1' site. The substrate specificity of the enzyme was studied by using natural and synthetic peptides of known amino acid sequence. The identification of 11 cleavage sites indicates that the enzyme preferentially cleaves peptides at the amino side when hydrophobic residues are in the P1' site and basic amino acid residues in the P2' and P3' sites. In addition, tyrosine residues are commonly found at the P1 site. Hydrolysis is not, however, restricted to these residues. These results have allowed the synthesis of a model peptide, H2N-L-I-A-Y-L-K-K-A-T-COOH, which is cleaved by PSP between the tyrosine and leucine residues with a kcat/Km ratio of 1.8 X 10(6) M-1 s-1. Furthermore, a synthetic nonapeptide overlapping the last four amino acids of the prosequence and the first five residues of mature PSP was found to be cleaved by the protease at the expected site to release the mature enzyme. This result suggests a possible autocatalytic mechanism for the activation of the protease. Finally, the hydroxamate-derivatized dipeptide Cbz-Tyr-Leu-NHOH was shown to inhibit PSP competitively with a KI of 17 microM.     

Fluorescein-based amino acids for solid phase synthesis of fluorogenic protease substrates

An efficient synthesis of new type fluorescent amino acids is described. The Fmoc-protected dyes can be prepared in a four-step procedure with approximately 30% overall yield from aminofluoresceins and other inexpensive commercially available precursors. The dyes are much more photostable compared to fluorescein and exhibit constant pH-independent fluorescence that is advantageous in biological applications. The Fmoc-protected fluorescent amino acids are ready for use in solid phase peptide synthesis. As a proof of concept, a fluorogenic papain substrate was synthesized and employed for on-bead detection of the protease activity. By using a novel technique for quantitative analysis of bead fluorescence, a approximately 2.7-fold increase in mean bead brightness was measured and was attributed to substrate cleavage by papain. The new type fluorescent amino acids seem to be a promising tool for the synthesis of fluorescent peptide ligands and fluorogenic protease substrates.     

Protease A activity and nitrogen fractions released during alcoholic fermentation and autolysis in enological conditions

Determination of protease A activity during alcoholic fermentation of a synthetic must (pH 3.5 at 25°C) and during autolysis showed that a sixfold induction of protease A activity occurred after sugar exhaustion, well before 100% cell death occurred. A decrease in protease A activity was observed when yeast cell autolysis started. Extracellular protease A activity was detected late in the autolysis process, which suggests that protease A is not easily released. Evolution of amino acids and peptides was determined during alcoholic fermentation and during autolysis. Amino acids were released in early stationary phase. These amino acids were subsequently assimilated during the fermentation. The same pattern was observed for peptides; this has never been reported previously. During autolysis, the concentration of amino acids and peptides increased to reach a maximum of 20 and 40 mg N l⁻¹, respectively. This study supports the idea that although protease A activity seemed to be responsible for peptides release, there is no clear correlation among protease A activity, cell death, and autolysis. The amino acid composition of the peptides showed some variations between peptides released during alcoholic fermentation and during autolysis. Depending on aging time on yeast lees, the nature of the peptides present in the medium changed, which could lead to different organoleptic properties. Journal of Industrial Microbiology & Biotechnology (2001) 26, 235–240. Received 02 August 2000/ Accepted in revised form 15 December 2000     

Amino acid sequence of p15 from avian myeloblastosis virus complex

The complete amino acid sequence of the p15 gag protein from avian myeloblastosis virus (AMV) complex has been determined by sequential Edman degradation of the intact molecule and of peptide fragments generated by limited tryptic cleavage, cleavage with staphylococcal protease, and cyanogen bromide cleavage. AMV p15 is a single-chain protein containing 124 amino acids. The charged amino acids tend to be clustered in the primary structure. p15 contains a single cysteine at position 113 which may be essential for the p15 associated proteolytic activity. However, p15 shows no appreciable sequence homology with papain or other classical thiol proteases.     

Degradation of a signal peptide by protease IV and oligopeptidase A.

The degradation of the prolipoprotein signal peptide in vitro by membranes, cytoplasmic fraction, and two purified major signal peptide peptidases from Escherichia coli was followed by reverse-phase liquid chromatography (RPLC). The cytoplasmic fraction hydrolyzed the signal peptide completely into amino acids. In contrast, many peptide fragments accumulated as final products during the cleavage by a membrane fraction. Most of the peptides were similar to the peptides formed during the cleavage of the signal peptide by the purified membrane-bound signal peptide peptidase, protease IV. Peptide fragments generated during the cleavage of the signal peptide by protease IV and a cytoplasmic enzyme, oligopeptidase A, were identified from their amino acid compositions, their retention times during RPLC, and knowledge of the amino acid sequence of the signal peptide. Both enzymes were endopeptidases, as neither dipeptides nor free amino acids were formed during the cleavage reactions. Protease IV cleaved the signal peptide predominantly in the hydrophobic segment (residues 7 to 14). Protease IV required substrates with hydrophobic amino acids at the primary and the adjacent substrate-binding sites, with a minimum of three amino acids on either side of the scissile bond. Oligopeptidase A cleaved peptides (minimally five residues) that had either alanine or glycine at the P'1 (primary binding site) or at the P1 (preceding P'1) site of the substrate. These results support the hypothesis that protease IV is the major signal peptide peptidase in membranes that initiates the degradation of the signal peptide by making endoproteolytic cuts; oligopeptidase A and other cytoplasmic enzymes further degrade the partially degraded portions of the signal peptide that may be diffused or transported back into the cytoplasm from the membranes.     

RNA-binding domain of the key structural protein P7 for the Rice dwarf virus particle assembly

The Rice dwarf virus (RDV) P7 structural protein is the key protein in the RDV particle assembly. The P7 protein was digested partially or completely by Staphylococcus aureus V8 protease and/or Pseudomonasfragi Asp-N protease. The molecular mass and the N-terminal amino acid sequence of the polypeptide fragments of the P7 protein were determined by SDS-PAGE and the Edman degradation method, respectively. Then the polypeptides were located in the deduced amino acid sequence of the RDV P7 protein based on the nucleotide sequence information, with the knowledge of the specific cleavage sites of the Staphylococcus aureus V8 and Pseudomonasfragi Asp-N protease, and the two RNA-binding domains in the P7 protein were identified. Domain 1 was located in the residue 128-249 containing 122 amino acids and domain 2 was located in the residue 325-355 containing 31 amino acids. Thus, these two domains may play an important role in the virus particle assembly by contributing to the packaging of viral dsRNAs inside the particles. The two domains may be novel RNA-binding domains, because no amino acid sequences highly similar to the conservative sequences of known dsRNA-binding domains reported so far. The similarity between the motif of domain 1 and the motif of the DNA-binding protein suggests that the DNA-binding activity of the RDV P7 protein may be due to this sequence. The similarity between the motif of domain 1 and the motif of the RNA polymerase domain suggests that the P7 protein may also play a role in RNA synthesis, besides its function in the assembly and subsequent packaging of viral dsRNA into core particles.