Influence of the amino acid residue downstream of (Asp)4Lys on enterokinase cleavage of a fusion protein

We have studied the cleavage efficiency of the protease enterokinase (EK) using the novel vector pESP4. pESP4 is a yeast expression vector equipped with ligation-independent cloning sites, a GST purification tag, and a FLAG epitope tag. EK is used to cleave the FLAG and GST tags leaving the protein of interest without any extraneously added amino acids. We have found that EK is relatively permissive of the amino acid residue downstream of the recognition sequence (the P'1 position). This makes EK an ideal choice to use as a protease to cleave any protein of interest cloned within the pESP4 yeast expression vector.     

Recombinant enterokinase light chain with affinity tag: expression from Saccharomyces cerevisiae and its utilities in fusion protein technology.

Enterokinase and recombinant enterokinase light chain (rEK(L)) have been used widely to cleave fusion proteins with the target sequence of (Asp)(4)-Lys. In this work, we show that their utility as a site-specific cleavage agent is compromised by sporadic cleavage at other sites, albeit at low levels. Further degradation of the fusion protein in cleavage reaction is due to an intrinsic broad specificity of the enzyme rather than to the presence of contaminating proteases. To offer facilitated purification from fermentation broth and efficient removal of rEK(L) after cleavage reaction, thus minimizing unwanted cleavage of target protein, histidine affinity tag was introduced into rEK(L). Utilizing the secretion enhancer peptide derived from the human interleukin 1 beta, the recombinant EK(L) was expressed in Saccharomyces cerevisiae and efficiently secreted into culture medium. The C-terminal His-tagged EK(L) was purified in a single-step procedure on nickel affinity chromatography. It retained full enzymatic activity similar to that of EK(L), whereas the N-terminal His-tagged EK(L) was neither efficiently purified nor had any enzymatic activity. After cleavage reaction of fusion protein, the C-terminal His-tagged EK(L) was efficiently removed from the reaction mixture by a single passage through nickel-NTA spin column. The simple affinity tag renders rEK(L) extremely useful for purification, post-cleavage removal, recovery, and recycling and will broaden the utility and the versatility of the enterokinase for the production of recombinant proteins.     

Immobilization of enterokinase on magnetic supports for the cleavage of fusion proteins

Magnetic nanobiocatalysts for tag cleavage on fusion proteins have been prepared by immobilizing enterokinase (EK) onto iron oxide magnetic nanoparticles coated with biopolymers. Two different chemistries have been explored for the covalent coupling of EK, namely carbodiimide (EDC coupling) and maleimide activation (Sulfo coupling). Upon immobilization, EK initial activity lowered but EDC coupling lead to higher activity retention. Regarding the stability of the nanobiocatalysts, these were recycled up to ten times with the greater activity losses observed in the first two cycles. The immobilized EK also proved to cleave a control fusion protein and to greatly simplify the separation of the enzyme from the reaction mixture.     

A pathway sensor for genome-wide screens of intracellular proteolytic cleavage

Protein cleavage is a central event in many regulated biological processes. We describe a system for detecting intracellular proteolysis based on non-conventional secretion of Gaussia luciferase (GLUC). GLUC exits the cell without benefit of a secretory leader peptide, but can be anchored in the cell by fusion to β-actin. By including protease cleavage sites between GLUC and β-actin, proteolytic cleavage can be detected. Using this assay, we have identified regulators of autophagy, apoptosis and β-actin cleavage.     

Mutational Analysis of the Pseudomonas aeruginosa Myovirus ?KZ Morphogenetic Protease gp175

Pseudomonas aeruginosa myovirus ϕKZ has a 270-kb genome within a T=27 icosahedral capsid that contains a large, unusual, and structurally well-defined protein cylindrical inner body (IB) spanning its interior. Proteolysis forms a pivotal stage in ϕKZ head and IB morphogenesis, with the protease gp175 cleaving at least 19 of 49 different head proteins, including the major capsid protein and five major structural IB proteins. Here we show that the purified mature form of gp175 is active and cleaves purified IB structural proteins gp93 and gp89. Expression vector synthesis and purification of the zymogen/precursor yielded an active, mature-length protease, showing independent C-terminal gp175 self-cleavage autoactivation. Mutation of either the predicted catalytic serine or histidine inactivated mature gp175, supporting its classification as a serine protease and representing the first such direct biochemical demonstration with purified protease and substrate proteins for any phage protease. These mutations also blocked self-cleavage of the precursor while allowing intermolecular gp175 processing. To confirm the cleavage specificity of gp175, we mutated three cleavage sites in gp93, which blocked proteolysis at these sites. The N-terminal propeptide of gp93 was shown to undergo more extensive proteolysis than previously identified. We found that proteolysis in gp93 progressed from the N to C terminus, while blocking cleavage sites slowed but did not eliminate downstream proteolysis. These findings were shown by informatics to be relevant to the head morphogenesis of numbers of other related IB-containing giant phages as well as to T4 and herpesviruses, which have homologous proteases.     

The different cleavage DNA sequence specificity explains the camptothecin resistance of the human topoisomerase I Glu418Lys mutant

Yeast cells expressing the Glu418Lys human topoisomerase I mutant display a camptothecin resistance that slowly decreases as a function of time. Molecular characterization of the single steps of the catalytic cycle of the purified mutant indicates that it has a relaxation activity identical to the wild-type protein but a different DNA sequence specificity for the cleavage sites when compared to the wild-type enzyme, as assayed on several substrates. In particular the mutant has a low specificity for CPT sensitive cleavable sites. In fact, the mutant has, at variance of the wild-type enzyme, a reduced preference for cleavage sites having a thymine base in position −1 of the scissile strand. This preference, together with the strict requirement for a thymine base in position −1 for an efficient camptothecin binding, explains the temporary camptothecin resistance of the yeast cell expressing the mutant and points out the importance of the DNA sequence in the binding of the camptothecin drug.     

Complexity in orchestration of chemical groups near different cleavage sites in RNase P RNA mediated cleavage

RNase P mediated cleavage of the tRNA(His) precursor does not rely on the formation of the "+73/294 interaction" to give the correct cleavage product, i.e. cleavage at -1, while other tRNA precursors that are cleaved at the canonical site +1 do. A previous model, here referred to as the "2'OH-model", predicts that the 2'OH at the canonical cleavage site would affect cleavage at -1. Here we used model RNA hairpin substrates mimicking the structural architecture of the tRNA(His) precursor cleavage site to investigate the role of 2'OH with respect to ground state binding and rate of cleavage in the presence and absence of the +73/294 interaction. Our data emphasize the importance of the 2'OH in the immediate vicinity of the scissile bond. Moreover, introduction of 2'H at the cleavage site did not affect cleavage at an alternative cleavage site to any significant extent. Our findings are therefore inconsistent with the 2'OH model. We favor a model where the 2'OH at the cleavage site influence Mg2+ binding in its vicinity, however we do not exclude the possibility that the 2'OH at the cleavage site interacts with RNase P RNA. Studying the importance of the 2'OH at different cleavage sites also indicated a higher dependence on the 2'OH at the cleavage site in the absence of the +73/294 interaction than in its presence. Finally, we provide data suggesting that N3 of U at position -1 in the substrate is most likely not involved in an interaction with RNase P RNA.     

Insights into the specificity of RNA cleavage by the Escherichia coli MazF toxin

The mazEF (chpA) toxin-antitoxin system of Escherichia coli is involved in the cell response to nutritional and antibiotic stresses as well as in bacterial-programmed cell death. Valuable information on the MazF toxin was derived from the determination of the crystal structure of the MazE/MazF complex and from in vivo data, suggesting that MazF promoted ribosome-dependent cleavage of messenger RNA. However, it was concluded from recent in vitro analyses using a MazF-(His6) fusion protein that MazF was an endoribonuclease that cleaved messenger RNA specifically at 5'-ACA-3' sites situated in single-stranded regions. In contrast, our work reported here shows that native MazF protein cleaves RNA at the 5' side of residue A in 5'-NAC-3' sequences (where N is preferentially U or A). MazF-dependent cleavage occurred at target sequences situated either in single- or double-stranded RNA regions. These activities were neutralized by a His6-MazE antitoxin. Although essentially consistent with previous in vivo reports on the substrate specificity of MazF, our results strongly suggest that the endoribonuclease activity of MazF may be modulated by additional factors to cleave messenger and other cellular RNAs.     

Preparation of recombinant thioredoxin fused N-terminal proCNP: Analysis of enterokinase cleavage products reveals new enterokinase cleavage sites

C-type natriuretic peptide (CNP) acts as a paracrine hormone to dilate blood vessels and is also required for the growth of long bones. In vivo, CNP is produced by cleavage from the C-terminal end of a larger proCNP peptide. The remaining N-terminal proCNP fragment (NT-proCNP) escapes into the circulation where its concentration is much higher than that of CNP due presumably to a lower clearance rate. Our strategy to obtain large quantities of pure NT-proCNP for further physiological investigations was to express it as a fusion protein with His(6)-tagged thioredoxin followed by cleavage using enterokinase to yield NT-proCNP alone. We have successfully designed and artificially synthesized the coding sequence specifying both mouse and human NT-proCNP with built-in codon bias towards Escherichia coli codon preference. An enterokinase recognition sequence was incorporated immediately upstream of the NT-proCNP coding sequence to allow the fusion protein to be cleaved without leaving any extra residues on the NT-proCNP peptide. High levels of fusion proteins were obtained, constituting 50-58% of total bacterial proteins. Greater than 90% of recombinant thioredoxin/NT-proCNP was expressed in the soluble form and purified to near homogeneity in a single chromatographic step using nickel as the metal ion in IMAC. A time course analysis of the products released from enterokinase cleavage of the recombinant proteins by ESI-MS revealed three sensitive secondary cleavage sites: two were located on vector-associated sequences linking the thioredoxin moiety and NT-proCNP, and one at the C-terminal end of NT-proCNP. Clearly, substrate specificity of both the native and recombinant forms of enterokinase for the recognition sequence DDDDK was by no means exclusive. Hydrolysis at the unexpected LKGDR site located towards the carboxyl end on NT-proCNP was significantly more efficient than at the internally sited DDDDK target sequence. However, when this same sequence was sited internally replacing the DDDDK in another construct of thioredoxin/mouse NT-proCNP, it was found to be poorly processed by enterokinase. Our results showed that non-target sequences can be preferentially recognized over the canonical DDDDK sequence when located accessibly at the ends of proteins.     

Transition-state stabilization in Escherichia coli ribonuclease P RNA-mediated cleavage of model substrates

We have used model substrates carrying modified nucleotides at the site immediately 5′ of the canonical RNase P cleavage site, the −1 position, to study Escherichia coli RNase P RNA-mediated cleavage. We show that the nucleobase at −1 is not essential but its presence and identity contribute to efficiency, fidelity of cleavage and stabilization of the transition state. When U or C is present at −1, the carbonyl oxygen at C2 on the nucleobase contributes to transition-state stabilization, and thus acts as a positive determinant. For substrates with purines at −1, an exocyclic amine at C2 on the nucleobase promotes cleavage at an alternative site and it has a negative impact on cleavage at the canonical site. We also provide new insights into the interaction between E. coli RNase P RNA and the −1 residue in the substrate. Our findings will be discussed using a model where bacterial RNase P cleavage proceeds through a conformational-assisted mechanism that positions the metal(II)-activated H₂O for an in-line attack on the phosphorous atom that leads to breakage of the phosphodiester bond.     

Specific anti‐integrase abzymes from HIV‐infected patients: a comparison of the cleavage sites of intact globular HIV integrase and two 20‐mer oligopeptides corresponding to its antigenic determinants

HIV‐infected patients possess anti‐integrase (IN) IgGs and IgMs that, after isolation by chromatography on IN‐Sepharose, unlike canonical proteases, specifically hydrolyze only IN but not many other tested proteins. Hydrolysis of intact globular IN first leads to formation of many long fragments of protein, while its long incubation with anti‐IN antibodies, especially in the case of abzymes (Abzs) with a high proteolytic activity, results in the formation of short and very short oligopeptides (OPs). To identify all sites of IgG‐mediated proteolysis corresponding to known AGDs of integrase, we have used a combination of reverse‐phase chromatography, matrix‐assisted laser desorption/ionization spectrometry, and thin‐layer chromatography to analyze the cleavage products of two 20‐mer OPs corresponding to these AGDs. Both OPs contained 9–10 mainly clustered major, medium, and minor sites of cleavage. The main superficial cleavage sites of the AGDs in the intact IN and sites of partial or deep hydrolysis of the peptides analyzed do not coincide. The active sites of anti‐IN Abzs are localized on their light chains, whereas the heavy chains are responsible for the affinity of protein substrates. Interactions of intact globular proteins with both light and heavy chains of Abzs provide high specificity of IN hydrolysis. The affinity of anti‐IN Abzs for intact integrase was ~1000‐fold higher than for the OPs. The data suggest that both OPs interact mainly with the light chains of different monoclonal Abzs of the total pool of IgGs, which possesses lower affinity for substrates; and therefore, depending on the oligopeptide sequences, their hydrolysis may be less specific and remarkably different in comparison with the cleavage of intact globular IN. Copyright © 2013 John Wiley & Sons, Ltd.     

Functional interplay between caspase cleavage and phosphorylation sculpts the apoptotic proteome

Caspase proteases are principal mediators of apoptosis, where they cleave hundreds of proteins. Phosphorylation also plays an important role in apoptosis, although the extent to which proteolytic and phosphorylation pathways crosstalk during programmed cell death remains poorly understood. Using a quantitative proteomic platform that integrates phosphorylation sites into the topographical maps of proteins, we identify a cohort of over 500 apoptosis-specific phosphorylation events and show that they are enriched on cleaved proteins and clustered around sites of caspase proteolysis. We find that caspase cleavage can expose new sites for phosphorylation, and, conversely, that phosphorylation at the +3 position of cleavage sites can directly promote substrate proteolysis by caspase-8. This study provides a global portrait of the apoptotic phosphoproteome, revealing heretofore unrecognized forms of functional crosstalk between phosphorylation and caspase proteolytic pathways that lead to enhanced rates of protein cleavage and the unveiling of new sites for phosphorylation.     

An effective system for detecting protein-protein interaction based on in vivo cleavage by PPV NIa protease

Detection of protein-protein interaction can provide valuable information for investigating the biological function of proteins. The current methods that applied in protein-protein interaction, such as co-immunoprecipitation and pull down etc., often cause plenty of working time due to the burdensome cloning and purification procedures. Here we established a system that characterization of protein-protein interaction was accomplished by co-expression and simply purification of target proteins from one expression cassette within E. coli system. We modified pET vector into co-expression vector pInvivo which encoded PPV NIa protease, two cleavage site F and two multiple cloning sites that flanking cleavage sites. The target proteins (for example: protein A and protein B) were inserted at multiple cloning sites and translated into polyprotein in the order of MBP tag-protein A-site F-PPV NIa protease-site F-protein B-His₆ tag. PPV NIa protease carried out intracellular cleavage along expression, then led to the separation of polyprotein components, therefore, the interaction between protein A-protein B can be detected through one-step purification and analysis. Negative control for protein B was brought into this system for monitoring interaction specificity. We successfully employed this system to prove two cases of reported protien- protein interaction: RHA2a/ANAC and FTA/FTB. In conclusion, a convenient and efficient system has been successfully developed for detecting protein-protein interaction.     

An SRLLR motif downstream of the scissile bond enhances enterokinase cleavage efficiency

In a previous paper, we reported more efficient enterokinase cleavage at a C-terminal non-target LKGDR(201) site compared with an internally sited canonical recognition site, DDDDK(156). When this non-target site was placed internally to replace DDDDK(156) between the thioredoxin moiety and mouse NT-proCNP(1-50), this site was poorly processed leading us to conclude that efficient processing at LKGDR(201) in the first instance was due to its accessibility at the C-terminus of the fusion protein. Subsequently, we reasoned that treatment of thioredoxin-fused NT-proCNP(1-81) would allow us to retrieve full-length NT-proCNP(1-81) without undue processing at the LKGDR(201) site since this non-target site would now be located internally about 36 residues away from the C-terminus and hence not be hydrolyzed efficiently. Surprisingly, ESI-MS data showed that the LKGDR site in thioredoxin-fused human NT-proCNP(1-81) was still very efficiently cleaved and revealed a new but slow hydrolysis site with the sequence RVDTK/SRAAW to yield a peptide consistent with NT-proCNP(58-81). The evidence obtained from these experiments led us to postulate that efficient cleavage at the non-target LKGDR(201) site was not merely influenced by steric constraints but also by the sequence context downstream of the scissile bond. Hence, we constructed variants of thioredoxin-mouse NT-proCNP(1-50) where SRLLR residues (i.e. those immediately downstream from the LKGDR(201) site in NT-proCNP(1-50)) were systematically added one at a time downstream of the internal DDDDK(156) site. To evaluate the relative effects of site accessibility and downstream sequence context on the efficiency of enterokinase cleavage, we have also replaced the native LKGDR(201) sequence with DDDDK(201). Our results showed that incremental addition of SRLLR residues led to a steady increase in the rate of hydrolysis at DDDDK(156). Further variants comprising DDDDK(156)SS, DDDDK(156)SD and DDDDK(156)RR showed that the minimal critical determinants for enhanced enterokinase cleavage are serine in the P1' position followed by a serine or a basic residue, lysine or arginine, in the P2' position. Our data provided conclusive evidence that the influence of downstream sequences on recombinant light chain enterokinase activity was greater than accessibility of the target site at the terminus region of the protein. We further showed that the catalytic efficiency of the native holoenzyme was influenced primarily by residues on the N-terminal side of the scissile bond while being neutral to residues on the C-terminal side. Finally, we found that cleavage of all nine fusion proteins reflects accurate hydrolysis at the DDDDK(156) and DDDDK(201) sites when recombinant light chain enterokinase was used while non-specific processing at secondary sites were observed when these fusion proteins were treated with the native holoenzyme.     

Cross talk between the +73/294 interaction and the cleavage site in RNase P RNA mediated cleavage

To monitor functionally important metal ions and possible cross talk in RNase P RNA mediated cleavage we studied cleavage of substrates, where the 2′OH at the RNase P cleavage site (at −1) and/or at position +73 had been replaced with a 2′ amino group (or 2′H). Our data showed that the presence of 2′ modifications at these positions affected cleavage site recognition, ground state binding of substrate and/or rate of cleavage. Cleavage of 2′ amino substituted substrates at different pH showed that substitution of Mg²⁺ by Mn²⁺ (or Ca²⁺), identity of residues at and near the cleavage site, and addition of C5 protein influenced the frequency of miscleavage at −1 (cleavage at the correct site is referred to as +1). From this we infer that these findings point at effects mediated by protonation/deprotonation of the 2′ amino group, i.e. an altered charge distribution, at the site of cleavage. Moreover, our data suggested that the structural architecture of the interaction between the 3′ end of the substrate and RNase P RNA influence the charge distribution at the cleavage site as well as the rate of cleavage under conditions where the chemistry is suggested to be rate limiting. Thus, these data provide evidence for cross talk between the +73/294 interaction and the cleavage site in RNase P RNA mediated cleavage. We discuss the role metal ions might play in this cross talk and the likelihood that at least one functionally important metal ion is positioned in the vicinity of, and use the 2′OH at the cleavage site as an inner or outer sphere ligand.     

Matrix metalloproteinase-1 cleavage site recognition and binding in full-length human type III collagen

Matrix metalloproteinases (MMPs) are essential for normal collagen turnover, recovery from fibrosis, and vascular permeability. In fibrillar collagens, MMP-1, MMP-8, and MMP-13 cleave a specific glycine–isoleucine or glycine–leucine bond, despite the presence of this sequence in other parts of the protein. This cut site specificity has been hypothesized to arise from a unique, relaxed super-secondary structure in this area due to local hydroxyproline poor character. In this study we examined the mechanism of interaction and cleavage of human type III collagen by fibroblast MMP-1 by using a panel of recombinant human type III collagens (rhCIIIs) containing engineered sequences in the vicinity of the cleavage site. Native and recombinant type III collagens had similar biochemical and structural characteristics, as indicated by transmission electron microscopy, circular dichroism spectropolarimetry, melting temperature and hydroxyproline analysis. A single amino acid change at the I785 cleavage site to proline resulted in partial MMP-1 resistance, but cuts were found in novel sites in the original cleavage region. However, the replacement of five Y-position residues by proline in this region, regardless of I785 variation, conferred complete resistance to MMP-1, MMP-8, MMP-13, trypsin, and elastase. MMP-1 had a decreased specific activity towards and reduced cleavage rate of rhCIII I785P but a K_m similar to wild-type. Despite the reductions in protease sensitivity, MMP-1 bound to all of the engineered rhCIIIs with comparable affinity, indicating that MMP-1 binding is not sufficient for cleavage. The relaxed tertiary structure in the MMP cleavage region may permit local collagen unwinding by MMP-1 that enables site-specific proteolysis.     

The C-terminal loop of the homing endonuclease I-CreI is essential for site recognition, DNA binding and cleavage

Meganucleases are sequence-specific endonucleases with large cleavage sites that can be used to induce efficient homologous gene targeting in cultured cells and plants. These enzymes open novel perspectives for genome engineering in a wide range of fields, including gene therapy. A new crystal structure of the I-CreI dimer without DNA has allowed the comparison with the DNA-bound protein. The C-terminal loop displays a different conformation, which suggests its implication in DNA binding. A site-directed mutagenesis study in this region demonstrates that whereas the C-terminal helix is negligible for DNA binding, the final C-terminal loop is essential in DNA binding and cleavage. We have identified two regions that comprise the Ser138–Lys139 and Lys142–Thr143 pairs whose double mutation affect DNA binding in vitro and abolish cleavage in vivo. However, the mutation of only one residue in these sites allows DNA binding in vitro and cleavage in vivo. These findings demonstrate that the C-terminal loop of I-CreI endonuclease plays a fundamental role in its catalytic mechanism and suggest this novel site as a region to take into account for engineering new endonucleases with tailored specificity.     

NAR Breakthrough Article: Sequence-specific cleavage of dsRNA by Mini-III RNase

Ribonucleases (RNases) play a critical role in RNA processing and degradation by hydrolyzing phosphodiester bonds (exo- or endonucleolytically). Many RNases that cut RNA internally exhibit substrate specificity, but their target sites are usually limited to one or a few specific nucleotides in single-stranded RNA and often in a context of a particular three-dimensional structure of the substrate. Thus far, no RNase counterparts of restriction enzymes have been identified which could cleave double-stranded RNA (dsRNA) in a sequence-specific manner. Here, we present evidence for a sequence-dependent cleavage of long dsRNA by RNase Mini-III from Bacillus subtilis (BsMiniIII). Analysis of the sites cleaved by this enzyme in limited digest of bacteriophage Φ6 dsRNA led to the identification of a consensus target sequence. We defined nucleotide residues within the preferred cleavage site that affected the efficiency of the cleavage and were essential for the discrimination of cleavable versus non-cleavable dsRNA sequences. We have also determined that the loop α5b-α6, a distinctive structural element in Mini-III RNases, is crucial for the specific cleavage, but not for dsRNA binding. Our results suggest that BsMiniIII may serve as a prototype of a sequence-specific dsRNase that could possibly be used for targeted cleavage of dsRNA.     

Conformational analysis and proteolytic processing of synthetic pre-pro-GnRH/GAP protein

Homogeneous pre-pro-GnRH/GAP protein was recently synthesized in 100 mg quantities by solid-phase methods and surprisingly, the synthetic pre-pro-protein, which normally does not escape the endoplasmic recticulum, was found to inhibit the release of prolactin from cultured pituitary cells. This is the first demonstration of significant biological activity associated with a precursor protein and provides the rationale for its further study. We now report the results of our initial examination of the conformational properties of pre-pro-GnRH/GAP protein as a prelude to solving its solution phase conformation by homonuclear¹H-NMR protocols. Thermal andpH titration fluorescence and circular dichroism spectroscopies reveal that the protein is resistant to thermal-induced conformational changes but is particularly sensitive topH-induced conformational changes; while Asp/Glu and Arg residues may contribute to structural stability, His and Lys residues predominate. Pre-pro-GnRH/GAP is about 30% helix in the range of 2–40°C; however, even at 90°C, the peptide retains nearly 50% of its helix character. There is no evidence for a cooperative transition; for this reason, differential scanning calorimetry failed to yield a defined transition thermogram. Pre-pro-GnRH/GAP apparently does not pass through a transition state as a function of temperature but appears to flex and retain a high percentage of helix structure, resulting in subtle changes in secondary structure. There is no discernible isodichroic point. On either side of the neutralpH range, however, there are dramatic changes in structure that result in nonreversible denaturation of the protein. Relative to N(Ac)Trp-amide, the emission position of intrinsic Trp fluorescence of pre-pro-GnRH/GAP is blue shifted to 338 nm, indicating that the microenvironment(s) encompassing the 2 Trp residues are buried within the protein structure. Synthetic pre-pro-GNRH/GAP is a substrate for GAP-releasing enzyme (the proposed physiologically relevant processing enzyme of the precursor protein) and yields GAP peptide (D14–I69). Of the other serine proteinases tested (trypsin, plasmin, kallikrein), only GAP-releasing enzyme shows this specificity of cleavage. Hierarchical cleavage observed in the time course of proteolysis with trypsin, however, suggests that other peptide products might be formed from GAP once it is processed from the precursor protein by cleavage at sites other than the primary processing site catalyzed by enzymes other than GAP-releasing enzyme. The primary processing site for GAP-releasing enzyme (GLRPGGKR) is thus accessible in the precursor protein, consistent with our hypothesis that the recognition sequence is located at the surface of the protein and acts as a recognition element for the processing endoproteinase. The conformation of the precursor protein is dynamic, supporting the idea that intracellular (and/or intragranular) conditions may play a role in regulation of endoproteolysis. Conformational flexing of the pro-hormone in response to intracellular conditions may serve to differentially expose various processing sites which may help explain tissue specificity of processing.     

Identification of three sites of proteolytic cleavage in the hinge region between the two domains of the beta 2 subunit of tryptophan synthase of Escherichia coli or Salmonella typhimurium

The beta 2 subunit of tryptophan synthase is composed of two independently folding domains connected by a hinge segment of the polypeptide that is particularly susceptible to limited proteolysis by trypsin [H?gberg-Raibaud, A., & Goldberg, M. (1977) Biochemistry 16, 4014-4019]. Since tryptic cleavage in the hinge region inactivates the beta 2 subunit, the spatial relationship between the two domains is important for enzyme activity. However, it was not previously known whether inactivation results from cleavage of the chain or from the loss of internal fragment(s) subsequent to cleavage at two or more sites. We now report comparative studies of limited proteolysis by three proteinases: trypsin and endoproteinases Lys-C and Arg-C. Our key finding that endoproteinase Arg-C inactivates the beta 2 subunit by cleavage at a single site (Arg-275) demonstrates the important role of the hinge peptide for enzymatic activity. We have also identified the sites of cleavage and the time course of proteolysis by trypsin at Arg-275, Lys-283, and Lys-272 and by endoproteinase Lys-C at Lys-283 and Lys-272. Sodium dodecyl sulfate gel electrophoresis, Edman degradation, and carboxypeptidases B and Y have been used to identify the several fragments and peptides produced. Our finding that the beta 2 subunit and F1 fragments have a heterogeneous amino terminus (Met-1 or Thr-2) indicates that the amino-terminal methionine is incompletely removed during posttranslational modification. Our results show that Edman degradation can be effectively used with a protein of known sequence to analyze proteolytic digests that have at least four different amino-terminal sequences.(ABSTRACT TRUNCATED AT 250 WORDS)