IMPROVEMENT OF T-PA PROPERTIES BY MEANS OF SITE DIRECTED MUTAGENESIS

1987 ◽  
Author(s):  
N Haigwood ◽  
E-P Pâques ◽  
G Mullenbach ◽  
G Moore ◽  
L DesJardin ◽  
...  
Keyword(s):  
Amino Acids ◽  
Cleavage Site ◽  
Specific Activity ◽  
Biological Properties ◽  
Site Directed Mutagenesis ◽  
Directed Mutagenesis ◽  
Mutant Proteins ◽  
C Terminus

The clinical relevance of tissue-plasminogen-activator (t-PA) as a potent thrombolytic agent has recently been established. It has however been recognized that t-PA does not fulfill all conditions required for an ideal thrombolytic pharmaceutical agent; for example, its physiological stability and its short half life in vivo necessitate the use of very large clinical doses. We have therefore attempted to develop novel mutant t-PA proteins with improved properties by creating mutants by site-directed mutagenesis in M13 bacteriophage. Seventeen mutants were designed, cloned, and expressed in CHO cells. Modifications were of three types: alterations to glycosylation sites, truncations of the N- or C-termini, and amino acids changes at the cleavage site utilized to generate the two chain form of t-PA. The mutant proteins were analyzed in vitro for specific activity, fibrin dependence of the plasminogen activation, fibrin affinity, and susceptibility to inhibition by PAI.In brief, the results are: 1) some unglycosylated and partially glycosylated molecules obtained by mutagenesis are characterized by several-fold higher specific activity than wild type t-PA; 2) truncation at the C-terminus by three amino acids yields a molecule with increased fibrin specificity; 3) mutations at the cleavage site lead zo a decreased inhibition by PAI; and 4) recombinants of these genes have been constructed and the proteins were shown to possess multiple improved properties. The use of site directed mutagenesis has proved to be a powerful instrument to modulate the biological properties of t-PA.

scholarly journals Specificity of human rhinovirus 2Apro is determined by combined spatial properties of four cleavage site residues

2013 ◽  
Vol 94 (7) ◽  
pp. 1535-1546 ◽  
Author(s):  
David Neubauer ◽  
Martina Aumayr ◽  
Irene Gösler ◽  
Tim Skern
Keyword(s):  
Amino Acids ◽  
Substrate Specificity ◽  
Cleavage Site ◽  
Antiviral Agents ◽  
Genetic Group ◽  
Human Rhinovirus ◽  
C Terminus ◽  
Group B

The 2A proteinase (2Apro) of human rhinoviruses cleaves the virally encoded polyprotein between the C terminus of VP1 and its own N terminus. Poor understanding of the 2Apro substrate specificity of this enzyme has hampered progress in developing inhibitors that may serve as antiviral agents. We show here that the 2Apro of human rhinovirus (HRV) 1A and 2 (rhinoviruses from genetic group A) cannot self-process at the HRV14 (a genetic group B rhinovirus) cleavage site. When the amino acids in the cleavage site of HRV2 2Apro (Ile-Ile-Thr-Thr-Ala*Gly-Pro-Ser-Asp) were singly or doubly replaced with the corresponding HRV14 residues (Asp-Ile-Lys-Ser-Tyr*Gly-Leu-Gly-Pro) at positions from P3 to P2′, HRV1A and HRV2 2Apro cleavage took place at WT levels. However, when three or more positions of the HRV1A or 2 2Apro were substituted (e.g. at P2, P1 and P2′), cleavage in vitro was essentially eliminated. Introduction of the full HRV14 cleavage site into a full-length clone of the HRV1A and transfection of HeLa cells with a transcribed RNA did not give rise to viable virus. In contrast, revertant viruses bearing cysteine at the P1 position or proline at P2′ were obtained when an RNA bearing the three inhibitory amino acids was transfected. Reversions in the enzyme affecting substrate specificity were not found in any of the in vivo experiments. Modelling of oligopeptide substrates onto the structure of HRV2 2Apro revealed no appreciable differences in residues of HRV2 and HRV14 in the respective substrate binding sites, suggesting that the overall shape of the substrate is important in determining binding efficiency.

scholarly journals The SKHR Motif Is Required for Biological Function of the VirR Response Regulator from Clostridium perfringens

2003 ◽  
Vol 185 (20) ◽  
pp. 6205-6208 ◽  
Author(s):  
Sheena McGowan ◽  
Jennifer R. O'Connor ◽  
Jackie K. Cheung ◽  
Julian I. Rood
Keyword(s):  
Amino Acids ◽  
Dna Binding ◽  
Clostridium Perfringens ◽  
Biological Function ◽  
Response Regulator ◽  
Site Directed Mutagenesis ◽  
Directed Mutagenesis ◽  
Toxin Gene

ABSTRACT The response regulator VirR and its cognate sensor histidine kinase, VirS, are responsible for toxin gene regulation in the human pathogen Clostridium perfringens. The C-terminal domain of VirR (VirRc) contains the functional FxRxHrS motif, which is involved in DNA binding and is conserved in many regulatory proteins. VirRc was cloned, purified, and shown by in vivo and in vitro studies to comprise an independent DNA binding domain. Random and site-directed mutagenesis was used to identify further amino acids that were required for the functional integrity of the protein. Random mutagenesis identified a unique residue, Met-172, that was required for biological function. Site-directed mutagenesis of the SKHR motif (amino acids 216 to 219) revealed that these residues were also required for biological activity. Analysis of the mutated proteins indicated that they were unable to bind to the DNA target with the same efficiency as the wild-type protein.

scholarly journals Mutational Analysis of the MutH Protein fromEscherichia coli

2000 ◽  
Vol 276 (15) ◽  
pp. 12113-12119 ◽  
Author(s):  
Tamalette Loh ◽  
Kenan C. Murphy ◽  
Martin G. Marinus
Keyword(s):  
Amino Acids ◽  
Dna Binding ◽  
Mutational Analysis ◽  
Site Directed Mutagenesis ◽  
Mutant Proteins ◽  
Flexible Loop ◽  
Loop Regions ◽  
Binding Residues

Site-directed mutagenesis was performed on several areas of MutH based on the similarity of MutH andPvuII structural models. The aims were to identify DNA-binding residues; to determine whether MutH has the same mechanism for DNA binding and catalysis asPvuII; and to localize the residues responsible for MutH stimulation by MutL. No DNA-binding residues were identified in the two flexible loop regions of MutH, although similar loops inPvuII are involved in DNA binding. Two histidines in MutH are in a similar position as two histidines (His-84 and His-85) inPvuII that signal for DNA binding and catalysis. These MutH histidines (His-112 and His-115) were changed to alanines, but the mutant proteins had wild-type activity bothin vivoandin vitro. The results indicate that the MutH signal for DNA binding and catalysis remains unknown. Instead, a lysine residue (Lys-48) was found in the first flexible loop that functions in catalysis together with the three presumed catalytic amino acids (Asp-70, Glu-77, and Lys-79). Two deletion mutations (MutHΔ224 and MutHΔ214) in the C-terminal end of the protein, localized the MutL stimulation region to five amino acids (Ala-220, Leu-221, Leu-222, Ala-223, and Arg-224).

scholarly journals The consequences of deglycosylation of recombinant intra-melanosomal domain of human tyrosinase

2017 ◽  
Vol 399 (1) ◽  
pp. 73-77 ◽  
Author(s):  
Monika B. Dolinska ◽  
Yuri V. Sergeev
Keyword(s):  
Oculocutaneous Albinism ◽  
Site Directed Mutagenesis ◽  
Directed Mutagenesis ◽  
Multiple Site ◽  
Insect Larvae ◽  
Melanin Biosynthesis ◽  
Human Tyrosinase

AbstractTyrosinase, a melanosomal glycoenzyme, catalyzes initial steps of the melanin biosynthesis. While glycosylation was previously studiedin vivo, we present three recombinant mutant variants of human tyrosinase, which were obtained using multiple site-directed mutagenesis, expressed in insect larvae, purified and characterized biochemically. The mutagenesis demonstrated the reduced protein expression and enzymatic activity due to possible loss of protein stability and protein degradation. However, the complete deglycosylation of asparagine residuesin vitro, including the residue in position 371, interrupts tyrosinase function, which is consistent with a melanin loss in oculocutaneous albinism type 1 (OCA1) patients.

scholarly journals Site-directed mutagenesis at aspartate and glutamate residues of xylanase from Bacillus pumilus

1992 ◽  
Vol 288 (1) ◽  
pp. 117-121 ◽  
Author(s):  
E P Ko ◽  
H Akatsuka ◽  
H Moriyama ◽  
A Shinmyo ◽  
Y Hata ◽  
...  
Keyword(s):  
Amino Acids ◽  
Reaction Mechanism ◽  
Amino Acid ◽  
Bacillus Pumilus ◽  
Specific Activity ◽  
Sequence Similarity ◽  
Site Directed Mutagenesis ◽  
Dimensional Structure ◽  
Amino Acid Sequence Similarity ◽  
Directed Mutagenesis

To elucidate the reaction mechanism of xylanase, the identification of amino acids essential for its catalysis is of importance. Studies have indicated the possibility that the reaction mechanism of xylanase is similar to that of hen's egg lysozyme, which involves acidic amino acid residues. On the basis of this assumption, together with the three-dimensional structure of Bacillus pumilus xylanase and its amino acid sequence similarity to other xylanases of different origins, three acidic amino acids, namely Asp-21, Glu-93 and Glu-182, were selected for site-directed mutagenesis. The Asp residue was altered to either Ser or Glu, and the Glu residues to Ser or Asp. The purified mutant xylanases D21E, D21S, E93D, E93S, E182D and E182S showed single protein bands of about 26 kDa on SDS/PAGE. C.d. spectra of these mutant enzymes show no effect on the secondary structure of xylanase, except that of D21E, which shows a little variation. Furthermore, mutations of Glu-93 and Glu-182 resulted in a drastic decrease in the specific activity of xylanase as compared with mutation of Asp-21. On the basis of these results we propose that Glu-93 and Glu-182 are the best candidates for the essential catalytic residues of xylanase.

DIRECTED MUTAGENESIS IN THE STUDY OF THE REQUIREMENTS FOR FACTOR VIII ACTIVITY IN VITRO AND IN VIVO

1987 ◽  
Author(s):  
Randal J Kaufman ◽  
Debra D Pittman ◽  
Louise C Wasley ◽  
W Barry Foster ◽  
Godfrey W Amphlett ◽  
...  
Keyword(s):  
Amino Acids ◽  
Factor Viii ◽  
Cleavage Site ◽  
Factor Xa ◽  
Cleavage Sites ◽  
Thrombin Cleavage ◽  
Terminal Fragment ◽  
Cofactor Activity

Factor VIII is a high molecular weight plasma glycoprotein that functions in the blood clotting cascade as the cofactor for factor DCa proteolytic activation of factor X. Factor VIII does not function proteolytically in this reaction hut itself can be proteolytically activated by other coagulation enzymes such as factor Xa and thrombin. In the plasma, factor VIII exists as a 200 kDa amino-terminal fragment in a metal ion stabilized complex with a 76 kDa carboxy-terminal fragment. The isolation of the cENA for human factor VIII provided the deduced primary amino acid sequence of factor VIIT and revealed three distinct structural domains: 1) a triplicated A domain of 330 amino acids which has homology to ceruloplasmin, a plasma copper binding protein, 2) a duplicated C domain of 150 amino acids, and 3) a unique B domain of 980 amino acids. These domains are arranged as shown below. We have previously reported the B domain is dispensible far cofactor activity in vitro (Toole et al. 1986 Proc. Natl. Acad 5939). The in vivo efficacy of factor VIII molecules harboring the B domain deletion was tested by purification of the wildtype and modified forms and infusion into factor VIII deficient, hemophilic, dogs. The wildtype and the deleted forms of recombinant derived factor VIII exhibited very similar survival curves (Tl/2 = 13 hrs) and the cuticle bleeding times suggested that both preparations appeared functionally equivalent. Sepharose 4B chromatography indicated that both factor VIII molecules were capable of binding canine plasma vWF.Further studies have addressed what cleavages are necessary for activation of factor VIII. The position of the thrombin, factor Xa, and activated protein C (AFC) cleavage sites within factor VIII are presented below, site-directed ENA medicated mutagenesis has been performed to modify the arginine at the amino side of each cleavagesite to an soleucine. In all cases this modification resulted in molecules that were resistant to cleavage by thrombin at the modified site. Modification of the thrombin cleavage sites at 336 and 740 and modification of the factor Xa cleavage site at 1721 resulted in no loss of cofactor activity. Modification of the thrombin cleavage site at either 372 or 1689 destroyed oofactor activity. Modification of the thrombin cleavage site at 336 resulted in a factor VIII having an increased activity, possibly due to resistance to inactivation. These results suggest the requirement of cleavage at residues 372 and 1689 for cofactor activity.

Proteinsynthese in grünen Blättern

1964 ◽  
Vol 19 (3) ◽  
pp. 235-248 ◽  
Author(s):  
Benno Parthier
Keyword(s):  
Amino Acids ◽  
Specific Activity ◽  
Subcellular Fractions ◽  
Mechanical Destruction ◽  
Cell Organization ◽  
Specific Activities ◽  
Short Time ◽  
Natural Cell

In the green leaves of Nicotiana rustica, protein synthesis of various subcellular fractions has been investigated in vivo after 14CO2-photosynthesis and also in vitro by incorporation of radioactive amino acids. Following photosynthesis, homogenization of the tissues, and differential centrifugation of the homogenates, the results show that all structural particles of the cell are able to use photosynthetically formed amino acids for the incorporation into their proteins. The proteins with the highest specific activities are found in the mitochondria-rich fractions, and with the lowest in the soluble cytoplasma supernatant. High specific activities are also observed in the ribosomal-rich fraction in short-time experiments, and also in the chloroplasts after exposure of the leaves to light. After an osmotic-mechanical destruction of the isolated 14C-labelled chloroplasts, the specific activities of lamellar proteins exceed the colourless soluble proteins of the chloroplasts. A green fraction, sedimented at 1,000 g, and perhaps mainly consisting of broken and leached chloroplasts, shows the highest specific activity of all chloroplast fractions. Obviously, due to the destruction of the natural cell organization, in vitro experiments give not only drastically decreased specific activities but also another distribution of the incorporated amino acids between the subcellular fractions, compared with experiments in vivo.

scholarly journals The C-Terminal 88 Amino Acids of the Sendai Virus P Protein Have Multiple Functions Separable by Mutation

2002 ◽  
Vol 76 (1) ◽  
pp. 68-77 ◽  
Author(s):  
Jeffery Tuckis ◽  
Sherin Smallwood ◽  
Joyce A. Feller ◽  
Sue A. Moyer
Keyword(s):  
Amino Acids ◽  
Protein Interactions ◽  
Sendai Virus ◽  
Intact Cell ◽  
Binding Domain ◽  
Site Directed Mutagenesis ◽  
Genome Replication ◽  
Multiple Functions

ABSTRACT The Sendai virus P-L polymerase complex binds the NP-encapsidated nucleocapsid (NC) template through a P-NP interaction. To identify P amino acids responsible for binding we performed site-directed mutagenesis on the C-terminal 88 amino acids in the NC binding domain. The mutant P proteins expressed from plasmids were assayed for viral RNA synthesis and for various protein-protein interactions. All the mutants formed P oligomers and bound to L protein. While two mutants, JT3 and JT8, retained all P functions at or near the levels of wild-type (wt) P, three others—JT4, JT6, and JT9—were completely defective for both transcription and genome replication in vitro. Each of the inactive mutants retained significant NC binding but had a different spectrum of other binding interactions and activities, suggesting that the NC binding domain also affects the catalytic function of the polymerase. NC binding was inhibited by combinations of the inactive mutations. The remaining P mutants were active in transcription but defective in various aspects of genome replication. Some P mutants were defective in NP0 binding and abolished the reconstitution of replication from separate P-L and NP0-P complexes. In some of these cases the coexpression of the wt polymerase with the mutant NP0-P complex could rescue the defect in replication, suggesting an interaction between these complexes. For some P mutants replication occurred in vivo, but not in vitro, suggesting that the intact cell is providing an unknown function that cannot be reproduced in extracts of cells. Thus, the C-terminal region of P is complex and possesses multiple functions besides NC binding that can be separated by mutation.

scholarly journals S. mansoni SmKI-1 Kunitz-domain: Leucine point mutation at P1 site generates enhanced neutrophil elastase inhibitory activity

2021 ◽  
Vol 15 (1) ◽  
pp. e0009007
Author(s):  
Fábio Mambelli ◽  
Bruno P. O. Santos ◽  
Suellen B. Morais ◽  
Enrico G. T. Gimenez ◽  
Duana C. dos S. Astoni ◽  
...  
Keyword(s):  
Inhibitory Activity ◽  
Neutrophil Elastase ◽  
Point Mutations ◽  
Point Of View ◽  
Mutant Proteins ◽  
Inflammatory Parameters ◽  
C Terminus ◽  
Kunitz Domain

The Schistosoma mansoni SmKI-1 protein is composed of two domains: a Kunitz-type serine protease inhibitor motif (KD) and a C-terminus domain with no similarity outside the genera. Our previous work has demonstrated that KD plays an essential role in neutrophil elastase (NE) binding blockage, in neutrophil influx and as a potential anti-inflammatory molecule. In order to enhance NE blocking capacity, we analyzed the KD sequence from a structure-function point of view and designed specific point mutations in order to enhance NE affinity. We substituted the P1 site residue at the reactive site for a leucine (termed RL-KD), given its central role for KD’s inhibition to NE. We have also substituted a glutamic acid that strongly interacts with the P1 residue for an alanine, to help KD to be buried on NE S1 site (termed EA-KD). KD and the mutant proteins were evaluated in silico by molecular docking to human NE, expressed in Escherichia coli and tested towards its NE inhibitory activity. Both mutated proteins presented enhanced NE inhibitory activity in vitro and RL-KD presented the best performance. We further tested RL-KD in vivo in an experimental model of monosodium urate (MSU)-induced acute arthritis. RL-KD showed reduced numbers of total cells and neutrophils in the mouse knee cavity when compared to KD. Nevertheless, both RL-KD and KD reduced mice hypernociception in a similar fashion. In summary, our results demonstrated that both mutated proteins showed enhanced NE inhibitory activity in vitro. However, RL-KD had a prominent effect in diminishing inflammatory parameters in vivo.

scholarly journals Activation of GPR116/ADGRF5 by its tethered agonist requires key amino acids in extracellular loop 2 of the transmembrane domain

2021 ◽  
Author(s):  
James P Bridges ◽  
Caterina Safina ◽  
Bernard Picard ◽  
Kari Brown ◽  
Alyssa Filuta ◽  
...  
Keyword(s):  
Amino Acids ◽  
Transmembrane Domain ◽  
Receptor Activation ◽  
Site Directed Mutagenesis ◽  
Peptide Sequence ◽  
Knock Out ◽  
Autocatalytic Cleavage ◽  
Knock Out Mice

The mechanistic details of the tethered agonist mode of activation for adhesion GPCRs has not been completely deciphered. We set out to investigate the physiologic importance of autocatalytic cleavage upstream of the agonistic peptide sequence, an event necessary for NTF displacement and subsequent receptor activation. To examine this hypothesis, we characterized tethered agonist-mediated activation of GPR116 in vitro and in vivo. A knock-in mouse expressing a non-cleavable GPR116 mutant phenocopies the pulmonary phenotype of GPR116 knock-out mice, demonstrating that tethered agonist-mediated receptor activation is indispensable for function in vivo. Using site-directed mutagenesis and species swapping approaches we identified key conserved amino acids for GPR116 activation in the tethered agonist sequence and in extracellular loops 2/3 (ECL2/3). We further highlight residues in transmembrane7 (TM7) that mediate stronger signaling in mouse versus human GPR116 and recapitulate these findings in a model supporting tethered agonist:ECL2 interactions for GPR116 activation.

Sign in / Sign up

Export Citation Format

Share Document