Analysis of the putative cleavage site in human lactasephlorizin hydrolase

1995 ◽  
Vol 23 (2) ◽  
pp. 305S-305S ◽  
Author(s):  
RALF JACOB ◽  
HASSAN Y. NAIM
Keyword(s):  
Cleavage Site ◽  
Putative Cleavage Site

scholarly journals In SilicoAnalysis of ORF1ab in Coronavirus HKU1 Genome Reveals a Unique Putative Cleavage Site of Coronavirus HKU1 3C-Like Protease

2005 ◽  
Vol 49 (10) ◽  
pp. 899-908 ◽  
Author(s):  
Patrick C.Y. Woo ◽  
Yi Huang ◽  
Susanna K.P. Lau ◽  
Hoi-wah Tsoi ◽  
Kwok-yung Yuen
Keyword(s):  
Cleavage Site ◽  
Putative Cleavage Site

scholarly journals Fusogenic Activity of Hepadnavirus Peptides Corresponding to Sequences Downstream of the Putative Cleavage Site

Virology ◽  
1999 ◽  
Vol 261 (1) ◽  
pp. 133-142 ◽  
Author(s):  
Ignacio Rodríguez-Crespo ◽  
Elena Núñez ◽  
Belén Yélamos ◽  
Julián Gómez-Gutiérrez ◽  
Juan P. Albar ◽  
...  
Keyword(s):  
Cleavage Site ◽  
Putative Cleavage Site ◽  
Fusogenic Activity

scholarly journals Characterization of the R572T Point Mutant of a Putative Cleavage Site in Human Foamy Virus Env

2000 ◽  
Vol 74 (6) ◽  
pp. 2949-2954 ◽  
Author(s):  
Anju Bansal ◽  
Kit L. Shaw ◽  
Bradley H. Edwards ◽  
Paul A. Goepfert ◽  
Mark J. Mulligan
Keyword(s):  
Cleavage Site ◽  
Foamy Virus ◽  
Syncytium Formation ◽  
Single Amino Acid ◽  
Human Foamy Virus ◽  
Putative Cleavage Site ◽  
Virus Maturation ◽  
Cell Surface Biotinylation ◽  
Proteolytic Site ◽  
And Function

ABSTRACT A putative cleavage site of the human foamy virus (HFV) envelope glycoprotein (Env) was altered. Transient env expression revealed that the R572T mutant Env was normally expressed and modified by asparagine-linked oligosaccharide chains. However, this single-amino-acid substitution was sufficient to abolish all detectable cleavage of the gp130 precursor polyprotein. Cell surface biotinylation demonstrated that the uncleaved mutant gp130 was transported to the plasma membrane. The uncleaved mutant protein was incapable of syncytium formation. Glycoprotein-driven virion budding, a unique aspect of HFV assembly, occurred despite the absence of Env cleavage. We then substituted the R572T mutant env into a replication-competent HFV molecular clone. Transfection of the mutant viral DNA into BHK-21 cells followed by viral titration with the FAB (foamy virus-activated β-galactosidase expression) assay revealed that proteolysis of the HFV Env was essential for viral infectivity. Wild-type HFV Env partially complemented the defective virus phenotype. Taken together, these experimental results established the location of the HFV Env proteolytic site; the effects of cleavage on Env transport, processing, and function; and the importance of Env proteolysis for virus maturation and infectivity.

scholarly journals An S101P Substitution in the Putative Cleavage Motif of the Human Metapneumovirus Fusion Protein Is a Major Determinant for Trypsin-Independent Growth in Vero Cells and Does Not Alter Tissue Tropism in Hamsters

2005 ◽  
Vol 79 (16) ◽  
pp. 10678-10689 ◽  
Author(s):  
Jeanne H. Schickli ◽  
Jasmine Kaur ◽  
Nancy Ulbrandt ◽  
Richard R. Spaete ◽  
Roderick S. Tang
Keyword(s):  
Amino Acid ◽  
Respiratory Tract ◽  
Amino Acid Substitution ◽  
Cleavage Site ◽  
Vero Cells ◽  
Human Metapneumovirus ◽  
Single Amino Acid ◽  
Putative Cleavage Site ◽  
Single Amino Acid Change ◽  
Fusion Glycoprotein

ABSTRACT Human metapneumovirus (hMPV), a recently described paramyxovirus, is a major etiological agent for lower respiratory tract disease in young children that can manifest with severe cough, bronchiolitis, and pneumonia. The hMPV fusion glycoprotein (F) shares conserved functional domains with other paramyxovirus F proteins that are important for virus entry and spread. For other paramyxovirus F proteins, cleavage of a precursor protein (F0) into F1 and F2 exposes a fusion peptide at the N terminus of the F1 fragment, a likely prerequisite for fusion activity. Many hMPV strains have been reported to require trypsin for growth in tissue culture. The majority of these strains contain RQSR at the putative cleavage site. However, strains hMPV/NL/1/00 and hMPV/NL/1/99 expanded in our laboratory contain the sequence RQPR and do not require trypsin for growth in Vero cells. The contribution of this single amino acid change was verified directly by generating recombinant virus (rhMPV/NL/1/00) with either proline or serine at position 101 in F. These results suggested that cleavage of F protein in Vero cells could be achieved by trypsin or S101P amino acid substitution in the putative cleavage site motif. Moreover, trypsin-independent cleavage of hMPV F containing 101P was enhanced by the amino acid substitution E93K. In hamsters, rhMPV/93K/101S and rhMPV/93K/101P grew to equivalent titers in the respiratory tract and replication was restricted to respiratory tissues. The ability of these hMPV strains to replicate efficiently in the absence of trypsin should greatly facilitate the generation, preclinical testing, and manufacturing of attenuated hMPV vaccine candidates.

scholarly journals Proteolysis ofmecARepressor Is Essential for Expression of Methicillin Resistance by Staphylococcus aureus

2013 ◽  
Vol 57 (4) ◽  
pp. 2001-2002 ◽  
Author(s):  
Pedro Arêde ◽  
Duarte C. Oliveira
Keyword(s):  
Staphylococcus Aureus ◽  
Point Mutation ◽  
Cleavage Site ◽  
Methicillin Resistance ◽  
The Novel ◽  
Putative Cleavage Site ◽  
Component System ◽  
Content Type ◽  
Regulatory Locus ◽  
Gene Coding

ABSTRACTRecently, we have demonstrated that the cognate regulatory locus of themecAgene in methicillin-resistantStaphylococcus aureus(MRSA) is in fact a three-component system containing the novelmecR2gene coding for an antirepressor. MecR2 interacts with the repressor MecI, disturbing its binding to themecApromoter and fostering its proteolysis. Here, we engineered a point mutation in the putative cleavage site of MecI and demonstrated that MecI proteolysis is strictly required for the optimal expression of β-lactam resistance.

scholarly journals Identification of the Murine Coronavirus MP1 Cleavage Site Recognized by Papain-Like Proteinase 2

2003 ◽  
Vol 77 (13) ◽  
pp. 7376-7382 ◽  
Author(s):  
Amornrat Kanjanahaluethai ◽  
Dalia Jukneliene ◽  
Susan C. Baker
Keyword(s):  
Cleavage Site ◽  
Edman Degradation ◽  
Putative Cleavage Site ◽  
Amino Terminal ◽  
Cleavage Assay ◽  
Degradation Reaction ◽  
Methionine Residues ◽  
Identification And Characterization ◽  
Murine Coronavirus

ABSTRACT The replicase polyprotein of murine coronavirus is extensively processed by three proteinases, two papain-like proteinases (PLPs), termed PLP1 and PLP2, and a picornavirus 3C-like proteinase (3CLpro). Previously, we established a trans-cleavage assay and showed that PLP2 cleaves the replicase polyprotein between p210 and membrane protein 1 (MP1) (A. Kanjanahaluethai and S. C. Baker, J. Virol. 74:7911-7921, 2000). Here, we report the results of our studies identifying and characterizing this cleavage site. To determine the approximate position of the cleavage site, we expressed constructs that extended various distances upstream from the previously defined C-terminal end of MP1. We found that the construct extending from the putative PLP2 cleavage site at glycine 2840-alanine 2841 was most similar in size to the processed MP1 replicase product generated in a trans-cleavage assay. To determine which amino acids are critical for PLP2 recognition and processing, we generated 14 constructs with amino acid substitutions upstream and downstream of the putative cleavage site and assessed the effects of the mutations in the PLP2 trans-cleavage assay. We found that substitutions at phenylalanine 2835, glycine 2839, or glycine 2840 resulted in a reduction in cleavage of MP1. Finally, to unequivocally identify this cleavage site, we isolated radiolabeled MP1 protein and determined the position of [35S]methionine residues released by Edman degradation reaction. We found that the amino-terminal residue of MP1 corresponds to alanine 2841. Therefore, murine coronavirus PLP2 cleaves the replicase polyprotein between glycine 2840 and alanine 2841, and the critical determinants for PLP2 recognition and processing occupy the P6, P2, and P1 positions of the cleavage site. This study is the first report of the identification and characterization of a cleavage site recognized by murine coronavirus PLP2 activity.

Properties of Chimeric (Tissue-Type/Urokinase-Type) Plasminogen Activators Obtained by Fusion at the Plasmin Cleavage Site

1993 ◽  
Vol 69 (05) ◽  
pp. 466-472 ◽  
Author(s):  
M Colucci ◽  
L G Cavallo ◽  
G Agnelli ◽  
A Mele ◽  
R Bürgi ◽  
...  
Keyword(s):  
Molecular Weight ◽  
Plasminogen Activator ◽  
Cleavage Site ◽  
Plasminogen Activators ◽  
Tissue Type ◽  
Low Molecular Weight ◽  
Single Chain ◽  
Type Plasminogen Activator ◽  
Kringle 2 ◽  
Fibrin Monomers

SummaryTwo hybrid plasminogen activators (K2tu-PA and FK2tu-PA), linking the kringle 2 domain or the finger plus the kringle 2 domains of tissue-type plasminogen activator (t-PA) to the catalytic domain of single-chain urokinase-type plasminogen activator (scu-PA) were studied. At variance with similar constructs previously reported, they were obtained by fusion of the t-PA and scu-PA derived portions at their plasmin cleavage site (between Arg275 of t-PA and Ile159 of scu-PA), thus eliminating from scu-PA the two peptide bonds (Glu143-Leu144 and Arg156-Phe157) that lead to low molecular weight scu-PA and to thrombin-inactivated tcu-PA. The specific activities of K2tu-PA and FK2tu-PA, as measured by fibrin plate were 2.5 × 106 and 1.0 × 106 t-PA equivalent units/mg, respectively. Activation of plasminogen by hybrid PAs was stimulated by both CNBr-digested fibrinogen (40- and 80-fold) and Des-A-fibrin monomers (6- and 12-fold). The relatively weak stimulation of chimeric PAs by minimally degraded fibrin monomers was consistent with their reduced fibrin binding capacity. Like scu-PA, the chimeric PAs, in the single-chain form, were insensitive to inhibition, as they retained full activity after prolonged incubation in plasma and did not interact with SDS-reactivated recombinant PAI-1. The concentration producing 50% lysis of blood clots in 3 h was 0.5 μg/ml for K2tu-PA and 1 μg/ml for FK2tu-PA, as compared to 0.5 μg/ml and >2 μg/ml for t-PA and scu-PA, respectively. Plasminogen and α2-antiplasmin consumption induced by the hybrid PAs in clot-free plasma was comparable to (K2tu-PA) or lower than (FK2tu-PA) that induced by either t-PA or scu-PA. When exposed to plasmin, the hybrids were completely converted into two-chain molecules with full enzymatic activity. At variance with u-PA, however, the two-chain recombinant activators still required fibrin for full expression of activity. These data indicate that the products of such “artificial” fusion behave like true chimeras without loss of biological activity. The insensitivity to thrombin inactivation and to the proteolytic cleavage leading to low molecular weight scu-PA might confer enhanced stability to the molecules, especially at thrombus level. Moreover, if the thrombolytic activity observed in vitro is maintained in vivo, the prolonged half life of these hybrids should result in higher plasma levels of activator and thus in more extensive and rapid lysis.

RNA interference tolerates 2'-fluoro modifications at the Argonaute2 cleavage site

2008 ◽  
Author(s):  
Tuula Tennilä ◽  
Pirkko Muhonen ◽  
Elena Azhayeva ◽  
H. Kalervo Väänänen ◽  
Alex Azhayev ◽  
...  
Keyword(s):  
Rna Interference ◽  
Cleavage Site
Sign in / Sign up

Export Citation Format

Share Document