Mutational and functional analysis of the Leishmania surface metalloproteinase GP63: similarities to matrix metalloproteinases

Parasitology ◽  
1994 ◽  
Vol 108 (S1) ◽  
pp. S29-S36 ◽  
Author(s):  
W. R. McMaster ◽  
C. J. Morrison ◽  
M. H. Macdonald ◽  
P. B. Joshi
Keyword(s):  
Matrix Metalloproteinases ◽  
Life Stage ◽  
Expression System ◽  
Baculovirus Expression System ◽  
Site Directed Mutagenesis ◽  
Surface Glycoprotein ◽  
Proteinase Activity ◽  
Mammalian Host ◽  
Targeted Gene Deletion ◽  
Major Surface

SUMMARYThe major surface glycoprotein of Leishmania, referred to as GP63, is a zinc metalloproteinase of 63000 Mr present on promastigotes and amastigotes from diverse species of Leishmania. GP63 shares several characteristics with the members of the matrix metalloproteinase family including degradation of at least one component of the extracellular matrix, location at the cell surface, requirement for Zn2+ for proteinase activity and inhibition of the proteinase activity by chelating agents and α2–macroglobulin. Site-directed mutagenesis of the cloned L. major GP63 genes was carried out to determine whether the proposed active site of Leishmania GP63 was homologous to those of other zinc metalloproteinases. The codon encoding the catalytic glutamic acid was modified to encode an aspartic acid and when expressed in COS–7 cells the resulting mutant GP63 had no demonstrable proteinase activity compared to wild type GP63. GP63 was predicted to be synthesized as a precursor protein containing a pro region at the NH2–terminus of GP63 implicated to be involved with the regulation of proteinase activity. As with many other proteinases, including matrix metalloproteinases, these enzymes are synthesized as latent proteinases that require activation for full proteinase activity. L. major recombinant GP63 (rGP63) has been produced in the baculovirus expression system where rGP63 was secreted as a latent proteinase. To study the activation of baculovirus rGP63, purified rGP63 was incubated with the mercurial compound, HgCl2, at concentrations previously shown to result in activation of other latent matrix degrading metalloproteinases and resulted in a significant enhancement of GP63 proteinase activity. The similarity of GP63 to the family of matrix-degrading proteinases suggests that the proteinase activity of GP63 maybe involved with the pathology of lesion formation in the mammalian host and may also be involved with the promastigote life stage in the sandfly vector. To study the functional role of GP63 proteinase, mutant strains of L. major, deficient in the expression of GP63, are currently being derived by targeted gene deletion. Using this strategy results have demonstrated the deletion of an entire L. major GP63 locus, containing in total six GP63 genes. Strategies to delete the second GP63 gene locus are developed and will determine whether deletion of both loci results in viable promastigotes. L. major strains deficient in the expression of GP63 may then be used to address the function of GP63 glycoprotein in the life cycle of Leishmania.

scholarly journals Diversity and complexity of the large surface protein family in the compacted genomes of various Pneumocystis species

2019 ◽  
Author(s):  
Liang Ma ◽  
Zehua Chen ◽  
Da Wei Huang ◽  
Ousmane H. Cissé ◽  
Jamie L. Rothenburger ◽  
...  
Keyword(s):  
Antigenic Variation ◽  
Life Stage ◽  
Surface Protein ◽  
Opportunistic Pathogen ◽  
Surface Proteins ◽  
Surface Glycoprotein ◽  
Mammalian Host ◽  
Expression Site ◽  
Phylogeny And Evolution ◽  
Major Surface

AbstractPneumocystis, a major opportunistic pathogen in patients with a broad range of immunodeficiencies, contains abundant surface proteins encoded by a multi-copy gene family, termed the major surface glycoprotein (Msg) gene superfamily. This superfamily has been identified in all Pneumocystis species characterized to date, highlighting its important role in Pneumocystis biology. In this report, through a comprehensive and in-depth characterization of 459 msg genes from 7 Pneumocystis species, we demonstrate, for the first time, the phylogeny and evolution of conserved domains in Msg proteins, and provide detailed description of the classification, unique characteristics and phylogenetic relatedness of five Msg families. We further describe the relative expression levels of individual msg families in two rodent Pneumocystis species, the substantial variability of the msg repertoires in P. carinii from laboratory and wild rats, and the distinct features of the expression site for the classic msg genes in Pneumocystis from 8 mammalian host species. Our analysis suggests a wide variety of functions for this superfamily, not only conferring antigenic variation to allow immune evasion but also mediating life-stage development, optimizing cell mobility and adhesion, and adapting to specific host niches or environmental conditions. This study provides a rich source of information that lays the foundation for the continued experimental exploration of the functions of the Msg superfamily in Pneumocystis biology.

scholarly journals Variable Surface Glycoprotein from Trypanosoma brucei Undergoes Cleavage by Matrix Metalloproteinases: An in silico Approach

Pathogens ◽  
2019 ◽  
Vol 8 (4) ◽  
pp. 178
Author(s):  
Cláudia Jassica Gonçalves Moreno ◽  
Taffarel Torres ◽  
Marcelo Sousa Silva
Keyword(s):  
Matrix Metalloproteinases ◽  
Trypanosoma Brucei ◽  
Cleavage Site ◽  
Surface Protein ◽  
Matrix Metalloproteases ◽  
Surface Glycoprotein ◽  
Mammalian Host ◽  
Variable Surface Glycoprotein ◽  
Biological Studies ◽  
Variable Surface

In order to survive as extracellular parasites in the mammalian host environment, Trypanosoma brucei has developed efficient mechanisms of immune system evasion, which include the abundant expression of a variable surface glycoprotein (VSG) coat. VSGs are anchored in the parasite membrane by covalent C-terminal binding to glycosylphosphatidylinositol and may be periodically removed by a phospholipase C (PLC) and a major surface protein (TbMSP). VSG molecules show extraordinary antigenic diversity and a comparative analysis of protein sequences suggests that conserved elements may be a suitable target against African trypanosomiasis. However, the cleavage mechanisms of these molecules remain unclear. Moreover, in protozoan infections, including those caused by Trypanosoma brucei, it is possible to observe an increased expression of the matrix metalloproteinases (MMPs). To address the cleavage mechanism of VSGs, the PROSPER server was used for the identification of VSG sequence cleavage sites. After data compilation, it was observed that 64 VSG consensus sequences showed a high conservation of hydrophobic residues, such as valine (V), methionine (M), leucine (L) and isoleucine (I) in the fifth position—the exact location of the cleavage site. In addition, the PROSPER server identified conserved cleavage site portions of VSG proteins recognized by three matrix metalloproteases (gelatinases: MMP-2, MMP-3 and MMP-9). However, further biological studies are needed in order to analyze and confirm this prediction.

scholarly journals Trypanosoma brucei Interaction with Host: Mechanism of VSG Release as Target for Drug Discovery for African Trypanosomiasis

2019 ◽  
Vol 20 (6) ◽  
pp. 1484 ◽  
Author(s):  
Cláudia Moreno ◽  
Adriana Temporão ◽  
Taffarel Torres ◽  
Marcelo Sousa Silva
Keyword(s):  
Drug Discovery ◽  
Phospholipase C ◽  
Trypanosoma Brucei ◽  
Antigenic Variation ◽  
Surface Glycoprotein ◽  
Mammalian Host ◽  
African Trypanosomiasis ◽  
Variable Surface Glycoprotein ◽  
Variable Surface ◽  
Major Surface

The protozoan Trypanosoma brucei, responsible for animal and human trypanosomiasis, has a family of major surface proteases (MSPs) and phospholipase-C (PLC), both involved in some mechanisms of virulence during mammalian infections. During parasitism in the mammalian host, this protozoan is exclusively extracellular and presents a robust mechanism of antigenic variation that allows the persistence of infection. There has been incredible progress in our understanding of how variable surface glycoproteins (VSGs) are organised and expressed, and how expression is switched, particularly through recombination. The objective of this manuscript is to create a reflection about the mechanisms of antigenic variation in T. brucei, more specifically, in the process of variable surface glycoprotein (VSG) release. We firstly explore the mechanism of VSG release as a potential pathway and target for the development of anti-T. brucei drugs.

scholarly journals Immunodominant surface epitopes power immune evasion in the African trypanosome

2021 ◽  
Author(s):  
Anastasia Gkeka ◽  
Francisco Aresta-Branco ◽  
Gianna Triller ◽  
Evi P Vlachou ◽  
Mirjana Lilic ◽  
...  
Keyword(s):  
Immune Evasion ◽  
Antigenic Variation ◽  
Cross Reactivity ◽  
Surface Glycoprotein ◽  
Mammalian Host ◽  
African Trypanosome ◽  
Repetitive Nature ◽  
Variable Surface ◽  
Major Surface ◽  
Immunodominant Epitopes

The African trypanosome survives the immune response of its mammalian host by antigenic variation of its major surface antigen (the Variable Surface Glycoprotein, or VSG). Here we describe the antibody repertoires elicited by different VSGs. We show that the repertoires are highly restricted, directed predominantly to epitopes on the surface of the VSGs. They are also highly discriminatory: minor alterations within these exposed epitopes confer antigenically-distinct properties to these VSGs and elicit different repertoires. We propose that the patterned and repetitive nature of the VSG coat focuses host immunity to a restricted set of immunodominant epitopes per VSG, eliciting a highly stereotyped response, minimizing cross reactivity between different VSGs and facilitating prolonged immune evasion through epitope variation.

scholarly journals Identification of an important cysteine residue in human glutamate–cysteine ligase catalytic subunit by site-directed mutagenesis

1998 ◽  
Vol 336 (3) ◽  
pp. 675-680 ◽  
Author(s):  
Zhongheng TU ◽  
M. W. ANDERS
Keyword(s):  
Expression System ◽  
Baculovirus Expression System ◽  
Cysteine Residue ◽  
Catalytic Subunit ◽  
Site Directed Mutagenesis ◽  
Regulatory Subunit ◽  
Cysteine Residues ◽  
Directed Mutagenesis ◽  
Wild Type ◽  
Glutamate Cysteine Ligase

Glutamate–cysteine ligase (GLCL) catalyses the rate-limiting step in glutathione biosynthesis. To identify cysteine residues in GLCL that are involved in its activity, eight conserved cysteine residues in human GLCL catalytic subunit (hGLCLC) were replaced with glycine residues by PCR-based site-directed mutagenesis. Both recombinant hGLCLC and hGLCL holoenzyme were expressed and purified with a baculovirus expression system. The activity of purified hGLCL holoenzyme with the mutant hGLCLC-C553G was 110±12 µmol/h per mg of protein compared with 370±20 µmol/h per mg of protein for the wild-type. Holoenzymes with hGLCLC-C52G, -C248G, -C249G, -C295G, -C491G, -C501G or -C605G showed activities similar to the wild type. The Km values of hGLCL containing hGLCLC-C553G were slightly lower than those of the wild type, indicating that the replacement of cysteine-553 with Gly in hGLCLC did not significantly affect substrate binding by the enzyme. hGLCLC-C553G was more easily dissociated from hGLCLR than the wild-type hGLCLC. GLCL activity increased by 11% after hGLCLC-C553G was incubated with an equimolar amount of purified hGLCL regulatory subunit (hGLCLR) at room temperature for 30 min, but increased by 110% after wild-type hGLCLC was incubated with hGLCLR for 10 min. These results indicate that cysteine-553 in hGLCLC is involved in heterodimer formation between hGLCLC and hGLCLR.

scholarly journals Cysteine-86 is not needed for the enzymic activity of glutathione S-transferase 3-3

1991 ◽  
Vol 278 (1) ◽  
pp. 293-297 ◽  
Author(s):  
J C Hsieh ◽  
S C Huang ◽  
W L Chen ◽  
Y C Lai ◽  
M F Tam
Keyword(s):  
Spodoptera Frugiperda ◽  
Expression System ◽  
Baculovirus Expression System ◽  
Cysteine Residue ◽  
Enzymic Activity ◽  
Site Directed Mutagenesis ◽  
Terminal Sequence ◽  
Glutathione S Transferase ◽  
Baculovirus Expression ◽  
Modified Peptides

Recombinant glutathione S-transferase 3-3 expressed in Spodoptera frugiperda (SF9) cells with the use of a baculovirus expression system was modified with 1 mM-iodoacetamide. Amino acid analysis indicated that 0.79 +/- 0.15 cysteine residue was modified per enzyme subunit. The S-carbaminomethylated protein retains the GSH-conjugating activity. Glutathione S-transferase 3-3 modified with iodo[14C]acetamide was digested with Achromobacter proteinase I and the resulting peptides were separated by h.p.l.c. The modified peptides were pooled and further digested with Staphylococcus aureus V8 proteinase. Isotope-labelled peptides were isolated and collected for N-terminal sequence analysis. By this procedure, cysteine-86 was identified as the major S-carbaminomethylated residue. Verification of this findings came from the use of site-directed mutagenesis in which this cysteine was replaced by serine (C86S mutant). The C86S mutant is enzymically active. Therefore cysteine-86 is not needed for the conjugation of GSH with electrophilic compounds on glutathione S-transferase 3-3.

Biochemical Chgaracterization of Recombinant Baculovirus 373 K/E p53 Protein

2018 ◽  
Vol 9 (03) ◽  
pp. 20204-20223
Author(s):  
Maghsoudi, Hossein ◽  
U Pati
Keyword(s):  
Dna Binding ◽  
P53 Protein ◽  
Expression System ◽  
Gel Filtration ◽  
Recombinant Baculovirus ◽  
Baculovirus Expression System ◽  
Cross Linking ◽  
Mobility Shift ◽  
Dna Complexes ◽  
P53 Antibodies

In this study, we expressed and purified the recombinant baculovirus 373 K/E p53 protein in a baculovirus expression system to characterize this mutant and compare it with wild type p53. Gel- filtration chromatography and chemical cross-linking experiments indicated that purified recombinant baculovirus 373 K/E p53 protein assembles into multimeric forms ranging from tetramers to polymers. Gel-mobility shift assays and protein-DNA cross-linking studies demonstrated that the recombinant protein binds, to a consensus DNA target as a dimer but that additional p53 mutant molecules may then associate with the preformed p53-dimer-DNA complexes to form a larger p53_DNA complexes. These observations suggest that the p53 mutant tetramers and polymers that forms the minimal p53 mutant complex in solution dissociated upon DNA binding to form p53 mutant dimmer DNA complexes. The DNA binding activity of this mutant was then investigated using electrophoretic mobility shift assays as well as supershift assay with anti-p53 antibodies. Binding of the anti-p53 antibody PAb421to the oligomerization promoting domain on p53 stimulated the sequential formation of both the p53_dimer DNA and larger p53-DNA complexes

scholarly journals Expression of functional G protein beta gamma dimers of defined subunit composition using a baculovirus expression system.

1992 ◽  
Vol 267 (19) ◽  
pp. 13123-13126 ◽  
Author(s):  
S.G. Graber ◽  
R.A. Figler ◽  
V.K. Kalman-Maltese ◽  
J.D. Robishaw ◽  
J.C. Garrison
Keyword(s):  
G Protein ◽  
Expression System ◽  
Baculovirus Expression System ◽  
Subunit Composition ◽  
Baculovirus Expression

scholarly journals Production, processing and partial purification of functional G protein βγ subunits in baculovirus-infected insect cells

1992 ◽  
Vol 286 (3) ◽  
pp. 677-680 ◽  
Author(s):  
J D Robishaw ◽  
V K Kalman ◽  
K L Proulx
Keyword(s):  
G Protein ◽  
G Proteins ◽  
Insect Cells ◽  
Expression System ◽  
Baculovirus Expression System ◽  
Partial Purification ◽  
Baculovirus Expression ◽  
Gamma Subunits ◽  
Infected Insect ◽  
Beta 2

As a result of the inability to resolve the heterogeneous mixture of G protein beta gamma subunits present in tissues, it has not been possible to compare different beta gamma subunits of the G proteins in terms of their proposed roles in receptor-effector coupling. This study was undertaken to establish the utility of the baculovirus expression system in producing homogeneous beta gamma subunits of defined composition for the comparative analysis of these subunits in reconstitution systems. In this study we report the expression, and appropriate post-translational processing, of recombinant beta 2, gamma 2 and gamma 3 subunits. In addition, we show that the recombinant beta gamma subunits can be readily purified, and can functionally interact with the alpha subunits of the G proteins.

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