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Growth inhibition by bacterial Cas2Em proteins expressed in mammalian cells.

ABSTRACT: BACKGROUND:Clustered regularly interspaced short palindromic repeats (CRISPRs) and the CRISPR-associated (Cas) proteins are bacterial adaptive immune system for survival. In our previous study, we demonstrate that polyploid giant bacterial cells (PGBC) induced by Cas2 protein is a step required by new spacer acquisition reaction catalyzed by Cas1/Cas2 complex. We also demonstrated that a carboxyl terminal domain on Cas2Em (the protein Cas2 cloned from Elizabethkingia meningoseptica) is sufficient and enough for PGBC. Thus, the potential role of Cas2Em in microbial-host interaction was explored in this study. METHODS:The impacts of Cas2Em on growth of both CHO-K1 and Hela cells were investigated. The subcellular localization and potential molecular target of Ca2Em were studied. RESULTS:The growth of mammalian cells were inhibited by Cas2Em protein via G1 arresting and apoptosis. In addition, we also demonstrated that Cas2Em was tightly associated with nuclear outer membrane and could be immunoprecipitated with 14-3-3? through a 30 amino acid domain (homology of CLK2). CONCLUSION:Cas2Em significantly suppressed the growth of mammalian cells indicating Cas2 proteins play an important role in mammalian cells.

SUBMITTER: Yu X

PROVIDER: S-EPMC7344093 | biostudies-literature | 2020

REPOSITORIES: biostudies-literature

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Growth inhibition by bacterial Cas2Em proteins expressed in mammalian cells.

Yu Xin X Wang Lei L Zou Lin L Li Mengjie M Li Tiansheng T Hou Linlin L Guo Yameng Y Shen Danfeng D Sun Guiqin G Qu Di D Cheng Xunjia X Chen Li L

American journal of translational research 20200615 6

<h4>Background</h4>Clustered regularly interspaced short palindromic repeats (CRISPRs) and the CRISPR-associated (Cas) proteins are bacterial adaptive immune system for survival. In our previous study, we demonstrate that polyploid giant bacterial cells (PGBC) induced by Cas2 protein is a step required by new spacer acquisition reaction catalyzed by Cas1/Cas2 complex. We also demonstrated that a carboxyl terminal domain on Cas2Em (the protein Cas2 cloned from <i>Elizabethkingia meningoseptica</i ...[more]

PMID: 32655787

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Project description:BackgroundThere is very little reported in the literature about the relationship between modifications of bacterial proteins and their secretion by mammalian cells that synthesize them. We previously reported that the secretion of the bacterial enzyme Chondroitinase ABC by mammalian cells requires the strategic removal of at least three N-glycosylation sites. The aim of this study was to determine if it is possible to enhance the efficacy of the enzyme as a treatment for spinal cord injury by increasing the quantity of enzyme secreted or by altering its cellular location.Methodology/principal findingsTo determine if the efficiency of enzyme secretion could be further increased, cells were transfected with constructs encoding the gene for chondroitinase ABC modified for expression by mammalian cells; these contained additional modifications of strategic N-glycosylation sites or alternative signal sequences to direct secretion of the enzyme from the cells. We show that while removal of certain specific N-glycosylation sites enhances enzyme secretion, N-glycosylation of at least two other sites, N-856 and N-773, is essential for both production and secretion of active enzyme. Furthermore, we find that the signal sequence directing secretion also influences the quantity of enzyme secreted, and that this varies widely amongst the cell types tested. Last, we find that replacing the 3'UTR on the cDNA encoding Chondroitinase ABC with that of ?-actin is sufficient to target the enzyme to the neuronal growth cone when transfected into neurons. This also enhances neurite outgrowth on an inhibitory substrate.Conclusion/significanceSome intracellular trafficking pathways are adversely affected by cryptic signals present in the bacterial gene sequence, whilst unexpectedly others are required for efficient secretion of the enzyme. Furthermore, targeting chondroitinase to the neuronal growth cone promotes its ability to increase neurite outgrowth on an inhibitory substrate. These findings are timely in view of the renewed prospects for gene therapy, and of direct relevance to strategies aimed at expressing foreign proteins in mammalian cells, in particular bacterial proteins.

Project description:The development of a prophylactic vaccine for hepatitis C virus (HCV) remains a global health challenge. Cumulative evidence supports the importance of antibodies targeting the HCV E2 envelope glycoprotein to facilitate viral clearance. However, a significant challenge for a B cell-based vaccine is focusing the immune response on conserved E2 epitopes capable of eliciting neutralizing antibodies not associated with viral escape. We hypothesized that glycosylation might influence the antigenicity and immunogenicity of E2. Accordingly, we performed head-to-head molecular, antigenic, and immunogenic comparisons of soluble E2 (sE2) produced in (i) mammalian (HEK293) cells, which confer mostly complex- and high-mannose-type glycans; and (ii) insect (Sf9) cells, which impart mainly paucimannose-type glycans. Mass spectrometry demonstrated that all 11 predicted N-glycosylation sites were utilized in both HEK293- and Sf9-derived sE2, but that N-glycans in insect sE2 were on average smaller and less complex. Both proteins bound CD81 and were recognized by conformation-dependent antibodies. Mouse immunogenicity studies revealed that similar polyclonal antibody responses were generated against antigenic domains A to E of E2. Although neutralizing antibody titers showed that Sf9-derived sE2 induced moderately stronger responses than did HEK293-derived sE2 against the homologous HCV H77c isolate, the two proteins elicited comparable neutralization titers against heterologous isolates. Given that global alteration of HCV E2 glycosylation by expression in different hosts did not appreciably affect antigenicity or overall immunogenicity, a more productive approach to increasing the antibody response to neutralizing epitopes may be complete deletion, rather than just modification, of specific N-glycans proximal to these epitopes.IMPORTANCE The development of a vaccine for hepatitis C virus (HCV) remains a global health challenge. A major challenge for vaccine development is focusing the immune response on conserved regions of the HCV envelope protein, E2, capable of eliciting neutralizing antibodies. Modification of E2 by glycosylation might influence the immunogenicity of E2. Accordingly, we performed molecular and immunogenic comparisons of E2 produced in mammalian and insect cells. Mass spectrometry demonstrated that the predicted glycosylation sites were utilized in both mammalian and insect cell E2, although the glycan types in insect cell E2 were smaller and less complex. Mouse immunogenicity studies revealed similar polyclonal antibody responses. However, insect cell E2 induced stronger neutralizing antibody responses against the homologous isolate used in the vaccine, albeit the two proteins elicited comparable neutralization titers against heterologous isolates. A more productive approach for vaccine development may be complete deletion of specific glycans in the E2 protein.

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Growth inhibition by bacterial Cas2Em proteins expressed in mammalian cells.

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Growth inhibition by bacterial Cas2Em proteins expressed in mammalian cells.

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