Recombinant Human Deoxyribonuclease I

Lazarus, Robert A.; Wagener, Jeffrey S.

doi:10.1007/978-3-030-00710-2_22

Cited by 24 publications

(29 citation statements)

References 145 publications

(151 reference statements)

Supporting

Mentioning

Contrasting

Order By: Relevance

“…Human deoxyribonuclease I DNA (hDNase I) is an endonuclease that catalyzes the hydrolysis of extracellular DNA and is initially expressed as a precursor molecule that matures upon cleavage of its signal peptide and secretion . Recombinant hDNase I has been used for therapeutic treatment of CF patients to degrade the viscous sputum containing high concentrations of DNA derived from infiltrating leukocytes involved in fighting various airway infections .…”

Section: Introductionmentioning

confidence: 99%

“…3,6 Human deoxyribonuclease I DNA (hDNase I) is an endonuclease that catalyzes the hydrolysis of extracellular DNA and is initially expressed as a precursor molecule that matures upon cleavage of its signal peptide and secretion. 7 Recombinant hDNase I has been used for therapeutic treatment of CF patients to degrade the viscous sputum containing high concentrations of DNA derived from infiltrating leukocytes involved in fighting various airway infections. [8][9][10] There are also therapeutic implications for hDNase I in treating systemic lupus erythematosus (SLE) where anti-nuclear and anti-DNA antibodies are implicated in part of the disease pathogenesis, specifically nephritis due to their deposition in kidneys.…”

mentioning

confidence: 99%

See 1 more Smart Citation

Taming hyperactive hDNase I: Stable inducible expression of a hyperactive salt‐ and actin‐resistant variant of human deoxyribonuclease I in CHO cells

Lam¹,

Santell²,

Wilson³

et al. 2017

Biotechnology Progress

Self Cite

View full text Add to dashboard Cite

While the most common causes of clonal instability are DNA copy number loss and silencing, toxicity of the expressed protein(s) may also induce clonal instability. Human DNase I (hDNase I) is used therapeutically for the treatment of cystic fibrosis (CF) and may have potential benefit for use in systemic lupus erythematosus (SLE). hDNase I is an endonuclease that catalyzes degradation of extracellular DNA and is inhibited by both salt and G-actin. Engineered versions of hDNase I, bearing multiple point mutations, which renders them Hyperactive, Salt- and Actin-Resistant (HSAR-hDNase I) have been developed previously. However, constitutive expression of HSAR-hDNase I enzymes has been very challenging and, despite considerable efforts and screening thousands of clones, no stable clone capable of constitutive expression had been obtained. Here, we developed a regulated expression system for stable expression of an HSAR-hDNase I in Chinese Hamster Ovary (CHO) cells. The HSAR-hDNase I clones were stable and, upon induction, expressed enzymatically functional protein. Our findings suggest that degradation of host's DNA mediated by HSAR-hDNase I during cell division is the likely cause of clonal instability observed in cells constitutively expressing this protein. Purified HSAR-hDNase I was both hyperactive and resistant to inhibition by salt and G-actin, resulting in an enzyme having ca. 10-fold greater specific activity and the potential to be a superior therapeutic agent to wild type (WT) hDNase I. Furthermore, the ability to regulate hDNase I expression has enabled process development improvements that achieve higher cell growth and product titers while maintaining product quality. © 2017 American Institute of Chemical Engineers Biotechnol. Prog., 32:523-533, 2017.

show abstract

Section: Introductionmentioning

confidence: 99%

mentioning

confidence: 99%

Taming hyperactive hDNase I: Stable inducible expression of a hyperactive salt‐ and actin‐resistant variant of human deoxyribonuclease I in CHO cells

Lam¹,

Santell²,

Wilson³

et al. 2017

Biotechnology Progress

Self Cite

View full text Add to dashboard Cite

show abstract

“…The NETs release against P. brasiliensis have also been demonstrated in cutaneous lesions in patients with paracoccidioidomycosis ( Della Coletta et al., 2015 ), corroborating with the previous studies. The same study that raised the hypothesis that distinct NETs patterns induced by different isolates of the fungus (Pb18 and Pb265), could be related to a fungal DNase-like activity, once the enzyme (DNase) is an endonuclease that catalyzes the hydrolysis of extracellular DNA ( Lazarus and Wagener, 2019 ).…”

Section: Discussionmentioning

confidence: 99%

Paracoccidioides brasiliensis Releases a DNase-Like Protein That Degrades NETs and Allows for Fungal Escape

Zonta

Dezen

Coletta

et al. 2021

Front. Cell. Infect. Microbiol.

View full text Add to dashboard Cite

Paracoccidioidomycosis is a systemic fungal disease, considered endemic in Latin America. Its etiological agents, fungi of the Paracoccidioides complex, have restricted geographic habitat, conidia as infecting form, and thermo-dimorphic characteristics. Polymorphonuclear neutrophils (PMNs) are responsible for an important defense response against fungus, releasing Neutrophil Extracellular Traps (NETs), which can wrap and destroy the yeasts. However, it has been described that some pathogens are able to evade from these DNA structures by releasing DNase as an escape mechanism. As different NETs patterns have been identified in PMNs cultures challenged with different isolates of Paracoccidioides brasiliensis, the general objective of this study was to identify if different patterns of NETs released by human PMNs challenged with Pb18 (virulent) and Pb265 (avirulent) isolates would be correlated with fungal ability to produce a DNase-like protein. To this end, PMNs from healthy subjects were isolated and challenged in vitro with both fungal isolates. The production, release, and conformation of NETs in response to the fungi were evaluated by Confocal Microscopy, Scanning Microscopy, and NETs Quantification. The identification of fungal DNase production was assessed by DNase TEST Agar, and the relative gene expression for hypothetical proteins was investigated by RT-qPCR, whose genes had been identified in the fungal genome in the GenBank (PADG_11161 and PADG_08285). It was possible to verify the NETs release by PMNs, showing different NETs formation when in contact with different isolates of the fungus. The Pb18 isolate induced the release of looser, larger, and more looking like degraded NETs compared to the Pb265 isolate, which induced the release of denser and more compact NETs. DNase TEST Agar identified the production of a DNase-like protein, showing that only Pb18 showed the capacity to degrade DNA in these plates. Besides that, we were able to identify that both PADG_08528 and PADG_11161 genes were more expressed during interaction with neutrophil by the virulent isolate, being PADG_08528 highly expressed in these cultures, demonstrating that this gene could have a greater contribution to the production of the protein. Thus, we identified that the virulent isolate is inducing more scattered and loose NETs, probably by releasing a DNase-like protein. This factor could be an important escape mechanism used by the fungus to escape the NETs action.

show abstract

“…The principal differences between the proposed mechanisms are the number of divalent cations involved in catalysis, and the residues that directly participate in acid/base catalysis. The more recent single cation carboxylic acid model posits a single divalent cation and Asp-189 as the catalytic base 10,12 . In contrast, the more established double divalent cation model 13 predicts two cations and implicates His-155 as the catalytic acid and His-274 as the catalytic base 11 ; however, neither model is completely supported by experimental data.…”

Section: Mainmentioning

confidence: 99%

Structural features of Dnase1L3 responsible for serum antigen clearance

McCord¹,

Engavale²,

Masoumzadeh³

et al. 2022

Preprint

View full text Add to dashboard Cite

Autoimmunity develops when extracellular DNA released from dying cells is not cleared from serum. While serum DNA is primarily digested by Dnase1 and Dnase1L3, Dnase1 does not rescue autoimmunity arising from Dnase1L3 deficiencies. Dnase1L3 uniquely degrades antigenic forms of cell-free DNA, including DNA complexed with lipids and proteins. The distinct activity of Dnase1L3 relies on its unique C-terminal Domain (CTD), but the mechanism is unknown. We used multiple biophysical techniques and functional assays to study the interplay between the core catalytic domain and the CTD. While the core domain resembles Dnase1, there are several key differences between the two enzymes. Dnase1L3 is not inhibited by actin due to multiple differences in the actin recognition site. The CTD augments the ability of the core to bind DNA, thereby facilitating the degradation of complexed DNA to prevent autoimmune pathology. Together, these structural insights will inform the development of Dnase1L3-based therapies for autoimmunity.

show abstract

Recombinant Human Deoxyribonuclease I

Cited by 24 publications

References 145 publications

Taming hyperactive hDNase I: Stable inducible expression of a hyperactive salt‐ and actin‐resistant variant of human deoxyribonuclease I in CHO cells

Taming hyperactive hDNase I: Stable inducible expression of a hyperactive salt‐ and actin‐resistant variant of human deoxyribonuclease I in CHO cells

Paracoccidioides brasiliensis Releases a DNase-Like Protein That Degrades NETs and Allows for Fungal Escape

Structural features of Dnase1L3 responsible for serum antigen clearance

Contact Info

Product

Resources

About