Proprotein Convertases Process and Thereby Inactivate Formylglycine-generating Enzyme*

Ennemann, Eva C.; Radhakrishnan, Karthikeyan; Mariappan, Malaiyalam; Wachs, Michaela; Pringle, Thomas H.; Schmidt, Bernhard; Dierks, Thomas

doi:10.1074/jbc.m112.405159

Cited by 10 publications

(12 citation statements)

References 39 publications

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“…Previous reports provide evidence that the NTE is required for ER retention through disulfide bond formation between Cys 50 and/or Cys 52 with ERp44 ( 32 ). The border between the NTE and the core is a proteolytic cleavage site for proprotein convertases such as furin and PACE ( 33 ). When FGE encounters these enzymes in the secretory pathway, the NTE is removed and the truncated core can be secreted.…”

Section: Resultsmentioning

confidence: 99%

“…We discovered that in vitro , the full-length FGE had a propensity to aggregate at high concentrations, which led us to prepare the truncated core (cFGE). We were unsure whether this approach would be successful because previous results showed that the NTE is required for FGE activity in vitro ( 33 ). In our hands, the specific activity of Hs -cFGE (1,143 pkatal mg −1 , 33.3 kDa) was similar to Hs -FGE that contained the NTE (850 pkatal mg −1 , 36.9 kDa).…”

Section: Resultsmentioning

confidence: 99%

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Reconstitution of Formylglycine-generating Enzyme with Copper(II) for Aldehyde Tag Conversion

Holder

Jones

Drake

et al. 2015

Journal of Biological Chemistry

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Background: Aerobic formylglycine-generating enzyme (FGE) converts cysteine to formylglycine in vivo.Results: Purified FGE requires preactivation with copper to convert cysteine to formylglycine in vitro.Conclusion: FGE is a metalloenzyme. It is also a useful biocatalyst for the production of proteins that contain aldehyde tags.Significance: Understanding FGE biochemistry informs research on sulfatases and enables expanded biotechnology applications of the aldehyde tag.

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Section: Resultsmentioning

confidence: 99%

Section: Resultsmentioning

confidence: 99%

Reconstitution of Formylglycine-generating Enzyme with Copper(II) for Aldehyde Tag Conversion

Holder

Jones

Drake

et al. 2015

Journal of Biological Chemistry

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show abstract

“…Approximately 20–30% of secreted FGE avoids proteolytic cleavage and remains active. 56 Secreted FGE can traffic back to the ER from the cell surface. Indeed, tissue-specific expression of FGE in FGE-null mice has been shown to activate sulfatases in nontransduced tissues, in this way acting as a paracrine agent.…”

Section: Mammalian Fge Activity Is Regulated By Protein Partnersmentioning

confidence: 99%

Formylglycine, a Post-Translationally Generated Residue with Unique Catalytic Capabilities and Biotechnology Applications

2014

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Formylglycine (fGly) is a catalytically essential residue found almost exclusively in the active sites of type I sulfatases. Formed by post-translational oxidation of cysteine or serine side chains, this aldehyde-functionalized residue participates in a unique and highly efficient catalytic mechanism for sulfate ester hydrolysis. The enzymes that produce fGly, formylglycine-generating enzyme (FGE) and anaerobic sulfatase-maturating enzyme (anSME), are as unique and specialized as fGly itself. FGE especially is structurally and mechanistically distinct, and serves the sole function of activating type I sulfatase targets. This review summarizes the current state of knowledge regarding the mechanism by which fGly contributes to sulfate ester hydrolysis, the molecular details of fGly biogenesis by FGE and anSME, and finally, recent biotechnology applications of fGly beyond its natural catalytic function.

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“…As a first step to address the variability of fGly conversion levels, we considered that differences in intracellular FGE levels might affect conversion yields. Wild-type human FGE is partially retained in the ER through interactions of its N -terminal region (called the N -terminal extension, NTE) with endoplasmic reticulum resident protein 44 (ERp44) [ 12 ]. This interaction can be abrogated by furin-mediated proteolytic cleavage at an internal site, allowing the remaining catalytically-active core enzyme to be secreted [ 11 ].…”

Section: Resultsmentioning

confidence: 99%

Generating aldehyde-tagged antibodies with high titers and high formylglycine yields by supplementing culture media with copper(II)

et al. 2016

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BackgroundThe ability to site-specifically conjugate a protein to a payload of interest (e.g., a fluorophore, small molecule pharmacophore, oligonucleotide, or other protein) has found widespread application in basic research and drug development. For example, antibody-drug conjugates represent a class of biotherapeutics that couple the targeting specificity of an antibody with the chemotherapeutic potency of a small molecule drug. While first generation antibody-drug conjugates (ADCs) used random conjugation approaches, next-generation ADCs are employing site-specific conjugation. A facile way to generate site-specific protein conjugates is via the aldehyde tag technology, where a five amino acid consensus sequence (CXPXR) is genetically encoded into the protein of interest at the desired location. During protein expression, the Cys residue within this consensus sequence can be recognized by ectopically-expressed formylglycine generating enzyme (FGE), which converts the Cys to a formylglycine (fGly) residue. The latter bears an aldehyde functional group that serves as a chemical handle for subsequent conjugation.ResultsThe yield of Cys conversion to fGly during protein production can be variable and is highly dependent on culture conditions. We set out to achieve consistently high yields by modulating culture conditions to maximize FGE activity within the cell. We recently showed that FGE is a copper-dependent oxidase that binds copper in a stoichiometric fashion and uses it to activate oxygen, driving enzymatic turnover. Building upon that work, here we show that by supplementing cell culture media with copper we can routinely reach high yields of highly converted protein. We demonstrate that cells incorporate copper from the media into FGE, which results in increased specific activity of the enzyme. The amount of copper required is compatible with large scale cell culture, as demonstrated in fed-batch cell cultures with antibody titers of 5 g · L−1, specific cellular production rates of 75 pg · cell−1 · d−1, and fGly conversion yields of 95–98 %.ConclusionsWe describe a process with a high yield of site-specific formylglycine (fGly) generation during monoclonal antibody production in CHO cells. The conversion of Cys to fGly depends upon the activity of FGE, which can be ensured by supplementing the culture media with 50 uM copper(II) sulfate.Electronic supplementary materialThe online version of this article (doi:10.1186/s12896-016-0254-0) contains supplementary material, which is available to authorized users.

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Proprotein Convertases Process and Thereby Inactivate Formylglycine-generating Enzyme*

Cited by 10 publications

References 39 publications

Reconstitution of Formylglycine-generating Enzyme with Copper(II) for Aldehyde Tag Conversion

Reconstitution of Formylglycine-generating Enzyme with Copper(II) for Aldehyde Tag Conversion

Formylglycine, a Post-Translationally Generated Residue with Unique Catalytic Capabilities and Biotechnology Applications

Generating aldehyde-tagged antibodies with high titers and high formylglycine yields by supplementing culture media with copper(II)

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