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Development of the formylglycine aldehyde tag

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The aldehyde tag is an artificial peptide tag recognized by the formylglycine-generating enzyme (FGE). Formylglycine is a glycine with a formyl group (-CHO, an aldehyde) at the α-carbon. The sulfatase motif is the basis for the sequence of the peptide which results in the site-specific conversion of a cysteine to a formylglycine residue. The peptide tag was engineered after studies on FGE recognizable sequences in sulfatases from different organisms. Carrico et al. discovered a high homology in the sulfatase motif in bacteria, archaea as well as eukaryotes [1].

Aldehydes and ketones find use as chemical reporters due to their strong electrophilic properties. This enables a reaction under mild conditions when using a strong nucleophilic coupling partner. Typically, hydrazides and aminooxy probes are used in bioconjugation. They form stabilized addition products with carbonyl groups that are favoured under the physiological reaction conditions. At neutral pH, the equilibrium of Schiff base formation, is lying far to the reactant’s side. To make more product named compounds are used to form stable hydrazones and oximes. Since the pH-optimum of 4 to 6 cannot be achieved by adding a catalyst due to associated toxicity, the reaction is slow in live cells. A typical reaction constant is 10-4 to 10-3 M-1 s-1. [2]

A carbonyl group is introduced into proteins as a chemical reporter using different techniques, including modern methods like stop codon suppression and the herein discussed aldehyde tag [3], [4]. Limiting the use of aldehydes and ketones is their restricted bioorthogonality in certain cellular environments. Limitations of aldehydes and ketones as chemical reporters are due to

  • competition with endogenous aldehydes or ketones in metabolites and cofactors. Lower yields and impaired specificity can occur.
  • side reactions like oxidation or unwanted addition of endogenous nucleophiles.
  • restrained set of probes that form sufficiently stable products [5], [6].

Aldehydes and ketones are therefore best used in compartments where such unwanted side reactions are decreased. For experiments with life cells , cell surfaces and extracellular space are typical fielding areas. Nevertheless, a feature of carbonyl groups is the vast number of organic reactions that involve them as electrophiles. Some of these reactions are readily convertible to ligations for probing aldehydes. A rather exotic reaction recently employed for bioconjugation by Agarwal et al. is the adaptation of the Pictet-Spengler-reaction as a ligation. The reaction is known from natural product biosynthetic pathways [7] and has the major advantage that a new carbon-carbon bond is formed. This guarantees long-term stability compared to carbon-heteroatom bonds at same reaction kinetics [8].

The modification of cysteine or, more rarely, serine [9] by FGE is a rather unusual posttranslational modification and was discovered already in the late 1990s [10]. Interestingly, the deficiency of FGE leads to an overall deficiency of functional sulfatases due to a lack of α-formylglycine formation vital for the sulfatases to perform their function. FGE is essential for protein modification and need of high specificity and conversion rate is given in the native setting, which makes this reaction interesting for chemical and synthetic biology [11].

Carrico et al. pioneered the insertion of the modified sulfatase motif peptide into proteins of interest in 2007 [12]. Such use of aldehydes and ketones as a chemical reporter in bioorthogonal applications has been applied in self-assembly of cell-lysing drugs [13], the targeting of proteins [14], [15] as well as glycans [16] and the preparation of heterobifunctional fusion proteins [17] since then.

Genetically encoding the aldehyde tag

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The formylglycine tag or aldehyde tag is a convenient 6- or 13-amino acids long tag fused to a protein of interest. The 6-mer tag represents the small core consensus sequence and the 13-mer tag the longer full motif. The experiments on the genetically encoded aldehyde tag by Carrico et al. clearly showed the high conversion efficiency with only the core consensus sequence present. Four proteins were produced recombinantly in E.coli with a 86 % efficiency of for the full-length motif and >90 % efficiency for the 6-mer determined by mass spectrometry. [18] The size of the sequence is analogous to the commonly used 6x His-Tag [19] and has the advantage that it can also be genetically encoded. The sequence is recognized in the ER solely depending on primary sequence and subsequently targeted by FGE [20]. Notably, in the setup of recombinant expression proteins in E. coli a coexpression of exogenous FGE aids full conversion [1], although E. coli has endogenous FGE-activity. [21] The introduction of an aldehyde tag as proposed by Carrico et al. has a workflow that consists of three segments: A the expression of the fusion protein, that carries the peptide tag derived from the sulfatase motif, B the enzymatic conversion of Cys to f(Gly) and C the bioorthogonal probing with hydrazides or alkoxy amines (Fig. 1).

Figure 1: Formylglycine aldehyde tag Carrico et al.[22]: A The aldehyde tag is genetically inserted into a protein of interest. In this example, the human growth hormone (hGH, PDB:1HUW), one of the four initially examined proteins,is shown. The N-terminus of the protein is fused to the formylglycine aldehyde tag. B The FGE recognizes the motif and the cysteine (Cys) residue is converted into the formylglycine residue [f(Gly)]. The chemical reporter is formed on location by an enzymatic reaction. C The carbonyl group is probed using typically hydrazide- or alkoxy amine-functionalized dyes or other compounds.

As seen in Fig. 1, the engineered aldehyde tag consists of six amino acids. A set of organisms from all domains of life was chosen and the sequence homology of the sulfatase motif was determined. The sequence used is the best consensus for sequences found in bacteria, archaea, worms and higher vertebrates. [23].

FGE-mechanism of cysteine-formylglycine conversion

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The catalytic mechanism of FGE is well studied. A multistep redox reaction with a covalent enzyme:substrate intermediate is proposed. The role of the cysteine residue for the occurring conversion was studied by mutating the cysteine to alanine. No conversion was found using mass spectrometry when the mutated peptide tag was used [24]. The mechanism shows the important role of the redox active thiol group of cysteine in the formation of f(Gly), as seen in Fig. 2. The key step of the catalytic cycle is the monooxidation of the cysteine residue of the enzyme, forming a reactive sulfenic acid intermediate. Subsequently, the hydroxyl group is transferred to the cysteine of the substrate and after hetero-analogous β-elimination of H2O, a thioaldehyde is formed. This compound is very reactive and easily hydrolyzed, releasing the aldehyde and a molecule of H2S [25], [26], [27].

Figure 2: Conversion of Cys to f(Gly) by FGE from Dierks et al. [28]: Substrate I binds to FGE, and disulfide isomerization occurs IIb. Cys341 of FGE is oxidized to a sulfenic acid IIc The hydroxyl group is transferred to the substrate and a substrate-sulfenic acid III is generated. β‑elimination of water leads to a thioaldehyde IV which is quickly hydrated to V and after elimination of H2S, the aldehyde VI is formed. The equilibrium lies far to the product side due to the high reactivity of the thioaldehyde IV compared to the aldehyde VI and its arising tendency to form the hydrate V. CysSubs = substrate protein cysteine embedded in sulfatase motif.

References

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  1. ^ [1] Carrico, I. S., Carlson, B. L. and Bertozzi, C. R. (2007) Introducing genetically encoded aldehydes into proteins. Nat Chem Biol 3, 321–322.
  2. ^ [2] Jencks, W. P. (1959) Studies on the Mechanism of Oxime and Semicarbazone Formation1. J. Am. Chem. Soc. 81, 475–481.
  3. ^ [1] Carrico, I. S., Carlson, B. L. and Bertozzi, C. R. (2007) Introducing genetically encoded aldehydes into proteins. Nat Chem Biol 3, 321–322.
  4. ^ [3] Zhang, Z., Smith, B. A. C., Wang, L., Brock, A., Cho, C. and Schultz, P. G. (2003) A New Strategy for the Site-Specific Modification of Proteins in Vivo†. Biochemistry 42, 6735–6746.
  5. ^ [4] Lim, R. K. V. and Lin, Q. (2010) Bioorthogonal Chemistry: Recent Progress and Future Directions. Chem Commun (Camb) 46, 1589–1600.
  6. ^ [5] Prescher, J. A. and Bertozzi, C. R. (2005) Chemistry in living systems. Nat Chem Biol 1, 13–21.
  7. ^ [6] Stöckigt, J., Antonchick, A. P., Wu, F. and Waldmann, H. (2011) Die Pictet-Spengler-Reaktion in der Natur und der organischen Chemie. Angew. Chem. 123, 8692–8719.
  8. ^ [7] Agarwal, P., van der Weijden, J., Sletten, E. M., Rabuka, D. and Bertozzi, C. R. (2013) A Pictet-Spengler ligation for protein chemical modification. Proc Natl Acad Sci U S A 110, 46–51.
  9. ^ [8] Miech, C., Dierks, T., Selmer, T., Figura, K. von and Schmidt, B. (1998) Arylsulfatase from Klebsiella pneumoniae Carries a Formylglycine Generated from a Serine. J. Biol. Chem. 273, 4835–4837.
  10. ^ [9] Dierks, T., Lecca, M. R., Schmidt, B. and von Figura, K. (1998) Conversion of cysteine to formylglycine in eukaryotic sulfatases occurs by a common mechanism in the endoplasmic reticulum. FEBS Letters 423, 61–65.
  11. ^ [10] Dierks, T., Dickmanns, A., Preusser-Kunze, A., Schmidt, B., Mariappan, M., von Figura, K., Ficner, R. and Rudolph, M. G. (2005) Molecular Basis for Multiple Sulfatase Deficiency and Mechanism for Formylglycine Generation of the Human Formylglycine-Generating Enzyme. Cell 121, 541–552.
  12. ^ [1] Carrico, I. S., Carlson, B. L. and Bertozzi, C. R. (2007) Introducing genetically encoded aldehydes into proteins. Nat Chem Biol 3, 321–322. [2] Jencks, W. P. (1959) Studies on the Mechanism of Oxime and Semicarbazone Formation1. J. Am. Chem. Soc. 81, 475–481.
  13. ^ [11] Rideout, D. (1994) Self-assembling drugs: a new approach to biochemical modulation in cancer chemotherapy. Cancer Invest. 12, 189–202; discussion 268–269.
  14. ^ [12] Chen, I., Howarth, M., Lin, W. and Ting, A. Y. (2005) Site-specific labeling of cell surface proteins with biophysical probes using biotin ligase. Nat Meth 2, 99–104.
  15. ^ [13] Zhang, Z., Smith, B. A. C., Wang, L., Brock, A., Cho, C. and Schultz, P. G. (2003) A New Strategy for the Site-Specific Modification of Proteins in Vivo†. Biochemistry 42, 6735–6746.
  16. ^ [14] Mahal, L. K., Yarema, K. J. and Bertozzi, C. R. (1997) Engineering Chemical Reactivity on Cell Surfaces Through Oligosaccharide Biosynthesis. Science 276, 1125–1128.
  17. ^ [15] Hudak, J. E., Barfield, R. M., de Hart, G. W., Grob, P., Nogales, E., Bertozzi, C. R. and Rabuka, D. (2012) Synthesis of Heterobifunctional Protein Fusions Using Copper-Free Click Chemistry and the Aldehyde Tag. Angew. Chem. Int. Ed. 51, 4161–4165.
  18. ^ [1] Carrico, I. S., Carlson, B. L. and Bertozzi, C. R. (2007) Introducing genetically encoded aldehydes into proteins. Nat Chem Biol 3, 321–322.
  19. ^ [16] Hochuli, E., Bannwarth, W., Döbeli, H., Gentz, R. and Stüber, D. (1988) Genetic Approach to Facilitate Purification of Recombinant Proteins with a Novel Metal Chelate Adsorbent. Nat Biotech 6, 1321–1325
  20. ^ [17] Dierks, T., Lecca, M. R., Schmidt, B. and von Figura, K. (1998) Conversion of cysteine to formylglycine in eukaryotic sulfatases occurs by a common mechanism in the endoplasmic reticulum. FEBS Letters 423, 61–65.
  21. ^ [18] Dierks, T., Miech, C., Hummerjohann, J., Schmidt, B., Kertesz, M. A. and Figura, K. von. (1998) Posttranslational Formation of Formylglycine in Prokaryotic Sulfatases by Modification of Either Cysteine or Serine. J. Biol. Chem. 273, 25560–25564.
  22. ^ [1] Carrico, I. S., Carlson, B. L. and Bertozzi, C. R. (2007) Introducing genetically encoded aldehydes into proteins. Nat Chem Biol 3, 321–322.
  23. ^ [1] Carrico, I. S., Carlson, B. L. and Bertozzi, C. R. (2007) Introducing genetically encoded aldehydes into proteins. Nat Chem Biol 3, 321–322.
  24. ^ [1] Carrico, I. S., Carlson, B. L. and Bertozzi, C. R. (2007) Introducing genetically encoded aldehydes into proteins. Nat Chem Biol 3, 321–322.
  25. ^ [19] Roeser, D., Preusser-Kunze, A., Schmidt, B., Gasow, K., Wittmann, J. G., Dierks, T., Figura, K. von and Rudolph, M. G. (2006) A general binding mechanism for all human sulfatases by the formylglycine-generating enzyme. PNAS 103, 81–86.
  26. ^ [20] Sase, S., Kakimoto, R. and Goto, K. (2014) Synthesis of a Stable Selenoaldehyde by Self-Catalyzed Thermal Dehydration of a Primary-Alkyl-Substituted Selenenic Acid. Angew. Chem. n/a–n/a.
  27. ^ [21] Dierks, T., Dickmanns, A., Preusser-Kunze, A., Schmidt, B., Mariappan, M., von Figura, K., Ficner, R. and Rudolph, M. G. (2005) Molecular Basis for Multiple Sulfatase Deficiency and Mechanism for Formylglycine Generation of the Human Formylglycine-Generating Enzyme. Cell 121, 541–552.
  28. ^ [21] Dierks, T., Dickmanns, A., Preusser-Kunze, A., Schmidt, B., Mariappan, M., von Figura, K., Ficner, R. and Rudolph, M. G. (2005) Molecular Basis for Multiple Sulfatase Deficiency and Mechanism for Formylglycine Generation of the Human Formylglycine-Generating Enzyme. Cell 121, 541–552.
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