Transglutaminase‐mediated methods for site‐selective modification of human growth hormone

Buchardt, Jens; Selvig, Helle; Nielsen, Per F.; Johansen, Niklas Dyrby

doi:10.1002/bip.21353

Cited by 20 publications

(10 citation statements)

References 21 publications

(22 reference statements)

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“…The requirement of chain flexibility also explains why only a Lys residue embedded in a large loop in an engineered alkaline phosphatase can be modified by TGase . Similarly, the TGase-mediated modification of human growth hormone occurs specifically at Lys145, this residue being embedded in a disordered region of the protein (residues 134–149) prone to limited proteolysis . Therefore, our results overall demonstrate that, besides Gln, Lys derivatization by TGase requires some flexibility of the protein substrate.…”

Section: Discussionmentioning

confidence: 57%

“…59 Similarly, the TGase-mediated modification of human growth hormone occurs specifically at Lys145, this residue being embedded in a disordered region of the protein (residues 134−149) prone to limited proteolysis. 60 Therefore, our results overall demonstrate that, besides Gln, Lys derivatization by TGase requires some flexibility of the protein substrate. The site-specific modification at Lys has not been exploited so far for preparing bioconjugates of proteins, but it seems that with some protein substrates Lys derivatization can provide the advantages already proven with the TGase modification at Gln.…”

Section: ■ Conclusionmentioning

confidence: 58%

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Local Unfolding Is Required for the Site-Specific Protein Modification by Transglutaminase

et al. 2012

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The transglutaminase (TGase) from Streptomyces mobaraensis catalyzes transamidation reactions in a protein substrate leading to the modification of the side chains of Gln and Lys residues according to the A-CONH(2) + H(2)N-B → A-CONH-B + NH(3) reaction, where both A and B can be a protein or a ligand. A noteworthy property of TGase is its susbstrate specificity, so that often only a few specific Gln or Lys residues can be modified in a globular protein. The molecular features of a globular protein dictating the site-specific reactions mediated by TGase are yet poorly understood. Here, we have analyzed the reactivity toward TGase of apomyoglobin (apoMb), α-lactalbumin (α-LA), and fragment 205-316 of thermolysin. These proteins are models of protein structure and folding that have been studied previously using the limited proteolysis technique to unravel regions of local unfolding in their amino acid sequences. The three proteins were modified by TGase at the level of Gln or Lys residues with dansylcadaverine or carbobenzoxy-l-glutaminylglycine, respectively. Despite these model proteins containing several Gln and Lys residues, the sites of TGase derivatization occur over restricted chain regions of the protein substrates. In particular, the TGase-mediated modifications occur in the "helix F" region in apoMb, in the β-domain in apo-α-LA in its molten globule state, and in the N-terminal region in fragment 205-316 of thermolysin. Interestingly, the sites of limited proteolysis are located in the same chain regions of these proteins, thus providing a clear-cut demonstration that chain flexibility or local unfolding overwhelmingly dictates the site-specific modification by both TGase and a protease.

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Section: Discussionmentioning

confidence: 57%

Section: ■ Conclusionmentioning

confidence: 58%

Local Unfolding Is Required for the Site-Specific Protein Modification by Transglutaminase

et al. 2012

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“…Engineering a specific site into hGH is an effective method to achieve site-specific mono-PEGylation. The method included free sulfhydryl PEGylation, 17 chemical modification of hGH for PEGylation, 18 the enzymatic modification approach with transglutaminase, 19 and genetic incorporation of a reactive moiety into hGH. 20 Selection of a fixed site (e.g., N-terminus) of native hGH is an alternative strategy to achieve site-specific mono-PEGylation.…”

Section: ■ Introductionmentioning

confidence: 99%

Phenyl Amide Linker Improves the Pharmacokinetics and Pharmacodynamics of N-Terminally Mono-PEGylated Human Growth Hormone

Shen

et al. 2014

Mol. Pharmaceutics

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Human growth hormone (hGH) suffers from a short plasma half-life of ∼15 min, necessitating frequent injections to maintain its physiological effect. PEGylation, conjugation of polyethylene glycol (PEG), is an effective strategy to prolong the plasma half-life of hGH. However, PEGylation can significantly decrease the bioactivity of hGH. Thus, a new PEGylation approach is desired to improve the pharmacokinetics (PK) and pharmacodynamics (PD) of the PEGylated hGH. In the present study, two N-terminally mono-PEGylated hGHs were prepared using aldehyde chemistry. Phenyl amide and ethyl moieties were used as the linkers between PEG and hGH, respectively. The hydrodynamic volume, proteolytic sensitivity, and immunogenicity of the PEGylated hGH with phenyl amide linker (hGH-phenyl-PEG) were lower than those of the one with propyl linker (hGH-prop-PEG). In addition, hGH-phenyl-PEG showed a higher in vitro bioactivity and better PK and PD than hGH-prop-PEG. The better PK of hGH-phenyl-PEG was mainly due to its lower proteolytic sensitivity and low immunogenicity. The better PD of hGH-phenyl-PEG was mainly due to its higher in vitro bioactivity. Thus, the phenyl amide linker can improve the overall pharmacological profiles of the PEGylated hGH. Our study is expected to advance the development of long-acting protein biotherapeutics with high therapeutic efficacy.

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“…103 For example, transglutaminase (TGase) has been used to transfer polymer chains or fluorescent probes to glutamine residues. 104,105 In addition to these approaches in which amino acid side chains are modified, the N-and C-termini of proteins have specifically been functionalized. 106,107 These site-specific modification strategies have mainly been exploited for the linkage of relatively small molecules, such as fluorescent probes, to proteins.…”

Section: Post-translational Selective Enzyme Conjugationmentioning

confidence: 99%

Multi-enzyme systems: bringing enzymes together in vitro

2012

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Transglutaminase‐mediated methods for site‐selective modification of human growth hormone

Cited by 20 publications

References 21 publications

Local Unfolding Is Required for the Site-Specific Protein Modification by Transglutaminase

Local Unfolding Is Required for the Site-Specific Protein Modification by Transglutaminase

Phenyl Amide Linker Improves the Pharmacokinetics and Pharmacodynamics of N-Terminally Mono-PEGylated Human Growth Hormone

Multi-enzyme systems: bringing enzymes together in vitro

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