Unique Substrate Specificities of Two Adjacent Glutamine Residues in EAQQIVM for Transglutaminase: Identification and Characterization of the Reaction Products by Electrospray Ionization Tandem Mass Spectrometry

Sato, Hiroji; Yamada, Naoyuki; Shimba, Nobuhisa; Takahara, Yoshiyuki

doi:10.1006/abio.2000.4551

Cited by 22 publications

(11 citation statements)

References 23 publications

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“…Remarkably, many other consensus peptide sequences have been reported as efficient substrates for M-TGase. However, while some of these are quite similar to the one reported here (see for example the consensus RLQQP described by Lee and colleagues, [1]), other are quite divergent (AQQIVM, [10]), further underlying the concept that enzyme recognition can be either sequence or structure-driven.…”

Section: Lqsp Is a Transglutaminase Substrate More Efficient Than Tqgamentioning

confidence: 68%

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The LQSP tetrapeptide is a new highly efficient substrate of microbial transglutaminase for the site‐specific derivatization of peptides and proteins

Caporale

Selis²,

Sandomenico

et al. 2014

Biotechnology Journal

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Transglutaminases catalyze transglutamination reactions on glutamines. Transglutaminases are largely exploited for modifying proteins in pharmaceutical, food, and other biotechnological applications. A library of synthetic peptides has been designed, prepared, and screened to identify new peptide substrates. The new substrates are then used in TGAse-mediated conjugation reactions to engraft synthons onto biomolecules. These peptide substrates confer the bioactive peptides and proteins with new properties. We have identified an optimized substrate named LQSP, which is recognized and processed by microbial TGAse with a strikingly higher efficiency compared to the well-known TQGA sequence. The new substrate has been used to selectively modify prototypical bioactive peptides and proteins with fluoresceine or recognition motifs. We show that, where a reactive lysine is available, proteins and peptides of relevant therapeutic interest, can be selectively and smoothly modified in order to incorporate new functions such as fluorescent labels, recognition units, or reactive groups.

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Section: Lqsp Is a Transglutaminase Substrate More Efficient Than Tqgamentioning

confidence: 68%

“…The substrate specificity of TGases has been investigated by using both synthetic peptides [10][11][12] and model proteins [13]. The literature show we still have much to learn about TGases [14].…”

Section: Biotech Methodsmentioning

confidence: 99%

The LQSP tetrapeptide is a new highly efficient substrate of microbial transglutaminase for the site‐specific derivatization of peptides and proteins

Caporale

Selis²,

Sandomenico

et al. 2014

Biotechnology Journal

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“…TG2-catalyzed dansylcadaverine incorporation into FN N-terminal constructs was compared with incorporation catalyzed by FXIIIa, which is known to modify Gln-3 (19) and may also utilize Gln-4, and Gln-16 as predicted by mutagenesis and studies of peptides mimicking the FN N-terminal sequence (21)(22)(23). After dansylcadaverine incorporation into N-3 F3t, 70K, or 29K, the proteins were subjected to reduction, alkylation, extensive trypsinization, chromatographic separation of tryptic peptides, and tandem MS analysis to map modified glutaminyl residues of transamidation.…”

Section: Ltq-ms/ms Analysis Of Tg2-and Fxiiia-catalyzed Dansylcadavermentioning

confidence: 99%

“…Gln-3 (numbering starting after processing of pre-propeptide sequences) is the major target of amine incorporation into FN by FXIIIa (19 -20). Gln-4 and Gln-16 have been predicted to be additional sites of modification by in vitro mutagenesis and studies of peptide mimics (14,(21)(22)(23). These glutamines are in the unstructured N-terminal tail of FN (24).…”

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confidence: 99%

Reactivity of the N-terminal Region of Fibronectin Protein to Transglutaminase 2 and Factor XIIIA

Annis

Mosher

2011

Journal of Biological Chemistry

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Transglutaminase 2 (TG2) is secreted by a non-classical pathway into the extracellular space, where it has several activities pertinent to fibronectin (FN), including binding to the gelatinbinding domain of FN and acting as an integrin co-receptor. Glutamines in the N-terminal tail of FN are known to be susceptible to transamidation by both TG2 and activated blood coagulation factor XIII (FXIIIa). We used immunoblotting, limited proteolysis, and mass spectrometry to localize glutamines within FN that are subject to TG2-catalyzed incorporation of dansylcadaverine in comparison to residues modified by FXIIIa. Such analysis of plasma FN indicated that Gln-3, Gln-7, and Gln-9 in the N-terminal tail and Gln-246 of the linker between fifth and sixth type I modules ( 5 F1 and 6 F1) are transamidated by both enzymes. Only minor incorporation of dansylcadaverine was detected elsewhere. Labeling of C-terminally truncated FN constructs revealed efficient TG2-or FXIIIa-catalyzed dansylcadaverine incorporation into the N-terminal residues of constructs as small as the 29-kDa fragment that includes 1-5 F1 and lacks modules from the adjacent gelatin-binding domain. However, when only 1-3 F1 were present, dansylcadaverine incorporation into the N-terminal residues of FN was lost and instead was in the enzymes, near the active site of TG2 and terminal domains of FXIIIa. Thus, these results demonstrate that FXIIIa and TG2 act similarly on glutamines at either end of 1-5 F1 and transamidation specificity of both enzymes is achieved through interactions with the intact 29K fragment.

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“…We recently reported three natural substrate proteins of MTG. [17] Having taken the recognition sequence 1 as as tarting point, we designed oligopeptide 2.Inorder to precisely mimic the functional Q298 loop in DAIP,which is bordered by G295 and G302 with aC a distance of about 4,f lanking cysteine residues were introduced to rigidify the b-turn upon oxidation into acovalent disulfide (Scheme 1, peptide 2). [16c] Sequencing from the amino terminus of the protein followed by sequencing of the whole genome of Streptomyces mobaraensis strain 40847 revealed the DAIPencoding gene.W ith this information in hand, the parent DAIP,w hich contains five glutamine residues,a sw ell as derivatives possessing multiple Gln-to-Asn exchanges,w ere recombinantly produced in E. coli cells.S uccessive MTGpromoted ligation with monobiotinylcadaverine resulted in the identification of three MTG modification sites (Q39, and Q298, and Q345) that enable exceptionally efficient enzyme performance.…”

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confidence: 99%

Locked by Design: A Conformationally Constrained Transglutaminase Tag Enables Efficient Site‐Specific Conjugation

et al. 2015

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Based on the crystal structure of a natural protein substrate for microbial transglutaminase, an enzyme that catalyzes protein crosslinking, a recognition motif for site-specific conjugation was rationally designed. Conformationally locked by an intramolecular disulfide bond, this structural mimic of a native conjugation site ensured efficient conjugation of a reporter cargo to the therapeutic monoclonal antibody cetuximab without erosion of its binding properties.

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Unique Substrate Specificities of Two Adjacent Glutamine Residues in EAQQIVM for Transglutaminase: Identification and Characterization of the Reaction Products by Electrospray Ionization Tandem Mass Spectrometry

Cited by 22 publications

References 23 publications

The LQSP tetrapeptide is a new highly efficient substrate of microbial transglutaminase for the site‐specific derivatization of peptides and proteins

The LQSP tetrapeptide is a new highly efficient substrate of microbial transglutaminase for the site‐specific derivatization of peptides and proteins

Reactivity of the N-terminal Region of Fibronectin Protein to Transglutaminase 2 and Factor XIIIA

Locked by Design: A Conformationally Constrained Transglutaminase Tag Enables Efficient Site‐Specific Conjugation

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