Covalent Protein Labeling by Enzymatic Phosphocholination

Abstract

We present a new protein labeling method based on the covalent enzymatic phosphocholination of a specific octapeptide amino acid sequence in intact proteins. The bacterial enzyme AnkX from Legionella pneumophila has been established to transfer functional phosphocholine moieties from synthetically produced CDP-choline derivatives to N-termini, C-termini, and internal loop regions in proteins of interest. Furthermore, the covalent modification can be hydrolytically removed by the action of the Legionella enzyme Lem3. Only a short peptide sequence (eight amino acids) is required for efficient protein labeling and a small linker group (PEG-phosphocholine) is introduced to attach the conjugated cargo.

Exploring the Substrate Scope of the Bacterial Phosphocholine Transferase AnkX for Versatile Protein Functionalization

“…We have recently introduced a new strategy for the site‐specific labeling and delabeling of proteins of interest (POIs) at the N or C terminus (Scheme C) . This approach is based on AnkX‐catalyzed enzymatic phosphocholination and uses functionalized CDP‐choline analogues to label POIs bearing an eight‐residue recognition tag (TITSSYYR).…”

“…We started our investigation into AnkX's substrate scope by testing the incorporation of synthesized derivatives 2 – 5 into AnkX's natural substrate protein Rab1b, thus allowing reliable comparison of synthetic CDP‐choline analogues. Model proteins for AnkX‐mediated labeling exhibit poorer labeling efficiency than Rab1b but show identical co‐substrate profiles . Thus, results relating to the performance of synthetic CDP‐choline derivatives that are recorded with Rab1b can be qualitatively adapted to the system of model proteins.…”

“…However, it is striking that Rab1b, which naturally contains the S‐Tag, is modified to a greater extent than the model protein MBP, in which the T‐Tag had been recombinantly introduced at the N terminus. This is in line with previous work that showed that MBP modified with an N‐terminal AnkX recognition sequence exhibits phosphocholination rates that are reduced by a factor of around 700 in relation to Rab1b . Nevertheless, this proof‐of‐principle experiment demonstrates that the alternate use of AnkX and Lem3 in combination with two different recognition tags (S‐tag and T‐Tag) can be exploited for site‐selective labeling of POIs with two distinct functionalities.…”

Building Peptide Bonds in Haifa: The Seventh Chemical Protein Synthesis (CPS) Meeting

“…Aymelt Itzen (Technical University Munich, Germany) presented collaborative work with Christian Hedberg's lab (Umeå University, Sweden) on the development of an enzymatic protein‐labeling approach that is based on functionalized nucleotide–choline derivatives. The enzyme AnkX utilizes these nucleotides to transfer a phosphocholine moiety bearing, for example, alkynes or azides to an octapeptide recognition sequence at protein termini or internal loop regions . A peptide‐template‐assisted ligation approach for the in vivo labeling of membrane receptors with small organic tags in living cells was discussed by Annette Beck‐Sickinger (University of Leipzig, Germany) .…”

“…The enzyme AnkX utilizes these nucleotides to transfer ap hosphocholine moiety bearing, for example, alkynes or azides to an octapeptide recognition sequencea tp rotein terminio ri nternall oop regions. [44] Ap eptide-template-assisted ligationa pproach for the in vivo labeling of membrane receptorsw ith small organict ags in living cells was discussed by AnnetteB eck-Sickinger (Universityo f Leipzig,G ermany). [45] By using two-color,p ulse-chase labeling experiments, the human neuropeptide Y2 receptorc ould be trackedi nt he same cell at different times, and it wasd emonstrated that internalizing vesiclesf use within few minutes inside the cell.…”