Arginyltransferase ATE1 catalyzes midchain arginylation of proteins at side chain carboxylates in vivo

Abstract

Arginylation is an emerging posttranslational modification mediated by Arg-tRNA-protein-transferase (ATE1). It is believed that ATE1 links Arg solely to the N terminus of proteins, requiring prior proteolysis or action by Met-aminopeptidases to expose the arginylated site. Here, we tested the possibility of Arg linkage to midchain sites within intact protein targets and found that many proteins in vivo are modified on the side chains of Asp and Glu by unconventional chemistry that targets the carboxy rather than the amino groups at the target sites. Such arginylation appears to be functionally regulated, and it can be directly mediated by ATE1, in addition to the more conventional ATE1-mediated linkage of Arg to the N-terminal alpha amino group. This midchain arginylation implies an unconventional mechanism of ATE1 action that likely facilitates its major biological role.

Figure 1. Arginylation on side chains of acidic residues in vivo (top) and in vitro (bottom)

Top, Mass spectrum of a naturally occurring arginylation on the side chain of Asp. Peptide sequence is indicated on top, with the arginylated residue marked by +156 (a mass of Arg). See Table 1 and Supplemental Table 1 for a full list of identified side-chain-arginylated proteins and Supplemental Table 2 for sequences and parameters of the identified peptides. Bottom, mass spectrum of a standard peptide, enzymatically arginylated on the side chain of Asp. Peptide sequence is indicated on top, with the arginylated residue marked by +166 (a mass of stable isotope labeled [¹³C,¹⁵N]-Arg used in the experiment). Numbers indicate the masses of b (red) and y (blue) ions.

Figure 2. Characterization of side chain and N-terminal arginylation

A. Normalized incorporation of [³H]-Arg into a standard peptide corresponding to the N-terminal sequence of beta actin mediating N-terminal arginylation (DDIAALVVDNGSGMCK; left) and the same peptide with an N-terminal Arg inserted into the sequence to facilitate side chain arginylation (RDDIAALVVDNGSGMCK, right) with 4 ATE1 isoforms. Error bars represent SEM (n=3 for DDIAALVVDNGSGMCK and 4 for RDDIAALVVDNGSGMCK). B. N-terminal arginylation requires the presence of both a carboxy and an amino group. Normalized incorporation of [³H]-Arg into a synthetic DN peptide (containing N-terminal Asp and no internal arginylation sites, see text) is greatly inhibited when the amino group of the N-terminal Asp is modified with a methyl group (DN-Me) or when the carboxyl group is an allyl ester (DN-All). Error bars represent SEM (n=3). C. N-terminal acetylation inhibits arginylation. Normalized incorporation of [³H]-Arg into a synthetic peptide corresponding to the actin-s N-terminus (DDDIAALC) is greatly diminished after the N-terminal amino group of this peptide is blocked by acetylation (AcDDDIAALC) despite the presence of multiple side chain carboxyl groups. Error bars represent SEM (n=3).

Figure 3. ATE1 mediates different Arg linkage in proteins and peptides

A. Left, Normalized incorporation of [³H]-Arg into angiotensin II in vitro indicate that it is an efficient ATE1 target. Error bars represent SEM (n=3). Middle, NMR spectrogram of [¹³C,¹⁵N]-Arg linked to angiotensin II indicates a shift at Arg alpha carbon atom, suggesting that this atom is engaged in the linkage to the peptide via the “conventional” peptide bond. Mass shifts are shown in the table on the right. B, Coomassie-stained gel (top) and audioradiograph (bottom) of BSA, in vitro arginylated using [¹⁴C]-Arg, incubated for 30 min alone or in the presence of aminopeptidase B (APB) or carboxypeptidase B (CPB) as indicated at the bottom. C, CPB-mediated dearginylation of BSA can be inhibited by CPBi. Coomassie-stained gel (top) and audioradiograph (bottom) of BSA, in vitro arginylated using [¹⁴C]-Arg, incubated for increasing intervals of time alone or in the presence of CPB with and without added inhibitor (CPBi). The chart shows the levels of radioactive label divided by BSA protein levels (quantified from gel scans) for each condition at each time point. Error bars represent SEM (n=3). D, CPB-mediated dearginylation of BSA is dose dependent. Coomassie-stained gel (top) and audioradiograph (bottom) of BSA, in vitro arginylated using [¹⁴C]-Arg, incubated with decreasing molar ratios of CPB to BSA as indicated. E. Proposed mechanisms for arginylation (see explanations in the text). Protein chains are denoted by freeform circles.