Substrates of the Arabidopsis thaliana protein isoaspartyl methyltransferase 1 identified using phage display and biopanning

Abstract

The role of protein isoaspartyl methyltransferase (PIMT) in repairing a wide assortment of damaged proteins in a host of organisms has been inferred from the affinity of the enzyme for isoaspartyl residues in a plethora of amino acid contexts. The identification of PIMT target proteins in plant seeds, where the enzyme is highly active and proteome long-lived, has been hindered by large amounts of isoaspartate-containing storage proteins. Mature seed phage display libraries circumvented this problem. Inclusion of the PIMT co-substrate, S-adenosylmethionine (AdoMet), during panning permitted PIMT to retain aged phage in greater numbers than controls lacking co-substrate or when PIMT protein binding was poisoned with S-adenosyl homocysteine. After four rounds, phage titer plateaued in AdoMet-containing pans, whereas titer declined in both controls. This strategy identified 17 in-frame PIMT target proteins, including a cupin-family protein similar to those identified previously using on-blot methylation. All recovered phage had at least one susceptible Asp or Asn residue. Five targets were recovered independently. Two in-frame targets were produced in Escherichia coli as recombinant proteins and shown by on-blot methylation to acquire isoAsp, becoming a PIMT target. Both gained isoAsp rapidly in solution upon thermal insult. Mutant analysis of plants deficient in any of three in-frame PIMT targets resulted in demonstrable phenotypes. An over-representation of clones encoding proteins involved in protein production suggests that the translational apparatus comprises a subgroup for which PIMT-mediated repair is vital for orthodox seed longevity. Impaired PIMT activity would hinder protein function in these targets, possibly resulting in poor seed performance.

FIGURE 1.

Urea concentration and relative rAtPIMT1 activity in solution or when bound to microtiter plates. a, recombinant A. thaliana PIMT1 (rAtPIMT1), recovered from inclusion bodies, was solubilized in 6 m urea, purified over a nickel column, and dialyzed to remove imidazole (see the inset) First lane, rAtPIMT1 from inclusion bodies; second lane, purified, dialyzed rAtPIMT1. The enzyme was assayed in different concentrations of the chaotrope. The rAtPIMT1 (1 μl of 0.05 μg·μl⁻¹) was most active in 1 m urea. Different lowercase letters above the bars indicate significantly different relative activities in different urea concentrations when compared using Scheffe's multiple pairwise comparison. b, 100 μl of rAtPIMT1 (0.01 μg·μl⁻¹) was introduced into ELISA plate wells in a variety of urea concentrations (0.1–2.5 m). The wells were then washed with × mTBS containing no or 1 m urea to eliminate unbound rAtPIMT1, and the bound enzyme was assayed in reaction mix with no or 1 m urea. Greatest bound rAtPIMT1 relative activity was obtained when the enzyme was introduced into the wells in 0.7 m urea and washed with 1× mTBS buffer without urea before assay. Significant deviation was determined using Scheffe's test after an analysis of variance. In b, rAtPIMT1 activity was greater regardless of urea concentration used for rAtPIMT1 introduction, when the wash was without urea. Lowercase letters over bars indicate significantly deviating means among urea concentrations used for rAtPIMT1 introduction when no urea was included in the wash. Uppercase letters indicate the same for rAtPIMT1 activity when 1 m urea was used in the wash buffer. (500 mm Tris).

FIGURE 2.

Blocking reagent and washing buffer optimization. a, different blocking solutions influence phage titer. PVP, polyvinylpyrolidine. The company product achieved high titers either by preserving rAtPIMT1 activity or by binding phage indiscriminately. Different lowercase letters over the bars indicate significantly deviating mean plaque forming units retained in the wells coated with different blocking agents. b, these alternatives were tested by washing wells to which rAtPIMT1 or no protein (buffer only) had been bound before blocking with BR. Effect of the number of washing steps before phage elution on phage titer during the biopanning is shown. BR preserves rAtPIMT1 activity rather than indiscriminately binding phage. Different lowercase letters above bars (within a washing regime) indicate significantly deviating means dependent on the presence of rAtPIMT1. c, shown is SDS-amended mTBST washing buffer and rAtPIMT1 activity. The inclusion of even small percentages of SDS in the mTBST washing buffer detrimentally influenced the rAtPIMT1 activity. Significantly deviating means identified using Scheffe's test after an analysis of variance.

FIGURE 3.

a, shown is the effect of AdoMet (SAM) and AdoHcy (SAH) on phage titer from successive rounds of biopanning. None, neither AdoMet nor AdoHcy was added to the phage before introduction into the microtiter plate wells. S-Adenosylmethionine, a PIMT co-substrate, was added to the phage library to 100 μm just before biopanning. S-Adenosyl homocysteine, a PIMT inhibitor, was added to the phage library to 100 μm just before biopanning. Significantly deviating means among the three treatments were identified using Scheffe's test within each of the four rounds and are represented by different lowercase letters over each bar. b, shown is the effect of AdoMet and AdoHcy on the retention of phage with inserts in successive rounds of biopanning. c, shown is a graphical depiction of the 111 hits retrieved from the biopans. Out-of-frame (O.O.F.) and in-frame (I.F.) hits have been divided into those clones that were recovered only once (or the same clone (identical sequence) obtained multiple times; shades of red-purple), and those hits that are represented by at least two different clones (different lengths and/or portions of the same cDNA; shades of blue and green). All clones, after four rounds of biopanning, had at least one Asp or Asn residue. d, shown is clone identity for those recovered only once or for those recovered more than once but from the same biopan. e, clone identity for those recovered from different biopans and/or those that were different in sequence length and/or position in the cDNA and/or those that were from different libraries is shown. Different clones of At5g66400 recovered from library II are depicted in blue text. In all instances, the numbers in the pie slice represent the number of clones recovered and sequenced in that category.

FIGURE 4.

A representation of the frequency of neighboring amino acids at positions from non-prime 4 to prime 4 on either side of the Asp (D) (A and B) or Asn (N) residues (c and d) from the in-frame hits (a and c) and from proteins (b and d) along a randomly chosen segment of the A. thaliana genome. An asterisk above a bar indicates a significantly greater abundance of one/some amino acids relative to that expected based on codon usage for the species. The n under the graphs represents the number of Asp or Asn amino acid positions evaluated.

FIGURE 5.

Two full-length PIMT substrates, protein fragments of which were identified through biopanning, were cloned into pET23 and expressed in E. coli BL21(DE3)RIL. a, the Coomassie-stained PRH75 protein gel includes molecular weight (M), a blank, lysate from uninduced cells at harvest (1), insoluble (2)and soluble protein (3) from induced cells after harvest and centrifugation, a nickel column wash after introduction of soluble protein (4), a post-wash before imidazole (5), elution (2 ml of 1 m imidazole) (6), blank, and protein post-dialysis (7). b, The PIRL8 recombinant protein was recovered from lysed cells as an inclusion body and solubilized in 6 m urea, and Ni-NTA column-purified. The Coomassie-stained gel includes lysate from uninduced cells (1), molecular weight markers (M), lysate from IPTG-induced cells (2), the insoluble pellet from the lysate after centrifugation (3), the soluble proteins post-centrifugation (4), protein from the inclusion body after urea solubilization and Ni-NTA column purification, undialyzed in 6 m urea and imidazole (5), purified inclusion body in 6 m urea dialyzed to remove imidazole (6). In all lanes, for both purifications, 10 μl of the lysate/eluate/dialyzed sample was mixed with 10 μl of SDS-containing loading dye, boiled, and loaded. c, both purified proteins were electrophoresed using SDS-PAGE and Coomassie-stained or assayed for isoaspartyl formation through on-blot methylation (fluorograph depicted here) and liquid assay after incubation at 37 °C (Table 2) using human recombinant PIMT (d). kDa, kDa of the Bio-Rad-prestained molecular weight markers.

FIGURE 6.

Homozygous insertional mutant lines for three of the genes whose proteins were identified as being PIMT1 targets in phage display were recovered as homozygotes. a, one insertional mutant in the KED gene was determined to be a severe knockdown. The chevron points to a nonspecific amplicon from cDNA of both wild type and the ked mutant. b, seeds of this mutant, along with wild type seeds, were tested at 25 °C on water in constant light without moist chilling. c and d, three independent mutants of an Arabidopsis LEA 4 protein, similar to seed maturation protein1 (SMP1) from Glycine tomentella were obtained. One of these, smp1-1 located in the 3′-UTR, did not reduce SMP1 transcript and showed no phenotype in the screens used on the seeds (data not shown). The other two insertions resulted in severe knockdowns (smp1-2 and smp1-3). e, mutant and wild type seeds were placed on water-saturated filter paper at 40 °C in light in a sealed plastic bag with soaked paper towels for 4 days before being placed at 25 °C in light to complete germination. The smp1 mutants completed germination to a greater percentage than WT without moist chilling. f, two independent insertional mutants of the PIRL8 were identified (pirl8-2 depicted) and determined to be severe knockdowns. After heat shock to induce secondary dormancy and after the seeds had completed germination at 25 °C to the extent possible, seeds were moist-chilled for 3 days and placed at 25 °C to complete germination to distinguish between dead and dormant seeds (final germination percentage is depicted to the right of the vertical dashed line over M.C. in e). When possible, primers were designed to span an intron. For each germination time point, an analysis of variance was conducted between mutant and wild type to distinguish between significantly deviating means. This is depicted in b where different lowercase letters distinguish those time points where the ked mutant seeds averaged a greater percentage germination than their WT. For the other mutant/wild type combination significance is obvious.