Characterization of O-phosphohydroxyproline in rat {alpha}-crystallin A

Abstract

Post-translational modifications have major importance for the structure and function of a multiplicity of proteins. Phosphorylation is a widespread phenomenon among eukaryotic proteins. Whereas O-phosphorylation on the side chains of serine, threonine, and tyrosine in proteins is well known and has been studied extensively, to our knowledge the endogenous phosphorylation of hydroxyproline has not previously been reported. In the present work, we provide evidence for the first time that O-phosphohydroxyproline (Hyp(P)) is a proteinogenic amino acid. To detect Hyp(P) in proteins we generated a Hyp(P)-specific polyclonal antibody. We could identify Hyp(P) in various proteins by Western blot analysis, and we characterized the sequence position of Hyp(P) in the protein α-crystallin A by electrospray ionization-tandem mass spectrometry. Our experiments clearly demonstrate hydroxylation and subsequent phosphorylation of a proline residue in α-crystallin A in the eye as well as in heart tissue of rat.

FIGURE 1.

Specificity of the polyclonal antibody tested by dot blot analysis. Each spot contained 1 μg of synthetic peptide. Sequences of the peptides are denoted to the right of the spots. Phosphoamino acids in the sequences are boldfaced. Hyp and all phosphoamino acids are denoted in three-letter code, all other amino acids are in one-letter code.

FIGURE 2.

Detection of Hyp(P)-containing proteins by Western blot analysis. SDS-PAGE (8–15% gradient) of total protein of heart, aorta, and eye from rat is shown. The Western blot was performed with a Hyp(P)-specific polyclonal antibody. Aliquots of total protein without pretreatment, pretreated with calcineurin, and pretreated with alkaline phosphatase were compared. Protein bands denoted with roman numerals were identified by MS/MS analysis as (I) β-crystallin B2, (II) β-crystallin A4, (III) α-crystallin B, (IV) α-crystallin A.

FIGURE 3.

MS spectrum of eye α-crystallin A after trypsin digestion. Peptide signals within the ellipsoid are shown in the inset. Mass differences of 8 m/z and 40 m/z of doubly charged ions indicate a possible peptide hydroxylation and phosphorylation, respectively.

FIGURE 4.

Comparison of the MS/MS spectra of the precursor ions with [M+H]²⁺ 504.33 m/z, [M+2H]²⁺ 512.33 m/z, and [M+2H]²⁺ 552.32 m/z. All spectra show complete y series. The three peptides have identical amino acid sequences. The mass shift between fragment ions y₅ and y₆ is characteristic for Pro (A), Hyp (B), and Hyp(P) (C) at the relevant sequence position.

FIGURE 5.

Isomers of Hyp(P).

FIGURE 6.

MS/MS spectra of synthetic peptides. The spectra show the fragmentation patterns of the peptide dephosphorylation. Peptides A–C corresponded to the short α-crystallin A sequence of the Hyp(P)-containing peptide. Peptides D–F had the same sequence but were elongated by an additional arginine at the N terminus. Tau peptides G–I corresponded to a completely different sequence (see Table 3). Peptides in the top row (A, D, and G) contained trans-4-Hyp(P); peptides in the middle row (B, E, and H) contained trans-3-Hyp(P); and the peptides in the bottom row (C, F, and I) contained cis-4-Hyp(P).