Characterization of O-Phosphohydroxyproline in Rat α-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 ␣-crystallin A by electrospray ionization-tandem mass spectrometry. Our experiments clearly demonstrate hydroxylation and subsequent phosphorylation of a proline residue in ␣-crystallin A in the eye as well as in heart tissue of rat.A wide range of biological processes are controlled by reversible protein phosphorylation, and conservative estimates indicate reversible phosphorylation targets of up to one-third of cellular proteins (1). To understand protein regulation through phosphorylation it is essential to find and characterize all sites and types of phosphorylation on proteins and to identify the kinases involved.Besides the three genetically encoded hydroxyamino acids serine (Ser), threonine (Thr), and tyrosine (Tyr), there are two other hydroxyamino acids, hydroxyproline (Hyp) 2 and hydroxylysine (Hyl). They are not encoded in the genome, although they can be identified in mature proteins. They are generated by enzymatic hydroxylation of proline (Pro) and lysine (Lys) residues (2). All genetically encoded hydroxyamino acids in polypeptide chains, such as serine, threonine, and tyrosine have been shown that they can undergo posttranslational phosphorylation (3, 4). Although phosphorylation of Hyl has been described in collagens (5) phosphohydroxyproline (Hyp(P)) has not been identified in native proteins.In addition to the O-phosphorylation of the hydroxyl group, O-glycosylation is another important modification. There is a growing speculation that phosphorylation and glycosylation share a reciprocal relationship. Both Hyp and Hyl are known to undergo glycosylation in a few proteins. In collagens up to 70% of Hyl residues are modified with galactose and glycosyl-galactose (6). Hyl was also found to be phosphorylated in collagens (5). In extensins (hydroxyproline-rich glycoproteins) of the cell wall in plants, ϳ50% of protein mass is glycosylated at Hyp residues by mono-, di-, tri-(132)-␤-linked arabinoses (7,8,9). Furthermore, cytoplasmic F box-binding protein SKP1 in Dictyostelium contains a pentasaccharide linked to Hyp (10). These findings suggest that subsequent modification of Hyp is widespread and that, in analogy to all other hydroxyamino acids, phosphorylation of Hyp should be possible in native proteins.Hydroxylation of Pro is the preco...

Large-Scale Differentiation and Site Specific Discrimination of Hydroxyproline Isomers by Electron Transfer/Higher-Energy Collision Dissociation (EThcD) Mass Spectrometry

“…In plants, the cell wall consists of hydroxyproline-rich glycoproteins, and the type and extent of O-glycosylation is related to the location and context of HyP residues 10,40 . Phosphorylation of hydroxyproline (O-phosphohydroxyproline) was recently found in the protein α-crystallin A of rat heart tissue, with undetermined position of phosphate group and HyP isomer 41,42 . Since EThcD has been successfully implemented for characterizing phosphoproteome and glycoproteome 43,44, 45,46 , we reason that our method is applicable to simultaneously capture various types of PTMs in a single experiment.…”

“…As an ancient and conserved modification, various unsolved mysteries and correlations have been found between HyP and other PTMs. In plants, the cell wall consists of hydroxyproline-rich glycoproteins, and the type and extent of O-glycosylation is related to the location and context of HyP residues. , Phosphorylation of hydroxyproline (O-phosphohydroxyproline) was recently found in the protein α-Crystallin A of rat heart tissue, with undetermined position of phosphate group and HyP isomer. , Since EThcD has been successfully implemented for characterizing phosphoproteome and glycoproteome, − we reason that our method is applicable to simultaneously capture various types of PTMs in a single experiment. With the capacity to uncover the interplay among different PTMs, our approach possesses the potential to enable a better understanding of the molecular and cellular processes inside the cell and translate various PTM cross-talks into valuable biological and mechanistic insights.…”

Phosphoproteomics—More than meets the eye

“…In addition, in 2010, Kühlberg et al. provided evidence for the existence of proteinogenic phosphohydroxproline , a fourth O ‐phosphomonoester, so far only observed by enzymatic conversion of synthetic hydroxyproline‐containing peptides . Although phosphorylation of proteins occurs via the transfer of a phosphate group from adenosine triphosphate or guanosine triphosphate to the target protein by protein kinases, dephosphorylation is catalyzed by phosphatases releasing inorganic phosphate .…”

“…Abbreviations: AC, affinity chromatography; ERLIC, electrostatic repulsion hydrophilic interaction chromatography; ETD, electron transfer dissociation; HCD, higher energy collision dissociation; IMAC, immobilized metal ion affinity chromatography; MD score, Mascot Delta Score; MOAC, metal oxide affinity chromatography; MRM, multiple reaction monitoring; pHis, phosphohistidine; pSer, phosphoserine; pThr, phosphothreonine; pTyr, phosphotyrosine; SCX, strong cation exchange; SIMAC, sequential elution of immobilized metal ion affinity chromatography can be divided into four subclasses depending on the involved amino acids: (i) O-phosphomonoesters on the hydroxyl amino acids serine (phosphoserine, pSer), threonine (phosphothreonine, pThr), and tyrosine (phosphotyrosine, pTyr), (ii) N-phosphoramidates on arginine, lysine, and histidine, (iii) S-phosphothioesters on cysteine, and (iv) phosphoanhydrides on glutamic and aspartic acid, respectively [3]. In addition, in 2010, Kühlberg et al provided evidence for the existence of proteinogenic phosphohydroxproline [4], a fourth O-phosphomonoester, so far only observed by enzymatic conversion of synthetic hydroxyproline-containing peptides [5]. Although phosphorylation of proteins occurs via the transfer of a phosphate group from adenosine triphosphate or guanosine triphosphate to the target protein by protein kinases, dephosphorylation is catalyzed by phosphatases releasing inorganic phosphate [6,7].…”