Identification of the posttranslational modifications of bovine lens αB‐crystallins by mass spectrometry

Abstract

A combination of mass spectrometric techniques has been used to investigate the amino acid sequence and posttranslational modifications of aB-crystallin isolated from bovine lenses by gel filtration chromatography and reversed-phase high performance liquid chromatography. Chromatographic fractions were analyzed by electrospray ionization mass spectrometry to determine the homogeneity and molecular weights of proteins in the fractions. The aB-crystallin primary gene product, its mono-and diphosphorylated forms, its N-and C-terminal truncated forms, as well as other lens proteins unrelated to the aB-crystallins were identified by their molecular weights.Detailed information about the sites of phosphorylation, as well as evidence supporting reassignment of Asn to Asp at position 80, was obtained by analyzing proteolytic digests of these proteins by fast atom bombardment mass spectrometry. Results of this investigation indicate that aB-crystallin is phosphorylated in vivo at Ser 45, Ser 59, and either Ser 19 or 21. From the specificity of phosphorylation of a-crystallins, it appears that there may be two different kinases responsible for their phosphorylation.Keywords: crystallins; lens proteins; mass spectrometry; protein phosphorylationThe lens crystallins are long-lived structural proteins of particular interest in understanding cataractogenesis (Harding, 1985) and chemical processes associated with aging (Harding & Dilley, 1976). There are three primary groups of lens crystallins, a-, P-, and y-crystallins, which can be separated by the size of aggregates they form. a-crystallins form aggregates with molecular weights over 500,000; &crystallin aggregates have molecular weights of 40,000-200,000; y-crystallins remain as monomers of 20,000. The molecular weights of the monomers of aand P-crystallins are also approximately 20,000. The nomenclature of the m-and P-crystalIins describes whether they are acidic (e.g., aA2, and PA3) or basic (aB2 and f3B2). Early investigators of the lens crystallins attributed the altered electrophoretic mobility of several a-crystallins to deamidation of asparagine (Bloemendal et al., 1972;Stauffer et al., 1974) and C-terminal degradation (Van Kleef et al., 1976). In 1985, Spector et al. (1985) showed that radiolabeled phosphate could be incorporated into both &A-and aB-crystallins, with evidence that serine was the only phosphorylated residue. Their as- sumption that electrophoretic behavior originally attributed to deamidation is due to phosphorylation (Chiesa et al., 1987(Chiesa et al., , 1988) was substantiated by quantification of phosphorus in a-crystallins (Voorter et al., 1989). The C-terminaI degradation and phosphorylation of aA-crystallins were confirmed recently by mass spectrometry . The fact that the a-crystallins could be phosphorylated suggested that they might have biological functions other than their contribution to lens structure. Using radioactively labeled phosphate Chiesa et al. (1987Chiesa et al. ( , 1988 determined that the phosphorylation of aBcrystal...

Spontaneous cleavage of proteins at serine and threonine is facilitated by zinc

“…Cleavage of long‐lived proteins on the N‐terminal side of Ser has been reported in proteins from a number of tissues (Table 1) (Smith et al ., 1992; Takemoto, 1995; Lampi et al ., 1998; Santhoshkumar et al ., 2008; Su et al ., 2010, 2012; Friedrich et al ., 2012) although this process has been most well documented in the lens. In the human lens, an abundant peptide is derived from the age‐related truncation of alpha B crystallin between residues 18 (His) and 19(Ser) (Santhoshkumar et al ., 2008).…”

“…To examine cleavage at Thr in more detail, Ac‐YPERTIPY was employed. Ac‐YPERTIPY incorporates a sequence region of alpha B crystallin (154–160) where age‐related truncation is known to occur at Thr 158 (Smith et al ., 1992). Its cleavage was compared with that of a Ser‐containing peptide derived from alpha A crystallin, Ac‐YEVRSDRDY.…”

“…Cleavage next to these residues has been often observed in long‐lived proteins (Smith et al ., 1992; Takemoto, 1995; Lampi et al ., 1998; Santhoshkumar et al ., 2008; Su et al ., 2010, 2012; Friedrich et al ., 2012). Our work extends a publication on dipeptides by Yahiro et al .…”

Small heat-shock proteins: important players in regulating cellular proteostasis

“…For example, Bc is phosphorylated at three serine residues, Ser19, Ser45 and Ser59 [118,120,121]; phosphorylation at Ser45 is mediated by p44/p42 MAPK, at Ser59 by MAPKAPK-2 [118,122], whilst the kinase responsible for phosphorylation at Ser19 remains to be identified. Similarly, Hsp27 has three serine residues (Ser15, Ser78 and Ser82) that undergo phosphorylation [123,124].…”

“…While purified αBc from bovine lens has been used to investigate of the effects of phosphorylation on αBc structure and function (as it is extensively phosphorylated with age [114,120,121,145,146]), such sources of sHsps do not afford homogeneous phosphorylated isoform. Instead these contain a mixture of nonphosphorylated, mono-, di-and/or tri-phosphorylated forms in the one oligomer.…”