Exp..Eye Res. (1995) 60, 465--469

Loss of the C-terminal Serine Residue from Bovine pB2-Crystallin

GREG W. KILBY, JOHN A. CARVER, J IUL IN L. ZHU, MARGARET M. SHEIL A N D ROGER J. W. TRUSCOTT*

Australian Cataract Research Foundation, The University of Wollongong, NSW 2522, Australia

(Received Oxford 16 August and accepted in revised form 17 December 1994)

Electrospray mass spectrometric (KS-MS) examination of bovine /?-crystallins showed a significant component corresponding in mass to flB2-crystaUin less one serine residue. Tryptic digestion, followed by isolation and characterisation of the C-terminal peptide, demonstrated that this new species has arisen by the loss of the C-terminal serine residue. This phenomenon appears to be age-related since no truncation was detected in flB2-crystallin from foetal lenses and the proportion of the truncated form, as judged by KS-MS, was lower in/?-crystallin isolated from calf lenses than that from the lenses of 3-year- old animals. This process therefore is similar to a recently reported loss of the C-terminal serine from ~zA- crystallin, which we have confirmed using KS-MS. Loss of a C-terminal serine from both crystallins may indicate the presence of carboxypeptidase-A-like activity in bovine lenses. KS-MS data provided no evidence for a significant degree of phosphorylation of ffBfcrystallin.

Keg words: electrospray mass spectrometry; flB~-crystallin; ~-crystallin; tandem mass spectrometry.

1. Introduction

The post-translational modification of soluble lens proteins has been well documented. For example, cleavage of crystallins at various sites has been shown to occur, predominantly in older lenses. Among a number of truncations observed in 0~-crystallin (reviewed in Harding, 1991), age-related cleavage in the C-terminal domain of the ~A subunit (Siezen and Hoenders, 1979) and loss of the C-terminal serine from the same polypeptide have been reported (Emmons and Takemoto, 1992). Post-translational changes to the/?-crystallins, however, have been less well studied. Approximately 40% of the crystallins in the bovine lens are/?-crystallins. The most abundant of the seven subunits which comprise fl-crystallin is /?B~-crystallin (Carver, Cooper and Truscott, 1993).

Recently we initiated a programme to examine aged human and bovine crystallins by electrospray mass spectrometry (KS-MS) (Fenn et al., 1990). Smith and coworkers also reported KS-MS analysis of crystallins from young human (Miesbauer et al., 1994) and bovine (Smith et al., 1991) lenses. The utility of KS-MS for the determination of extremely accurate (up to 0-001% mass accuracy) molecular weights of large fragile biomolecules, such as proteins, has been clearly demonstrated (Fenn et al., 1990). Owing to the nature of the ionization process, large proteins can be analysed on quadrupole mass spectrometers with limited mass ranges, at low concentrations (typically 1-20 pmol/zl-1). Since detection of a protein by KS-MS is independent of the nature of modifying groups, this

* For correspondence at: Australian Catarac]; R,~v,,o~ch _v_....~. dation, Department of Chemistry, The Universit~ of Wollongong m NSW 2522, Australia.

0014-4835/95/050465+05 $08.00/0

technique is an ideal tool to aid in the examination of post-translational modifications or age-related changes occurring in lens proteins that may not be detected using traditional methods. E,S-MS is a par- ticularly attractive technique for studying the ~t- and fl-crystafilns which have blocked N-termini.

During the examination of the/?-crystallins by ES- MS, a peak with a mass equivalent to the fiB2 subunit minus a serine residue was observed in the mass spectrum of flB2-crystallin isolated from aged bovine lenses. This paper describes the characterization of that species.

2. Materials and Methods

Bovine lenses were obtained from the Parish Meat Suppliers abattoir at Yallah, NSW, Australia. Human lenses were obtained from the Sydney Hospital Lions Eye Bank, NSW, Australia.

Lenses were homogenized in buffer containing 50 mM Tris-HC1 (pH 7.45), 5 n~l EDTA, 1 mM phenyl- methylsulfonylfluoride (PMSF), 1 mM 1,4-dithio- threitol, and 0-02% (w/v) sodium azide (Cooper, Carver and Truscott, 1993) (approx 1.5 ml per bovine lens). For each age examined, a minimum of four lenses were extracted. The homogenate was centri- fuged ( 1 2 0 0 0 r p m for 3 0 m in) to remove insoluble protein. The initial separation of ~- and fl-crystallins was performed on a Pharmacia Sephacryl $300 gel filtration column (1 m x 25 nun) using 0.05 M phos- phate buffer (pH 6"8) containing 0"05% NaN 3 as the eluent, l~B2-crystallin was purified by fast protein anion exchange liquid chromatography (FPLC) ac- cording to the method of Slingsby et al. (1990).

The pept~e corresponding to the last 13 amino of flB~-crystallin, MQWHQRGAFI-IPSS (residues

@ 1995 Academic Press Limited

4 6 6 G . W . K I L B Y E T A L .

192-204) , was synthesised by Auspep Pty Ltd, Parkville, Victoria, Australia. Tryptic digestion of this peptide plus the native/~B~ polypeptide was performed according to the method of Berbers et al. (1984). Carboxypeptidase A digestion of the tryptic peptide GAFHPS8 was used to generate a reference sample of GAFHPS. The digested peptides were separated on a Waters HPLC system using a Waters Bondapak C18 column at a flow rate of 0"8 ml min -1 and a gradient elution of 100% solvent A (aqueous 0"05% TFA) to 50% solvent B (50% aqueous acetonitrile containing 0.05 % TFA) over 60 min. Peaks were detected on a Waters absorbance detector (model 441) at 229 or 280 nm.

Electrospray mass spectrometry was carried out on a VG Quattro mass spectrometer (VG Biotech, Altrincham, U.K.) with the solvent being delivered via an ISCO SFC-500 microflow pump at a flow rate of 5 #l min -1. Samples ( ,,- 20 pmol #1-1 for the proteins) were dissolved in 50 % aqueous acetonitrile containing 1% formic acid and 10#1 was injected for each analysis. The skimmer potential was set at 50 V and the resolution to 1 Da. The mass spectrometer was scanned at a rate of 1 sec per 100 Da, and data from 10-20 scans were summed to obtain representative spectra. The ES mass spectra of the/~B~ polypeptides reported here have been transformed from a mass-to- charge scale to a mass scale using the Maxent software supplied by VG. For MS-MS experiments argon was used as the collision gas and the collision energy was 200 eV. The concentrations of the peptide samples were higher ( ~ 200pmol/z1-1) than for the MS experiments.

3. Results

Figure 1 (a) shows an electrospray mass spectrum of //B2-crystallin purified from three year-old bovine lenses. The major peak observed was at 23208 _ 2 Da and this corresponds exactly to the mass calculated from the sequence (23208.7Da) of bovine fiB 2- crystallin (Slingsby et al., 1988). A second peak of approximately 12 % relative intensity, had a mass of 23121 ± 2 Da which differs from fiB 2 by 87 Da. This is the mass of a serine residue. In the ES-MS of fiB 2- crystallin obtained from calf (3 months old) lenses, shown in Fig. l(b), this second peak was much reduced, being approximately 2-5 % relative to that for the intact/~2-crystallin. In the ES-MS of flB2-crystallin isolated from foetal lenses (not shown) the second species was absent. Thus, the presence of this species appeared to be related to the age of the lens. Repeat analyses on individual lenses showed that these results were consistent in each age group, i.e. no peak corresponding to serine loss was observed in any of the three individual foetal lenses (and an homogenate of eight foetal lenses), whereas analyses of three calf lenses ( ~ 3 months old) yielded an average of 3 ± 1%

100

100-

(a)

~B2 23209

13B 2 less serine

23122

i 23210

(b)

~k' , . . . . L , - : . , . . . . , . . . . , . .

L , , , I .... ,r,, m a s s 23250 23500 23750 24000 22500 22750 23000

FIG. 1. Electrospray mass spectrum of bovine/]B2-crystallin from (a) 3-year-old lenses, and (b) 3-month-old lenses.

serine loss, and analyses of five adult lens (,-~ 3 years old) homogenates (four lenses per homogenate) dis- played an average of 13 ___ 2 % serine loss with respect to the parent/~B 2 peak.

Bovine/~B2-crystallin has a C-terminal serine residue (Ser2°4), so it seemed most likely that the loss of the serine had arisen from the removal of this residue. A series of experiments were conducted to confirm that this was the case.

The C-terminal extension of bovine /~-crys ta l l in can be cleaved at Arg 197 using limited tryptic digestion releasing the peptide GAFHPSS (Berbers et al., 1984). The optimal digestion time with trypsin was found to be 10 min, i.e. little or no digestion of the globular domain was observed in this time. A similar rapid cleavage at Arg 197 had been noted by Berbers et al. (1984). Figure 2 shows the HPLC profile of the tryptic digest of the same 3-year-old flB2-crystallin sample from which we obtained the ES-MS spectrum in Fig. l(a). The peak labelled P1 at 11"5 min corresponds to the retention time of the intact tryptic peptide, GAFHPSS and the peak P2 at 12.8 min corresponds to the retention time of the corresponding peptide less serine, i,e. GAFHPS. The peptide standards were obtained from isolation of fragments from tryptic/ carboxypeptidase digestion on a synthetic peptide as discussed below.

LOSS OF SERINE RESIDUE FROM f lB2-CRYSTALLIN 467

E c o ¢o o4

tJ i -

£ 0 (n .Q <

P1

I I I I

10

P2 \

I I i I | I I I I

20

Retention Time (mins~

Fta. 2. Reversed-phase HPLC separation from a 10-min tryptic digestion of the purified flBz-crystallin used to obtain the ES-MS spectrum in Fig. l(a). Peak 1 (R r = 11"5 rain) corresponds to the tryptic fragment GAFHPSS (MW = 701.3 Da) and, peak 2 (RT = 12"8 min) corresponds to the tryptic fragment GAFHPS (MW = 614'3 Da).

TABLE I

A summary of the ES-MS/MS data obtained on peak 1 (GAFHPSS) and peak 2 (GAFHPS) collected from HPLC

of the digest of ~lB~-crystallin (see Fig. 2)

Molecular weight

P1 P2 Assignment

702"2 - - [GAFHPSS + H] + - - 615.4 [GAFHPS +HI + - - 512.9 X 4

412"9 412'9 B, 385"5 385"5 A 4 289.6 - - Y~' 276.1 - - B 3 234.9 - - [HP+H] +

- - 206.9 [HP less CO+H] + - - 203.2 Y~

193.3 - - Y~' 165.0 - - IF+H] + 156.5 - - [PS less CO+H] +

- - 129.2 B 2 120"2 120"3 I a 110"2 110"1 14

- - 100"8 A 2 70"3 70'1 15

B. = N - t e r m i n a l f r a g m e n t c o n t a i n i n g n res idues . A n = N - t e r m i n a l f r a g m e n t c o n t a i n i n g n res idues m i n u s CO. Y~ = C- t e rmina l f r a g m e n t c o n t a i n i n g n res idues . X n = C- t e rmina l f r a g m e n t c o n t a i n i n g n r e s idues m i n u s CO. I~ = I m m o n i u m ion (H ,N = CH-R) + c o r r e s p o n d i n g to n r e s idues

f r o m the N - t e r m i n u s .

A synthetic peptide incorporating the C-terminal extension of flB2-crystallin, f.e. MQWHQRGAFHPSS (residues 192-204) , was digested with trypsin to yield the peptide GAFHPSS. Treatment of this peptide with carboxypeptidase A (CPA) removed the C-terminal serine resulting in the peptide, GAFHPS. Both the latter two peptides were isolated using the same HPLC conditions as in Fig. 2. This approach provided authentic standards for comparison with peptides found in the tryptic digests of the flB~-crystallin and demonstrated that the latter sequence, as expected, is susceptible to limited attack by carboxypeptidase A. The identity of the synthetic peptides was confirmed by ES-MS and MS-MS experiments. For GAFHPSS an [M+H] + ion by ES-MS of 702.2 was obtained (calculated MW, 702"3), and for GAFHPS an [M+H] + ion of 615"4 was obtained (calculated MW, 615.3).

The identity of the peaks, P1 and P2, in the tryptic digest of flB2-crystallin (Fig. 2) was verified by isolation of these components using HPLC separation and subsequent analysis by ES-MS and tandem mass spectrometry (MS-MS) (Busch, Glish and McCluckey, 1989). In the ES mass spectra of P1 and P2, the major peaks were observed at 7 0 2 . 3 + 0 . 5 D a and 615-6__+0"5 Da respectively and these correspond to the respective protonated molecular ions of GAFHPSS and GAFHPS. Table I summarizes the MS-MS data obtained from experiments on the two peptides. In each case, ions consistent with the respective sequences were observed, thus providing confirmation of the identity of the peaks P1 and P2.

The intensity of P2, i.e. the peak due to the serine loss, as determined from the relative abundancies in

468 G.W. KILBY ET AL.

t~A 19834 lOO- (a)

~A/~hosphorylated

19915

¢xB 20082

°'"" i I l serine 19950 20163

I~9748 l ~ h L J ~ A IA~A Jki

19833 loo- (b)

19912

% 20079

20156

Ii+ ~ 1 ~ . . . . . ~ =1 , . ~ . . . . . . . . ~.A, . . l . ! , A J,.( • . ~ . ?,..5. m a s s

19400 19600 19800 20000 20200 20400

FIG. 3. Electrospray mass spectrum of bovine total a- crystallin from (a) 3-year-old-lenses, and (b) foetal lenses.

the HPLC chromatogram shown in Fig. 2, is approxi- mately 12 % of P1 which is consistent with the ES-MS data [Fig. l(a)]. The HPLC chromatogram obtained from a tryptic digest of foetal flB2-crystallin under the same conditions showed no evidence of the serine-loss peak, P2.

Recently, Emmons and Takemoto demonstrated, using antisera to the C-terminal peptide of a-crystallin, that there appears to be an age-dependent loss of the C-terminal Ser from the aA subunit (Emmons and Takemoto, 1992). We were able to confirm these findings by direct examination of the a-crystallin fraction via ES-MS. The ES mass spectrum of the total bovine a-crystal[in from a 3-year-old lens is shown in Fig. 3(a). The major components are the aA (19834-t- 2 Da) and ~B (20082 + 2 Da) subunits. This spectrum also clearly shows the existence of mono- phosphorylated aA- (19915_+2Da) and aB- (20163_+2 Da) crystal[ins. A similar profile for a- crystal[in has been obtained by Smith et al. (1991). In addition, a small peak corresponding to diphos- phorylated aB-crystallin (20244_+ 2 Da) and a com- ponent corresponding in mass with that of aA- crystal[in minus one serine residue (19748_+2 Da) was observed with about 9 % relative intensity. The latter component also increases with increasing age of the animal (data not shown) and is absent from the

ES-MS spectrum of the total a-crystallin obtained from a foetal lens shown in figure 3(b). A peak at 20035 _+ 2 Da in Fig. 3 which Roquemore et al. (1991) have shown arises from modification by O-linked N- acetylglucosamine also showed a relative increase with age of the animal.

4. Discussion

It is striking that two bovine lens polypeptides, aA- and/~B2-crystal[in, of unrelated origin, and with little

or no overall sequence homology, both terminate in the amino acid sequence ProSerSer. We have demon- strated by NMR spectroscopy that both these proteins have short and flexible C-terminal extensions of eight amino acids in aA-crystallin (Carver et al., 1992) and ten amino acids in/~B2-crystallin (Cooper et al., 1993). At 25 °C the C-terminal extension of aA-crystallin is not involved in interactions with other crystal[ins (Carver et al., 1994; Cooper et al., 1994) whereas the C-terminal extension of flB2-crystaflin is (Cooper et al., 1993, 1994; Carver et al., 1994), for example, in the flH (octamer) species. At a higher temperature (45°C) however, the Set 2°4 residue of the C-terminal extension of flB2-crystallin, in fill, becomes exposed (Cooper et al., 1993). Thus both extensions are presumably accessible to protease activity in the lens. In this paper we have shown that the C-terminal serine is lost in an apparently age-dependent fashion from fil~2-crystallin, in an analogous manner to that reported for aA- crystal[in (Emmons and Takemoto, 1992). This trunc- ation can be mimicked by incubation of the /~B 2 C- terminal peptide with CPA. We were also able to confirm, using similar procedures, the reported loss of serine from aA-crystallin. A CPA-like activity may therefore be present in the bovine lens such that over time, gradual cleavage of the C-terminal serine from both aA- and flB~-crystallin occurs. In both proteins, the presence of proline adjacent to the penultimate serine would prevent further attack by CPA. Such a cleavage is one of a plethora of post-translational cleavages detected in lenses which become more pronounced with age (Harding, 1991; van Kleef, Thijssen and Hoenders, 1976).

As yet it is unclear what functional significance, if any, is associated with these C-terminal truncations. Loss of the C-terminal serine from flB2-crystallin may, for example, affect the interaction of the C-terminal extension with other crystal[ins. The flexible C- terminal extensions of a-crystaLlin may simply act as spacers between the crystal[ins within the lens (Cooper et al., 1994). Since a-crystallin has solvent-exposed hydrophobic regions (Liang and Li, 1991), it is possible that a-crystaflin subnnits which have lost part or all of their flexible C-terminal extensions may tend to aggregate and reduce their ability to act as stabilizers of other unfolded crystal[ins (Horwitz, 1992).

There appeared to be no evidence for phosphoryl-

LOSS OF SERINE RESIDUE FROM flB2-CRYSTALLIN

ation of flB2-crystallin, either in the spectra shown in Fig. 1 following FPLC, or in ES-MS of the/?L or the/?H fraction (data not shown) obtained following gel permeation separation. Kleiman et al. (19 88) reported phosphorylation of flB2:crystallin at Set 2°3 using an in vitro experiment with a2p labelling, but, if present in the lens, it wou ld appear from our data to be a very minor component.

In the case of the h u m a n lens, Miesbauer et al. (1993) have published a sequence for h u m a n /713 z- crystallin with a C-terminal Asn rather than Ser 2°4. This postulated sequence was based on experiments involving a combination of carboxypeptidase digestion and mass spectrometry. The molecular weight of h u m a n /?B2-crystallin reported by these workers of 2 32 93 Da, using ES-MS, is consistent with our results. We have been able to confirm the C-terminal Asn by conventional amino acid sequencing of the C-terminal peptide from h u m a n /?Bz-crystallin derived by cy- anogen bromide cleavage at Met 192 (unpublished data).

Acknowledgements

Grants from the Australian Research Council, the Uni- versity of Wollongong and the Ramaciotti Foundation, which assisted in the purchase of the electrospray mass spectrometer, are gratefully acknowledged. Assistance from Andrew Aqui[ina in the purification of g-crystal[in is appreciated.

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