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Albumin Banks Peninsula: a new termination variant characterised by electrospray mass spectrometry
Biochimica et Biophysica Acta 1433 (1999) 321^326 www.elsevier.com/locate/bba
Albumin Banks Peninsula: a new termination variant characterised by electrospray mass spectrometry Stephen O. Brennan *, Andrew P. Fellowes, Peter M. George Molecular Pathology Laboratory, Canterbury Health Laboratories, Christchurch Hospital, Christchurch, New Zealand Received 8 April 1999; received in revised form 4 May 1999; accepted 4 June 1999
Abstract Albumin Banks Peninsula is an electrophoretically fast variant that is expressed at only 2% of the total serum albumin. Electrospray ionisation analysis indicated a mass decrease of 755 Da relative to normal albumin and carboxypeptidase A digestion, together with CNBr peptide mapping, indicated a C-terminal truncation. This was confirmed by PCR and DNA sequence analysis which showed the introduction of a new AG acceptor splice site near the 3P end of intron 13. Predictably this results in the replacement of the C-terminal GKKLVAASQAALGL sequence by SLCSG and would be associated with an 861 Da decrease in molecular mass. We surmised that the new Cys was most probably cysteinylated as this albumin species would have a mass decrease of 742 Da and be very close to the measured value of 755 Da. Cysteinylation was confirmed when a mass decrease of 863 Da was measured between the proteins after reduction of their disulfide bonds. ß 1999 Elsevier Science B.V. All rights reserved. Keywords: Serum albumin; Truncation; Electrospray mass spectrometry; Low expression
1. Introduction Human serum albumin, the most abundant plasma protein, consists of a single polypeptide chain of 585 amino acids [1]. It is synthesised in the liver as preproalbumin. The prepeptide is co-translationally cleaved in the endoplasmic reticulum and the propeptide is removed in Golgi vesicles before secretion from the hepatocyte [2]. By virtue of its concentration, albumin regulates plasma oncotic pressure, but it functions primarily as a transport protein, and reversibly binds a wide array of ligands such as fatty acids, steroids, bilirubin, tryptophan and copper [1,3].
* Corresponding author. Fax: +64-3-3640-750.
The single copy of the albumin gene is located on chromosome four and is organised into 15 exons and 14 introns, the 15th exon being untranslated [4]. Genetic variation, or bisalbuminaemia, is usually detected by electrophoresis of plasma collected for diagnostic reasons. Bisalbuminaemia, however, may also be acquired as a result of proteolytic cleavage associated with pancreatitis, or due to increased drug or bilirubin binding. Some 50 di¡erent genetic variants have been characterised by protein and/or DNA sequence analysis [5], and these have provided unique insights into the intracellular processing of albumin and its ligand binding sites. Of the mutations characterised, four di¡erent genetic events have been reported that give rise to low concentration truncated molecules. These include single nucleotide deletions and frameshifts in Al Baz-
0167-4838 / 99 / $ ^ see front matter ß 1999 Elsevier Science B.V. All rights reserved. PII: S 0 1 6 7 - 4 8 3 8 ( 9 9 ) 0 0 1 3 1 - 4
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zano [5] and Al Catania [6], a 25 bp deletion that results in the skipping of exon 14 in Al Venezia [6], and a GT to CT 5P splice site mutation in intron 13 of Al Rugby Park [7]. Here, using electrospray ionisation mass spectrometry (ESI MS), we identify a truncated albumin with a mass decrease of 755 Da. This new albumin, Al Banks Peninsula, makes up only about 2% of the total plasma albumin and results from the introduction of an alternative 3P splice site in intron 13. 2. Materials and methods
2.4. High-performance liquid chromatography (HPLC) CNBr digests were analysed by both reverse-phase and size-exclusion chromatography. Reverse-phase separations were conducted on a Phenomenex W-Porex C4 column at a £ow rate of 1 ml/min in a 0.5% TFA/acetonitrile solvent system [9]. Sizeexclusion chromatography was performed on a TSK G2000SW (1U60 cm) column in 0.1% TFA 20% acetonitrile and at a £ow rate of 0.5 ml/ min. 2.5. ESI MS
2.1. Albumin puri¢cation Citrate anticoagulated plasma (30 ml) was chromatographed on a 2.6U15 cm column of DEAE Sephadex A 50 equilibrated in 16 mM sodium acetate bu¡er (pH 5.2). Bound albumin was eluted with 600 ml of this bu¡er with a pH gradient from pH 5.2 to 4.4 at a £ow rate of 1 ml/min [7]. Peaks were analysed by agarose gel electrophoresis at pH 8.6 [8] and the albumin peaks were pooled and lyophilised. The variant fraction, which contained approximately 70% Al Banks Peninsula, was re-chromatographed on a 12U53-mm Uno Q6 anion exchange column (Bio Rad) using similar bu¡ers with a 30-min gradient and a £ow rate of 3 ml/min. 2.2. Carboxypeptidase digestion Albumin, at a concentration of 5 mg/ml in 50 Wl of 50 mM NH4 HCO3 , was incubated with 15 Wg of carboxypeptidase A. Aliquots (20 Wl) were lyophilised after 1 and 5 h and redissolved in 100 Wl of 50% acetonitrile 0.1% HCOOH, and analysed directly by ESI MS. 2.3. CNBr digestion After reduction with 15 mM dithiothreitol in 8 M urea, 5 mg of albumin was S-carboxamidomethylated and redissolved in 150 Wl of 70% HCOOH containing 3 mg of CNBr. After overnight incubation the digest was dried and redissolved in 300 Wl of 5% acetonitrile.
Twenty Wl of native albumin (1^2 mg/ml in 0.1% HCOOH 50% acetonitrile) was delivered to the ion source of a Micromass Platform II quadrupole analyser at 5 Wl/min. The probe was maintained at 60³C and +3500 V, and the m/z range (1100^2100) was scanned with a cone voltage gradient of 40^60 V every 2.5 s. Data were acquired and processed using MassLynx software and transformed onto a true mass scale using Max-Ent software [10]. No external calibration was applied to the raw data. Reduced albumin was analysed similarly except scans were between 700 and 1600 m/z. Reduction was carried out in 8 M urea, 0.1 M Tris^HCl (pH 8), 15 mM dithiothreitol, and the albumin recovered by reversephase chromatography in TFA/acetonitrile [9]. Puri¢ed CNBr peptides were examined under similar conditions though scans were acquired from 500^ 1700 m/z [9]. 2.6. DNA sequence analysis Genomic DNA was extracted from whole blood [11] and a 1687 bp fragment spanning exons 13 through 15 of the albumin gene was ampli¢ed by polymerase chain reaction (PCR) as previously described [7]. The PCR product was puri¢ed using a HiPure PCR puri¢cation kit (Boehringer Mannheim) and sequenced using 33 P-labelled dideoxy terminators and Thermosequenase (Amersham) in both directions using primers RPS3 [7], A26B, A27A and A28A [12].
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Fig. 1. Agarose gel electrophoresis (pH 8.6). (a) Lane 1, normal plasma; lane 2, plasma from proposita; lane 3, plasma from a heterozygote for Al Rugby Park. (b) lane 1, plasma from proposita; lane 2, DEAE Sephadex fraction enriched in Al Banks Peninsula; lane 3, puri¢ed variant; lane 4, puri¢ed normal albumin. Inset at top of panel b is 63 Ni2 autoradiograph.
The subject was a plasma donor and was identi¢ed by routine electrophoresis following plasmaphoresis. Albumin measurements on two occasions revealed plasma concentrations of 35 and 42 mg/ml (normal range 35 to 53). Agarose gel electrophoresis at pH 8.6 detected an additional minor protein migrating slightly more anodally than normal albumin (Fig. 1a, lane 2) and like normal albumin, the new protein bound 63 Ni2 (Fig. 1b, lanes 1^3), suggesting that it was a more negative form of albumin. However, unlike most genetic variants, which contribute 50% of the total, the new band made up only 2% of the total albumin. Indeed, apart from a slightly lower concentration of variant, the plasma electrophoretic pattern closely resembled that of an Al Rugby Park heterozygote (Fig. 1a, lane 3). Thirty ml of plasma was fractionated on DEAE Sephadex where the new component eluted in the trailing edge of the Al A peak. Integration again
Fig. 2. Transformed ESI mass spectra of puri¢ed normal (A) and variant albumin (C) from the proposita before and after (B and D) incubation with carboxypeptidase A. Panel B shows a 1-h incubation while panel D shows a 5-h time point. Raw m/z data were transformed using Max-Ent software.
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Fig. 3. (a) Size-exclusion chromatography of CNBr digests of carboxamidomethylated albumin on TSK G2000 SW. Upper trace, Al A; lower trace, Al Banks Peninsula. (b) Mass spectra of CNBr 4 peak from: Al A, upper panel; Al Banks Peninsula, lower panel. Note no evidence of new CNBr 7 peptide co-chromatographing with CNBr 4.
con¢rmed that it represented 2% of the total albumin. Electrophoretic analysis of the pooled aberrant peak indicated a purity of about 70% (Fig. 1b, lane 2). Further ion exchange chromatography on a Uno Q6 column yielded a product which was v 95% pure (Fig. 1b, lane 3). ESI MS analysis of the normal and variant albumin, in 0.1% HCOOH 50% acetonitrile, revealed a series of positive molecular ions extending from [M+60H] to [M+32H]. Normal Al A isolated from the proposita was heterogeneous with three components of mass 66 446, 66 570 and 66 702 Da (Fig. 2A). Since the 66 446 Da peak was present in highest proportion and was closest to the predicted average isotopic mass (66 438 Da), it was used as the basis of comparison in the ensuing discussion. Transformation of the raw data from Al Banks Peninsula showed three similar components with masses of 65 686, 56 806 and 65 950 Da (Fig. 2C). The mass di¡erence between the native normal and variant albumin was, in this instance 3760 Da and averaged 3755 Da in ¢ve repeat measurements. Similar analysis of albumin after reduction in 8 M urea yielded what was essentially a single peak and the mass difference between normal and variant was 3855 and 3870 Da in two independent measurements. Since the variant displayed normal 63 Ni2 binding (implying a normal N-terminal sequence), it seemed
that some form of C-terminal truncation could best explain its 755 Da mass decrease. This was con¢rmed by digestion with carboxypeptidase A. After a 1-h incubation the mass of Al A decreased by 110 Da and did not change further over the following 5 h (Fig. 2B). This is consistent with the known C-terminal sequence of ^Gly^Leu, since the loss of Leu
Fig. 4. DNA sequence of 5P end of intron 13 showing new cryptic AG splice site and predicted new protein sequence.
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Fig. 5. DNA and predicted protein sequence of intron 13 and £anking exon sequences of Al Banks Peninsula. The majority of intron 13 is represented by dots and invariant splice site dinucleotides are boxed. The new acceptor splice site is indicated by broken boxes.
would cause a decrease of 113 Da and the exposed Gly would be resistant to further cleavage. There was, however, no change in mass when Al Banks Peninsula was similarly incubated (5 h) with carboxypeptidase A (Fig. 2D). In an attempt to de¢ne the mutation, CNBr fragments were ¢ngerprinted by reverse-phase HPLC (not shown). The normal and variant pro¢les appeared identical except that CNBr 7, the C-terminal peptide, was missing from digests of Al Banks Peninsula. Surprisingly, no new peptide was detected so a separation was attempted based on size-exclusion chromatography (Fig. 3). Peak assignments were made based on measured masses, and again CNBr 7 was missing. The mass of CNBr 7A was 4093 Da, in good agreement with a predicted value of 4094 Da. Since the mass decrease of Al Banks Peninsula was 755 Da, it was expected that the new peptide might co-elute with CNBr 4 (predicted lactone mass 3354 Da). However, ESI MS analysis of this peak showed molecular ions only at 839.14 and 1118.33 ; these are derived from CNBr 4 and indicated a normal mass of 3353 Da for the peptide. The absence of signals from CNBr 7 BP suggests that the new peptide is insoluble, since there was no evidence of late eluting peaks that would result if there was a new cleavage point in the peptide. Since no aberrant peptide could be isolated, PCR was used to amplify the region of the albumin gene encoding CNBr 7. This fragment included exons 13 and 14, the untranslated exon 15, as well as introns 13 and 14. Sequencing with both the forward and reverse primers (A27A and A28A) revealed a single T to A point mutation in intron 13, 15 bases upstream of the intron 13/exon14 boundary (Fig. 4). This mutation creates a new AG dinucleotide, the invariant sequence encountered in all eukaryotic in-
tron acceptor splice sites. Comparison of the sequence adjacent to this dinucleotide reveals that it conforms to the consensus eukaryotic acceptor splice site sequence with pyrimidines in 7 of the 10 positions from 35 to 315 bases of the putative splice site [13]. This is the same number as seen in the normal intron 13 acceptor splice site. Thus it appears likely that this point mutation activates a cryptic acceptor splice site at the 3P end of intron 13. Predictably this would result in translation of exon 14Banks Peninsula as Ser^Leu^Cys^Ser^Gly before a TAA terminator is reached (Fig. 5). 4. Discussion Albumin Banks Peninsula is a new genetic variant arising from the introduction of a new AG acceptor splice site at the 3P end of intron 13. This ¢nding is in full accord with the structural characterisation of the new protein and its low circulatory level. The mutation predicts that the exon 14 encoded residues, GKKLVAASQAALGL, would be missing and replaced by a new SLCSG sequence (Fig. 5). The predicted C-terminal Gly is in keeping with the results of carboxypeptidase A digestion, which showed that the new protein was resistant to hydrolysis. A mutation in the C-terminal region had been suspected from a decreased mass associated with normal 63 Ni2 binding and the absence of CNBr 7. The measured 755 Da mass decrease in native Al Banks Peninsula compares with a predicted decrease of 861 Da for the replacement of the terminal GKKLVAASQAALGL sequence by SLCSG. Since the loss of Leu (3113 Da) from Al A can be quite accurately measured as a 110 Da decrease (Fig. 2), it would appear that this mass discrepancy of 106 Da is real and probably re£ects the attachment of cysteine
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to the new Cys residue at position 573. Should this occur, the expected mass decrease would be 742 Da, in close agreement with the measured value (755 Da). The possibility of cysteinylation is not surprising since some 30^60% of circulating albumin molecules normally have cysteine bound to the free thiol at position 34 [1]. Indeed, this modi¢cation at residue 34 is the basis of the major heterogeneity (66 446/ 66 570 and 66 336/66 458 Da) seen in Fig. 2A,B [11]. Cysteinylation of Cys 573 was con¢rmed by repeat mass measurements after reduction; here the measured decrease in going from normal to variant was 863 Da, in excellent agreement with the prediction of 861 Da. The low expression of the new albumin, some 2% of the total, appears to re£ect poor utilisation of the new AG splice site. There is no a priori reason why most of the variant mRNA would not continue to be spliced at the intact AG site at the 3P end of intron 13 and translate as normal albumin. This supposition is supported by the normal plasma albumin concentration. Albumin synthesis does, however, appear to be up regulated in obligate carriers of analbuminaemia [1,12] and also in the three cases of Al Rugby Park, since these are associated with normal circulatory levels. This variant should have minimal physiological impact since it is expressed at such low levels, and two of the other truncation variants (Al Catania and Al Venezia) have been shown to have normal laurate binding [14]. It seems that perturbation of the terminal helix of domain IIIB, helix 10, has little e¡ect on function. Interestingly, the third case of Al Rugby Park was detected during the course of this investigation (Fig. 1a). It was con¢rmed by PCR and restriction endonuclease digestion and has been included for comparison since it involves a complementary mutation in intron 13. The GTCCT mutation at the intron boundary abolishes its 5P splice site, and since the mutation results in the replacement of the GKKLVAASQAALGL sequence by LLQFSSF [7] the protein would be expected to have the same +2 electrophoretic mobility as Al Banks Peninsula. This was indeed the case; the only di¡erence between the electrophoretic patterns of the two was the expression levels of the variants (Fig. 1a). The measured mass decrease, however, suggested a new mutation in
Al Banks Peninsula, since decreases of 485, 140, 87 and 14 Da would be predicted for the known truncations in Al Rugby Park, Al Venezia, Al Catania and Al Bazzano [5^7], respectively. This investigation highlights the power of ESI MS in the characterisation of both genetic and post-translational modi¢cations. Acknowledgements We thank Dr. Stephen Gibbons for obtaining the variant plasma and Prof. Frank Putnam for providing primers. This investigation was supported by the Canterbury Medical Research Foundation, Lottery Health and the Health Research Council of New Zealand.
References [1] T. Peters Jr., All About Albumin: Biochemistry Genetics and Medical Applications, Academic Press, San Diego, CA, 1996. [2] S.O. Brennan, R.J. Peach, J. Biol. Chem. 266 (1991) 21504^ 21508. [3] U. Kragh-Hansen, Pharmacol. Rev. 33 (1981) 17^53. [4] P.P. Mingetti, D.E. Ru¡ner, W.-J. Kuang, O.E. Dennison, W.J. Hawkins, W.G. Beatie, A. Dugaiczyk, J. Biol. Chem. 261 (1986) 6747^6757. [5] J. Madison, M. Galliano, S. Watkins, L. Minchiotti, F. Porta, A. Rossi, F.W. Putnam, Proc. Natl. Acad. Sci. USA 91 (1994) 6476^6480. [6] S. Watkins, J. Madison, E. Davis, Y. Sakamoto, M. Galliano, L. Minchiotti, F.W. Putnam, Proc. Natl. Acad. Sci. USA 885 (1991) 959^5963. [7] R.J. Peach, A.P. Fellowes, S.O. Brennan, P.M. George, Biochim. Biophys. Acta 1180 (1992) 107^110. [8] S.O. Brennan, T. Myles, R.J. Peach, D. Donaldson, P.M. George, Proc. Natl. Acad. Sci. USA 87 (1990) 26^30. [9] E.K.M. Chua, S.O. Brennan, P.M. George, Biochim. Biophys. Acta 1382 (1997) 305^310. [10] S.O. Brennan, Clin. Chem. 44 (1998) 2264^2269. [11] T.A. Ciulla, R.M. Sklar, S.L. Hauser, Anal. Biochem. 174 (1988) 485^488. [12] S. Watkins, J. Madison, M. Galliano, L. Minchiotti, F.W. Putnam, Proc. Natl. Acad. Sci. USA 91 (1994) 2275^ 2279. [13] S.M. Mount, Nucleic Acids Res. 10 (1982) 459^472. [14] U. Kragh-Hansen, A.O. Pedersen, M. Balliano, L. Minchiotti, S.O. Brennan, A.L. Tarnoky, F.M. Salzan, Biochem. J. 320 (1996) 911^916.
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Albumin Banks Peninsula: a new termination variant characterised by electrospray mass spectrometry Stephen O. Brennan *, Andrew P. Fellowes, Peter M. George Molecular Pathology Laboratory, Canterbury Health Laboratories, Christchurch Hospital, Christchurch, New Zealand Received 8 April 1999; received in revised form 4 May 1999; accepted 4 June 1999
Abstract Albumin Banks Peninsula is an electrophoretically fast variant that is expressed at only 2% of the total serum albumin. Electrospray ionisation analysis indicated a mass decrease of 755 Da relative to normal albumin and carboxypeptidase A digestion, together with CNBr peptide mapping, indicated a C-terminal truncation. This was confirmed by PCR and DNA sequence analysis which showed the introduction of a new AG acceptor splice site near the 3P end of intron 13. Predictably this results in the replacement of the C-terminal GKKLVAASQAALGL sequence by SLCSG and would be associated with an 861 Da decrease in molecular mass. We surmised that the new Cys was most probably cysteinylated as this albumin species would have a mass decrease of 742 Da and be very close to the measured value of 755 Da. Cysteinylation was confirmed when a mass decrease of 863 Da was measured between the proteins after reduction of their disulfide bonds. ß 1999 Elsevier Science B.V. All rights reserved. Keywords: Serum albumin; Truncation; Electrospray mass spectrometry; Low expression
1. Introduction Human serum albumin, the most abundant plasma protein, consists of a single polypeptide chain of 585 amino acids [1]. It is synthesised in the liver as preproalbumin. The prepeptide is co-translationally cleaved in the endoplasmic reticulum and the propeptide is removed in Golgi vesicles before secretion from the hepatocyte [2]. By virtue of its concentration, albumin regulates plasma oncotic pressure, but it functions primarily as a transport protein, and reversibly binds a wide array of ligands such as fatty acids, steroids, bilirubin, tryptophan and copper [1,3].
* Corresponding author. Fax: +64-3-3640-750.
The single copy of the albumin gene is located on chromosome four and is organised into 15 exons and 14 introns, the 15th exon being untranslated [4]. Genetic variation, or bisalbuminaemia, is usually detected by electrophoresis of plasma collected for diagnostic reasons. Bisalbuminaemia, however, may also be acquired as a result of proteolytic cleavage associated with pancreatitis, or due to increased drug or bilirubin binding. Some 50 di¡erent genetic variants have been characterised by protein and/or DNA sequence analysis [5], and these have provided unique insights into the intracellular processing of albumin and its ligand binding sites. Of the mutations characterised, four di¡erent genetic events have been reported that give rise to low concentration truncated molecules. These include single nucleotide deletions and frameshifts in Al Baz-
0167-4838 / 99 / $ ^ see front matter ß 1999 Elsevier Science B.V. All rights reserved. PII: S 0 1 6 7 - 4 8 3 8 ( 9 9 ) 0 0 1 3 1 - 4
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zano [5] and Al Catania [6], a 25 bp deletion that results in the skipping of exon 14 in Al Venezia [6], and a GT to CT 5P splice site mutation in intron 13 of Al Rugby Park [7]. Here, using electrospray ionisation mass spectrometry (ESI MS), we identify a truncated albumin with a mass decrease of 755 Da. This new albumin, Al Banks Peninsula, makes up only about 2% of the total plasma albumin and results from the introduction of an alternative 3P splice site in intron 13. 2. Materials and methods
2.4. High-performance liquid chromatography (HPLC) CNBr digests were analysed by both reverse-phase and size-exclusion chromatography. Reverse-phase separations were conducted on a Phenomenex W-Porex C4 column at a £ow rate of 1 ml/min in a 0.5% TFA/acetonitrile solvent system [9]. Sizeexclusion chromatography was performed on a TSK G2000SW (1U60 cm) column in 0.1% TFA 20% acetonitrile and at a £ow rate of 0.5 ml/ min. 2.5. ESI MS
2.1. Albumin puri¢cation Citrate anticoagulated plasma (30 ml) was chromatographed on a 2.6U15 cm column of DEAE Sephadex A 50 equilibrated in 16 mM sodium acetate bu¡er (pH 5.2). Bound albumin was eluted with 600 ml of this bu¡er with a pH gradient from pH 5.2 to 4.4 at a £ow rate of 1 ml/min [7]. Peaks were analysed by agarose gel electrophoresis at pH 8.6 [8] and the albumin peaks were pooled and lyophilised. The variant fraction, which contained approximately 70% Al Banks Peninsula, was re-chromatographed on a 12U53-mm Uno Q6 anion exchange column (Bio Rad) using similar bu¡ers with a 30-min gradient and a £ow rate of 3 ml/min. 2.2. Carboxypeptidase digestion Albumin, at a concentration of 5 mg/ml in 50 Wl of 50 mM NH4 HCO3 , was incubated with 15 Wg of carboxypeptidase A. Aliquots (20 Wl) were lyophilised after 1 and 5 h and redissolved in 100 Wl of 50% acetonitrile 0.1% HCOOH, and analysed directly by ESI MS. 2.3. CNBr digestion After reduction with 15 mM dithiothreitol in 8 M urea, 5 mg of albumin was S-carboxamidomethylated and redissolved in 150 Wl of 70% HCOOH containing 3 mg of CNBr. After overnight incubation the digest was dried and redissolved in 300 Wl of 5% acetonitrile.
Twenty Wl of native albumin (1^2 mg/ml in 0.1% HCOOH 50% acetonitrile) was delivered to the ion source of a Micromass Platform II quadrupole analyser at 5 Wl/min. The probe was maintained at 60³C and +3500 V, and the m/z range (1100^2100) was scanned with a cone voltage gradient of 40^60 V every 2.5 s. Data were acquired and processed using MassLynx software and transformed onto a true mass scale using Max-Ent software [10]. No external calibration was applied to the raw data. Reduced albumin was analysed similarly except scans were between 700 and 1600 m/z. Reduction was carried out in 8 M urea, 0.1 M Tris^HCl (pH 8), 15 mM dithiothreitol, and the albumin recovered by reversephase chromatography in TFA/acetonitrile [9]. Puri¢ed CNBr peptides were examined under similar conditions though scans were acquired from 500^ 1700 m/z [9]. 2.6. DNA sequence analysis Genomic DNA was extracted from whole blood [11] and a 1687 bp fragment spanning exons 13 through 15 of the albumin gene was ampli¢ed by polymerase chain reaction (PCR) as previously described [7]. The PCR product was puri¢ed using a HiPure PCR puri¢cation kit (Boehringer Mannheim) and sequenced using 33 P-labelled dideoxy terminators and Thermosequenase (Amersham) in both directions using primers RPS3 [7], A26B, A27A and A28A [12].
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Fig. 1. Agarose gel electrophoresis (pH 8.6). (a) Lane 1, normal plasma; lane 2, plasma from proposita; lane 3, plasma from a heterozygote for Al Rugby Park. (b) lane 1, plasma from proposita; lane 2, DEAE Sephadex fraction enriched in Al Banks Peninsula; lane 3, puri¢ed variant; lane 4, puri¢ed normal albumin. Inset at top of panel b is 63 Ni2 autoradiograph.
The subject was a plasma donor and was identi¢ed by routine electrophoresis following plasmaphoresis. Albumin measurements on two occasions revealed plasma concentrations of 35 and 42 mg/ml (normal range 35 to 53). Agarose gel electrophoresis at pH 8.6 detected an additional minor protein migrating slightly more anodally than normal albumin (Fig. 1a, lane 2) and like normal albumin, the new protein bound 63 Ni2 (Fig. 1b, lanes 1^3), suggesting that it was a more negative form of albumin. However, unlike most genetic variants, which contribute 50% of the total, the new band made up only 2% of the total albumin. Indeed, apart from a slightly lower concentration of variant, the plasma electrophoretic pattern closely resembled that of an Al Rugby Park heterozygote (Fig. 1a, lane 3). Thirty ml of plasma was fractionated on DEAE Sephadex where the new component eluted in the trailing edge of the Al A peak. Integration again
Fig. 2. Transformed ESI mass spectra of puri¢ed normal (A) and variant albumin (C) from the proposita before and after (B and D) incubation with carboxypeptidase A. Panel B shows a 1-h incubation while panel D shows a 5-h time point. Raw m/z data were transformed using Max-Ent software.
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Fig. 3. (a) Size-exclusion chromatography of CNBr digests of carboxamidomethylated albumin on TSK G2000 SW. Upper trace, Al A; lower trace, Al Banks Peninsula. (b) Mass spectra of CNBr 4 peak from: Al A, upper panel; Al Banks Peninsula, lower panel. Note no evidence of new CNBr 7 peptide co-chromatographing with CNBr 4.
con¢rmed that it represented 2% of the total albumin. Electrophoretic analysis of the pooled aberrant peak indicated a purity of about 70% (Fig. 1b, lane 2). Further ion exchange chromatography on a Uno Q6 column yielded a product which was v 95% pure (Fig. 1b, lane 3). ESI MS analysis of the normal and variant albumin, in 0.1% HCOOH 50% acetonitrile, revealed a series of positive molecular ions extending from [M+60H] to [M+32H]. Normal Al A isolated from the proposita was heterogeneous with three components of mass 66 446, 66 570 and 66 702 Da (Fig. 2A). Since the 66 446 Da peak was present in highest proportion and was closest to the predicted average isotopic mass (66 438 Da), it was used as the basis of comparison in the ensuing discussion. Transformation of the raw data from Al Banks Peninsula showed three similar components with masses of 65 686, 56 806 and 65 950 Da (Fig. 2C). The mass di¡erence between the native normal and variant albumin was, in this instance 3760 Da and averaged 3755 Da in ¢ve repeat measurements. Similar analysis of albumin after reduction in 8 M urea yielded what was essentially a single peak and the mass difference between normal and variant was 3855 and 3870 Da in two independent measurements. Since the variant displayed normal 63 Ni2 binding (implying a normal N-terminal sequence), it seemed
that some form of C-terminal truncation could best explain its 755 Da mass decrease. This was con¢rmed by digestion with carboxypeptidase A. After a 1-h incubation the mass of Al A decreased by 110 Da and did not change further over the following 5 h (Fig. 2B). This is consistent with the known C-terminal sequence of ^Gly^Leu, since the loss of Leu
Fig. 4. DNA sequence of 5P end of intron 13 showing new cryptic AG splice site and predicted new protein sequence.
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Fig. 5. DNA and predicted protein sequence of intron 13 and £anking exon sequences of Al Banks Peninsula. The majority of intron 13 is represented by dots and invariant splice site dinucleotides are boxed. The new acceptor splice site is indicated by broken boxes.
would cause a decrease of 113 Da and the exposed Gly would be resistant to further cleavage. There was, however, no change in mass when Al Banks Peninsula was similarly incubated (5 h) with carboxypeptidase A (Fig. 2D). In an attempt to de¢ne the mutation, CNBr fragments were ¢ngerprinted by reverse-phase HPLC (not shown). The normal and variant pro¢les appeared identical except that CNBr 7, the C-terminal peptide, was missing from digests of Al Banks Peninsula. Surprisingly, no new peptide was detected so a separation was attempted based on size-exclusion chromatography (Fig. 3). Peak assignments were made based on measured masses, and again CNBr 7 was missing. The mass of CNBr 7A was 4093 Da, in good agreement with a predicted value of 4094 Da. Since the mass decrease of Al Banks Peninsula was 755 Da, it was expected that the new peptide might co-elute with CNBr 4 (predicted lactone mass 3354 Da). However, ESI MS analysis of this peak showed molecular ions only at 839.14 and 1118.33 ; these are derived from CNBr 4 and indicated a normal mass of 3353 Da for the peptide. The absence of signals from CNBr 7 BP suggests that the new peptide is insoluble, since there was no evidence of late eluting peaks that would result if there was a new cleavage point in the peptide. Since no aberrant peptide could be isolated, PCR was used to amplify the region of the albumin gene encoding CNBr 7. This fragment included exons 13 and 14, the untranslated exon 15, as well as introns 13 and 14. Sequencing with both the forward and reverse primers (A27A and A28A) revealed a single T to A point mutation in intron 13, 15 bases upstream of the intron 13/exon14 boundary (Fig. 4). This mutation creates a new AG dinucleotide, the invariant sequence encountered in all eukaryotic in-
tron acceptor splice sites. Comparison of the sequence adjacent to this dinucleotide reveals that it conforms to the consensus eukaryotic acceptor splice site sequence with pyrimidines in 7 of the 10 positions from 35 to 315 bases of the putative splice site [13]. This is the same number as seen in the normal intron 13 acceptor splice site. Thus it appears likely that this point mutation activates a cryptic acceptor splice site at the 3P end of intron 13. Predictably this would result in translation of exon 14Banks Peninsula as Ser^Leu^Cys^Ser^Gly before a TAA terminator is reached (Fig. 5). 4. Discussion Albumin Banks Peninsula is a new genetic variant arising from the introduction of a new AG acceptor splice site at the 3P end of intron 13. This ¢nding is in full accord with the structural characterisation of the new protein and its low circulatory level. The mutation predicts that the exon 14 encoded residues, GKKLVAASQAALGL, would be missing and replaced by a new SLCSG sequence (Fig. 5). The predicted C-terminal Gly is in keeping with the results of carboxypeptidase A digestion, which showed that the new protein was resistant to hydrolysis. A mutation in the C-terminal region had been suspected from a decreased mass associated with normal 63 Ni2 binding and the absence of CNBr 7. The measured 755 Da mass decrease in native Al Banks Peninsula compares with a predicted decrease of 861 Da for the replacement of the terminal GKKLVAASQAALGL sequence by SLCSG. Since the loss of Leu (3113 Da) from Al A can be quite accurately measured as a 110 Da decrease (Fig. 2), it would appear that this mass discrepancy of 106 Da is real and probably re£ects the attachment of cysteine
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to the new Cys residue at position 573. Should this occur, the expected mass decrease would be 742 Da, in close agreement with the measured value (755 Da). The possibility of cysteinylation is not surprising since some 30^60% of circulating albumin molecules normally have cysteine bound to the free thiol at position 34 [1]. Indeed, this modi¢cation at residue 34 is the basis of the major heterogeneity (66 446/ 66 570 and 66 336/66 458 Da) seen in Fig. 2A,B [11]. Cysteinylation of Cys 573 was con¢rmed by repeat mass measurements after reduction; here the measured decrease in going from normal to variant was 863 Da, in excellent agreement with the prediction of 861 Da. The low expression of the new albumin, some 2% of the total, appears to re£ect poor utilisation of the new AG splice site. There is no a priori reason why most of the variant mRNA would not continue to be spliced at the intact AG site at the 3P end of intron 13 and translate as normal albumin. This supposition is supported by the normal plasma albumin concentration. Albumin synthesis does, however, appear to be up regulated in obligate carriers of analbuminaemia [1,12] and also in the three cases of Al Rugby Park, since these are associated with normal circulatory levels. This variant should have minimal physiological impact since it is expressed at such low levels, and two of the other truncation variants (Al Catania and Al Venezia) have been shown to have normal laurate binding [14]. It seems that perturbation of the terminal helix of domain IIIB, helix 10, has little e¡ect on function. Interestingly, the third case of Al Rugby Park was detected during the course of this investigation (Fig. 1a). It was con¢rmed by PCR and restriction endonuclease digestion and has been included for comparison since it involves a complementary mutation in intron 13. The GTCCT mutation at the intron boundary abolishes its 5P splice site, and since the mutation results in the replacement of the GKKLVAASQAALGL sequence by LLQFSSF [7] the protein would be expected to have the same +2 electrophoretic mobility as Al Banks Peninsula. This was indeed the case; the only di¡erence between the electrophoretic patterns of the two was the expression levels of the variants (Fig. 1a). The measured mass decrease, however, suggested a new mutation in
Al Banks Peninsula, since decreases of 485, 140, 87 and 14 Da would be predicted for the known truncations in Al Rugby Park, Al Venezia, Al Catania and Al Bazzano [5^7], respectively. This investigation highlights the power of ESI MS in the characterisation of both genetic and post-translational modi¢cations. Acknowledgements We thank Dr. Stephen Gibbons for obtaining the variant plasma and Prof. Frank Putnam for providing primers. This investigation was supported by the Canterbury Medical Research Foundation, Lottery Health and the Health Research Council of New Zealand.
References [1] T. Peters Jr., All About Albumin: Biochemistry Genetics and Medical Applications, Academic Press, San Diego, CA, 1996. [2] S.O. Brennan, R.J. Peach, J. Biol. Chem. 266 (1991) 21504^ 21508. [3] U. Kragh-Hansen, Pharmacol. Rev. 33 (1981) 17^53. [4] P.P. Mingetti, D.E. Ru¡ner, W.-J. Kuang, O.E. Dennison, W.J. Hawkins, W.G. Beatie, A. Dugaiczyk, J. Biol. Chem. 261 (1986) 6747^6757. [5] J. Madison, M. Galliano, S. Watkins, L. Minchiotti, F. Porta, A. Rossi, F.W. Putnam, Proc. Natl. Acad. Sci. USA 91 (1994) 6476^6480. [6] S. Watkins, J. Madison, E. Davis, Y. Sakamoto, M. Galliano, L. Minchiotti, F.W. Putnam, Proc. Natl. Acad. Sci. USA 885 (1991) 959^5963. [7] R.J. Peach, A.P. Fellowes, S.O. Brennan, P.M. George, Biochim. Biophys. Acta 1180 (1992) 107^110. [8] S.O. Brennan, T. Myles, R.J. Peach, D. Donaldson, P.M. George, Proc. Natl. Acad. Sci. USA 87 (1990) 26^30. [9] E.K.M. Chua, S.O. Brennan, P.M. George, Biochim. Biophys. Acta 1382 (1997) 305^310. [10] S.O. Brennan, Clin. Chem. 44 (1998) 2264^2269. [11] T.A. Ciulla, R.M. Sklar, S.L. Hauser, Anal. Biochem. 174 (1988) 485^488. [12] S. Watkins, J. Madison, M. Galliano, L. Minchiotti, F.W. Putnam, Proc. Natl. Acad. Sci. USA 91 (1994) 2275^ 2279. [13] S.M. Mount, Nucleic Acids Res. 10 (1982) 459^472. [14] U. Kragh-Hansen, A.O. Pedersen, M. Balliano, L. Minchiotti, S.O. Brennan, A.L. Tarnoky, F.M. Salzan, Biochem. J. 320 (1996) 911^916.
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