Molecular and Cellular Biochemistry 98: 127-133, 1990. © 1990 Kluwer Academic Publishers. Printed in the Netherlands.

Revision of the amino acid sequence of human heart fatty acid-binding protein Torsten Brrchers 1, Peter H0jrup 2, S0ren U. Nielsen 3, Peter Roepstorff2, Friedrich Spener 1 and Jens Knudsen 4 1Department of Biochemistry, University of Miinster, 4400 Miinster, FRG; Departments of 2Molecular Biology and 4Biochemistry, University of Odense, 5230 Odense M, Denmark," 3Department of Medical Biochemistry, University of Aarhus, 8000 Aarhus C, Denmark

Key words: fatty acid-binding protein, mass spectrometry, sequence, human heart

Summary Cardiac-type fatty acid-binding protein (cFABP) from human heart muscle of three individuals was isolated and characterized as pI 5.3-cFABP. The proteins were structurally analyzed by tryptic peptide mapping, application of plasma desorption time-of-flight mass spectrometry and amino acid sequencing. All three preparations of human heart FABP, having 132 amino acids, differed from the published sequence [Offner et al. Biochem J 251: 191-198, 1988] in position 104, where Leu is found instead of Lys, and in position 124, where Cys is found instead of Ser.

Introduction The three types of fatty acid-binding proteins (FABPs) that have been distinguished with respect to their source of first isolation are intestinal, hepatic and cardiac FABP. This classification is justified by their immunological behavior as fatty acidbinding proteins from different species crossreact only within one type [1]. Cardiac-type FABP (cFABP) from human heart was first isolated and characterized by Unterberg et al. [2] and shown to be a 15 kDa protein with an isoelectrical point of 5.3 (pI 5.3-cFABP). Recently, Offner et al. [3] published the primary structure of this protein, which exhibited high homology to rat (89.4% identity, [4]) and bovine cFABP (87.9% identity, [5]). However, in contrast to the amino acid Composition given by Unterberg et al. [2], Offner et al. [3] did not report cysteine in this protein. As we had previously established peptide mapping for both cFABP isoforms of bovine heart

cytosol (pI 4.9- and pI 5.1-cFABP) [6] and had available an expeditious and reliable method for identification of peptides, namely plasma desorption time-of-flight mass spectrometry (PDMS) [7], we reconsidered the amino acid sequence of human heart FABP.

Experimental procedures Isolation and characterization of cFABPs. Human and bovine heart FABP were isolated as described in references [2] and [6], respectively, and characterized by SDS-PAGE (20%) with subsequent Western blotting as well as anion exchange HPLC on Mono Q (see below). Modification of cFABP with vinylpyridine, pI 5.3cFABP (20 nmol) was reduced with 2/~mol dithiothreitol (DTT) in 6 M guanidinium.HC1/20 mM Tris.HCl/1 mM EDTA, pH 7.5, and incubated with

128 2/~1 vinylpyridine (25 ° C, 10 min) in a final volume of 200/xl. Oxygen was excluded by flushing all media with nitrogen. Upon fivefold dilution with water a precipitate was formed which was then washed with 0.1 M N H 4 H C O 3 and digested with trypsin as described below.

Results

Characterization ofp15.3 cFABP

modified cFABP was taken up in water, subjected to heat denaturation and simultaneously suspended by sonication, and was then digested with 2% (w/w) trypsin in 0.1 M NH4HCO3, pH 8.0 (37°C, 2h).

Both human and bovine heart FABP had a similar molecular weight in SDS-PAGE and, after subsequent Western blotting, reacted with the polyclonal antibody against bovine cFABP [1]. They also behaved similar on the Mono Q ion exchange column, where bovine pI 4.9- and human pI 5.3cFABP exhibited identical retention times. In contrast, the isoforms of bovine cFABP, pI 4.9- and pI 5.1-cFABP, could be separated on this anion exchanger [6].

HPLCseparations. Chromatography was perform-

Peptide mapping

ed on a Kontron HPLC-system connected with a Mono Q HR 5/5 (Pharmacia) column for proteins and with the following reverse phase columns for peptides: Nucleosil RP 18 (10/xm, 300/~, 8 x 125mm), Nucleosil RP 4 (10/.Lm, 300A, 4.6× 125 mm, Macherey & Nagel) and Dynospheres PD 102-RE (4.6 x 30 mm, Dyno Particles), the latter especially suited for hydrophobic molecules.

Tryptic peptides of human and bovine cFABP were separated on a reverse phase C18 column. The elution pattern of pI 5.3-cFABP peptides (Fig. 1A) also indicated strong similarities to that of bovine pI 4.9-cFABP (data not shown), as expected from the high homology. Due to the characteristic absorbance at 280 nm (arrows in Fig. 1) the tryptophan and tyrosine containing peptides could be easily assigned to the known peptide map of bovine pI 4.9-cFABP, as published elsewhere [6]. In particular, Tyr ~28and Tyr 19containing peaks FI-1 and F1-9 exhibited the same retention time as their bovine counterparts. Yet the Trp 97containing peptide F1-10 eluted slightly and the Trp 8 containing N-terminal peptide F1-12 considerably later than the corresponding peptides from bovine cFABP. The latter was expected from the exchange of VaP in pI 4.9-cFABP against Leu, resulting in a more hydrophobic peptide.

Enzymic cleavage. Prior to digestion native or

Plasma desorption time-of-flight mass spectrometry (PDMS). Tryptic peptides from HPLC-runs were lyophilized and dissolved in 0.1% TFA/20% methanol. Aliquots ( - 2 0 0 pmol) were applied onto a nitrocellulose coated aluminized mylar foil by the spin technique [8] and bombarded with fission fragments from the 252Cf source of a BIO-ION 10 K mass spectrometer. The acceleration voltage was set at 16 kV and 500 000 primary ions were recorded. Reduction of disulfides was carried out in situ with 3 nmol DTT at 37° C for 2 h in a humid chamber. All masses are given as average molecular mass.

Sequence analysis and peptide nomenclature. Tryptic peptides were subjected to automated Edman degradation in a Knauer 810 sequencer. They were sequentially numbered according to primary structure (T-1 to T-14) or according to elution order in reverse phase H P L C of respective FABP-samples (F1-1 to F1-12 for FABP-1 and F2-0 to F2-11 for FABP-2).

Characterization of peptides In order to identify all tryptic peptides of pI 5.3cFABP unequivocally and to align them with the reported sequence [3], we employed plasma desorption time-of-flight mass spectrometry [7]. This method enabled us to determine the molecular mass of peptides with an accuracy of 0.1%, thus giving sufficient information to prove the overall amino acid composition of a certain peptide. The mass of the singly charged, protonated molecular ion of the N-terminal peptide (MH ÷ = 1077 Da) is

129 in accordance with the mass calculated from the sequence [3] and confirms the existence of an acetyl blocking group. Table 1 summarizes the masses of all peptides. To our surprise we found two deviations from expected masses, namely for peptides T-12 and T-14. The first deviation can be seen from the mass spectrum shown in Fig. 2A. For peptide T-12 we found a molecular mass of 1203 Da that could not be fitted into the sequence reported by Offner et al. [3]. But from the fragmentation peak MH+-130, which was due to the loss of the indol side chain of tryptophan, and from the molecular mass, which indicated a decapeptide, we concluded that this peptide represented positions 97 to 106. Upon automated Edman degradation the sequence was determined to WD

GQ

10

A220

9

571,9

ETTLVR

in full support of the mass found by PDMS. The mass of the corresponding peptide, as calculated from the published sequence [3], is 964 Da and no peptide with this mass was found (Table 1). The second deviation occurred in the peptide presumed to cover positions 113 to 126. Due to its very hydrophobic nature it could only be recovered by reverse phase HPLC on the Dynospheres column, where it eluted with the same retention time as peptide T-13 from bovine pI 4.9-cFABP (data not shown). The mass spectrum showed a molecular mass of 1496 Da and not 1482 Da as expected from the published sequence [3]. Furthermore a peak of nearly the same height with the mass 2995 Da was detected and tentatively interpreted as a dimer formed via disulfide bridge. This interpretation was supported by the observation that in situ reduction with DTT indeed caused the disappearance of the 2995 Da molecular ion (Fig. 2B). The molecular mass of monomeric T-14, its retention time in reverse phase HPLC, identical to the bovine counterpart, and the presence of a reducible dimer can only be explained by the presence of CysTM instead of Ser TM in human cFABP. Hence human cFABP peptide T-14 and bovine cFABP peptide T-13 are identical. Our results are summa-

io

10

20

30

40

tlminl

Fig. 1. Separation of tryptic peptides from pI 5.3-cFABP by reverse phase HPLC. A gradient of 50% isopropanol in 0.1% trifluoroacetic acid was applied (3min 0%, 45 min to 45%, 12 min to 60%, 1.5 ml/min). Detection at 220 nm (0.2 AUFS), arrows indicate peptides with strong absorbancy at 280nm (0.01AUFS). A, peptides from 150/xg FABP-1 on RP 18 column; B, peptides from 150/zg vinylpyridinylated FABP-2 on RP 4 column.

rized in Fig. 3, where the revised sequence and t h e alignment of the tryptic peptides are presented. At this stage the question arose, whether the differences observed between reported and revised sequences were due to individual polymorphism or not. To solve this problem, we examined human heart FABP from two other individuals (FABP-2, FABP-3). The masses of the corresponding tryptic peptides are also given in Table 1 and fitted into the sequence as specified in Fig. 3 for FABP-2. They dearly support the results obtained for FABP-1 and suggest that hUman heart FABP has, inde-

130 5585MH +

A

1204.1

E 2z92 0 -Trp - C O 2 o

o

o

~ ~ +Na

~

'66b.......... "i bOO"........ i 4bd ......... "i abd .......... 22'0b ......... ' 26iJ0 ..... ' ..... 3bbo M/Z 1413-

B

MH +

1497.0

MH + (M-M)H +

-CO2 ~q

70(

0

-CO 2

o

M/Z Fig. 2. Mass spectra of tryptic peptides from pI 5.3-cFABP. A, peptide T-12 from FABP-1; B, peptide T-14 from FABP-1, reduced in situ with 3 nmol DTT, the mass spectrum of the unreduced peptide T-14 is inserted.

pendently of the individual source, the amino acid sequence presented here. Further proof for Cys TM in T-14 was obtained by modification of the protein (FABP-2) with 4-vinyl-

pyridine. I n the c o r r e s p o n d i n g tryptic p e p t i d e m a p o n a reverse phase C4 c o l u m n (Fig. 1B) a n e w p e a k (F2-10) with a characteristic a b s o r b a n c y at 260 n m a n d a mass of 1604 D a e l u t e d , as e x p e c t e d for vinyl-

131 pyridinylated T-14. The sequence of this peptide, was determined to LILTLTHGTAVCTR corroborating the foregoing data.

Discussion

In the literature some controversy exists on the amino acid sequence of rat heart FABP. Hitherto three different sequences from five laboratories are available, even in a revised sequence [9] two discrepancies to the cDNA [4, 10] can be found. The cDNA derived sequence is supported by a recent report [11], in which rat cFABP peptides are examined by amino acid analysis. Here we demonstrate that the primary structure of human heart FABP from three different individuals differs in two positions from the published structure [3]. Namely in position 104 Leu is found instead of Lys. The misinterpretation may be caused by cleavage after Leu ~°4due to chymotryptic

activity of trypsin. This seems plausible, because FABPs are quite resistant to enzymic degradation, and large quantities of trypsin as well as long incubation times are often applied. Furthermore in position 124 we observed Cys instead of Ser. Since cysteine is difficult to detect in sequencing and amino acid analysis without prior alkylation, we modified pI 5.3-cFABP with 4-vinylpyridine. This modification was not applied by Offner et al. [3]. The presence of cysteine and its potential for disulfide bridge formation is supported by data of Nielsen et al. [12], who observed dimers of human cFABP in denaturing two-dimensional gel electrophoresis. These dimers could be monomerized upon reduction with DT-F. The revised sequence results in a higher homology of human cFABP to bovine pI 4.9-cFABP (89.4% identity). This particular bovine cFABP isoform also contains Asp in position 98 [6], whereas in the other isoform (pI 5.1 cFABP) of bovine heart cytosol Asn 98is found as the only difference. But, though both bovine isoforms can be separated upon anion exchange chromatography on Mono Q, bovine pI 4.9-cFABP and human pI 5.3-cFABP

Table 1. Summary of mass spectrometric data Peptide

T-1 T-2 T-3 T-4 T-5 T-6 T-7 T-8 T-9 T-10 T-11 T-12 T-13 T-14 T-15

Residues in sequence

1-9 10-14 15-21 22-30 31-44 45-52 53-58 59-65 66-78 82-90 91-96 97-106 107-112 113-126 127-130

Mr calc.

10771 561 932 907 1546 873 720 838 1467 889 737 1204 673 1498 539

xN-acetylated peptide. 2The higher M r is due to oxidation of Met (MH + + 16). 3Mr of the vinylpyridinylated T-14, ( M r vinylpyridine = 105.1). nd, not determined; nf,, not found.

Mr by PDMS FABP-f

FABP-2

FABP-3

1076 560 932 906 1546 nf 719 838 1466 888 736 1203 673 1496 539

1077 561 932 nf 15642 873 719 838 1467 889 736 1204 673 16043 540

nd nd nd nd nd nd nd nd nd nd nd 1204 nd 1498 nd

132

10

20

30

Ac-V O A F L G T W K L V D S K N F D D Y M K S L G V G F A T R Q V A F

1

-

F2-11

1 -

2

-

-

-

F 1 - 2 - -

- -

F

F2-O

- -

F2-5

- -

40

1

-

8 -

-

I EKNGD

-

70

-

F2-8

-

-

- -

F2-9

~

1

-

F2-4

-

110

F1-10 2

-

. . . . . 7

-

-

-

-

- -

F1-4

I S FKL

F1-3

-

-

F1-8

F2-O

- - . F 2 - 3

-

-

-

-

8

-

-

-

LQKWDG - - F 1 - 5 - - -

F2-2

120

QETTlVRELIDGKLI

F

-

90

F

100

-

I VTLDGGKLVH

F1-11

-

-

80

GV E F D E T T A D D R K V K S

-

F1-11

60

-

F2-6

_ _ _

-

I LTLKTHSTFKNTE

- F1-10

-

-

50

SMTKPTTI

-

-

-

130

LTLTHGTAVCTRTYQKEA -

-

F 2 - O - -

F1-D _

. . . . . . .

F2-10__--

--F1-1 . . . . . . .

- -

--F2-o--

Fig. 3. Revised sequence of pI 5.3-cFABP. Differences to published sequence [3] are shown in bold type. Aligned peptides from FABP-1 and FABP-2were characterized by PDMS ( ) and sequencing (---) and denoted accordingto their elution order as shown in Fig. tA. Peptide T-14was recovered from the Dynospheres column (F1-D). The four peptides from FABP-2not bound by the RP 4 column (Fig. 1B, peak 0) were rechromatographed on the RP 18 column prior to mass determination and are all denoted F2-0.

exhibit identical retention times. H e n c e the elution order seems to be dominated by the negative charge of the surface-located Asp 9s of those proteins. In contrast to bovine c F A B P , no isoforms were detected for h u m a n cFABP. In isoelectric focusing only one F A B P was found by Offner et al. (pI 5.3) [3] as well as by Paulussen et al. (pI 5.2) [13], in agreement with Unterberg et al. [2]. Based on this it is unlikely that the sequences presented here and in ref. [3] correspond to different isoforms.

Acknowledgements Part of this work was supported by a grant from the

Deutsche Forschungsgemeinschaft (SFB 310). T.B. acknowledges a fellowship from the state of Nordrhein-Westfalen (Graduiertenf6rderung). P.H. was supported by the Carlsberg Foundation and the mass spectrometer by grants from the Danish Technical Science Council.

References 1. Spener F, Unterberg C, B6rchers T, Grosse R: Characteristics of fatty acid-binding proteins and their relation to mammary derived growth inhibitor. Mol Cell Biochem98: 57-68 2. Unterbcrg C, Heidl G, yon BassewitzD-B, SpenerF: Isolation and characterization of the fatty acid-binding protein from human heart. J Lipid Res 27: 1287-1293, 1986

133 3. Offner GD, Brecher P, SawlivichWB, CosteUo CE, Troxler RF: Characterization and amino acid sequence of a fatty acid-binding protein from human heart. Biochem J 252: 191-198, 1988 4. Claffey KP, Herrera VL, Brecher P, Ruiz-Opazo N: Cloning and tissue distribution of rat heart fatty acid-binding protein mRNA: Identical forms in heart and skeletal muscle. Biochemistry 26: 7900-7904, 1987 5. Billich S, Wissel T, Kratzin H, Hahn U, Hagenhoff B, Lezius AG, Spener F: Cloning of a full-length complementary DNA for fatty acid-binding protein from bovine heart. Eur J Biochem 175: 549-556, 1988 6. Unterberg C, BSrchers T, Hcjrup P, Roepstorff P, Knudsen J, Spener F: Cardiac fatty acid-binding proteins. Isolation of a mitochondrial fatty acid-binding protein and its structural relationship with the cytosolic isoforms. J Biol Chem, in press 7. Sundkvist B, McFarlane RD: 252-Cf-plasma desorption mass spectrometry. Mass Spectrom Rev 4: 421-460, 1985 8. Nielsen PF, Klarskov K, H0jrup P, Roepstorff P: Optimization of sample preparation for plasma desorption mass spectrometry of peptides and proteins using a nitrocellulose matrix. Biomed Environ Mass Spectrom 17: 355-362, 1988 9. Gibson BW, Yu Z, Aberth W, Burlingame AL, Bass NM:

10.

11.

12.

13.

Revision of the blocked N terminus of rat heart fatty acidbinding protein by liquid secondary ion mass spectrometry. J Biol Chem 263: 4182-4185, 1988 Heuckeroth RO, Birkenmeier EH, Levin MS, Gordon JI: Analysis of the tissue-specific expression, developmental regulation, and linkage relationships of a rodent gene encoding heart fatty acid-binding protein. J Biol Chem 262: 9709-9717, 1987 Kimura H, Hitomi M, Odani S, Koide T, Arakawa M, Ono T: Rat heart fatty acid-binding protein. Evidence that supports the amino acid sequence predicted from the cDNA. Biochem J 260: 303-306, 1989 Nielsen SU, Pedersen AO, Vorum H, Brodersen R: FABP from human heart localized in native and denaturing twodimensional gels. Mol Cell Biochem 98: 121-126, 1990 Paulussen RJA, van der Logt CPE, Veerkamp JH: Characterization and binding properties of fatty acid-binding proteins from human, pig, and rat heart. Arch Biochem Biophys 264: 533-545, 1988

Address for offprints: F. Spener, Department of Biochemistry, University of Miinster, Wilhelm-Klemm-Str. 2, D-4400 Mtinster, FRG

Revision of the amino acid sequence of human heart fatty acid-binding protein.

Cardiac-type fatty acid-binding protein (cFABP) from human heart muscle of three individuals was isolated and characterized as pI 5.3-cFABP. The prote...
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