Hb Doha or α2β2[X-N-Met-1-(NA1)Val → Glu]; a new β-chain abnormal hemoglobin observed in a Qatari female

Biochimica etBiophvsicaActa 831 (1985) 257-260

257

Elsevier BBA 32283

H b D o h a or ot2fl 2 [ X - N - M e t - I ( N A 1 ) V a l

-=, Giu]; a n e w B-chain abnormal

h e m o g l o b i n observed in a Qatari f e m a l e

b,

K . K a m e l a, A. E 1 - N a j j a r B.B. W e b b e r c, S.S. C h e n c, J.B. W i l s o n c, A. K u t l a r c a n d T . H . J . H u i s m a n c,** a Department of Laboratory Medicine and Pathology, Hamad General Hospital and 6 Department of Obstetrics, Women's Hospital, Doha (Qatar) and c Laboratory of Protein Chemistry, Department of Cell and Molecular Biology *, Medical College of Georgia, Augusta, GA 30912 ( U. S. A.)

(ReceivedApril 22nd, 1985)

Key words: Amino acid sequence; Blockinggroup; Hemoglobinvariant; Methionine; N-terminal; (Human)

Structural analysis of a fast-moving hemoglobin variant, present in three members of a Qatari family, identified a Val -~ Glu substitution at position 1 (NAI) of the B-chain. The introduction of this glutamic acid residue prevents the removal of the initiator methione, thus extending the N-terminus by one residue to Met-Glu-His-Leu-Thr-. The methionine residue is blocked by an as yet not completely identified molecule. The presence of the variant in a heterozygote does not have clinical consequences.

Introduction Recently, we studied the hemoglobin (Hb) of a Qatari woman and of some of her relatives, and observed an electrophoretically fast-moving major variant which, upon analysis, was found to contain an abnormal B-chain with a Met-Glu-His-Leu Nterminus. A Val ~ Glu replacement at position fll apparently prevented the methionyl from being removed during the completion of the synthesis of the polypeptide chain. Moreover, the new N-terminal residue is blocked with an incompletely identified molecule. The present report provides details of this study.

Materials and Methods Blood samples from the proposita and four relatives were collected in vacutainers with EDTA as anticoagulant, and shipped by airmail, special * Contribution No. 0901. ** To whom correspondenceshould be addressed.

delivery, from Doha, Qatar, to Augusta, GA. Informed consent was obtained. Hematological data were collected at the local hospital using routine methodology and an advanced blood cell counter. Hb analyses included cellulose acetate electrophoresis [1], citrate agar electrophoresis [1], alkali denaturation [2], cation exchange high-performance liquid chromatography (HPLC) [3] and DEAE-cellulose chromatography [4]. The abnormal B-chain ( f i x ) was separated from the a-chain by CM-cellulose chromatography [5]. An appropriate quantity of the fix chain was digested with TPCK-trypsin (Worthington) for 6 h at room temperature and at pH 8.6. The resulting peptides were separated by reverse-phase HPLC [6]. Sequence analysis of some peptides made use of the manual ultramicro procedure developed by Chang et al. [7,8]. The isolated abnormal flXT-1 peptide was digested with thermolysin (Cal-Biochem) in 0.5% N H 4 H C O 3 (pH 8.4), for 1 h at 52°C. This solution was acidified with 1 N HC1 to a pH of 2.5-3.0 and lyophilized, whereafter the fragments were separated on a

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25.~

Vydac 218TP 54 Cls RP column with 0.1% trifluoroacetic acid in water and 0.1% trifluoroacetic acid in 75% acetonitrile and 25% H 2 0 as developers. A linear gradient between these two developers resulted in an increase in acetonitrile at a rate of 0.5%/rain at a flow-rate 1 m l / m i n . This method is essentially the same as that developed by Schroeder and his colleagues [9,10].

%DOHA

E CJ

T6 T-7 T-8 T-15

T13 i

T2 T-5 T-9

T'lX

T4

T-14

o

a~

<

1" 2'0 3'° ,'o Results and Discussion

do 6'0

70

810

910

I 100

I 110

I 120

Time in Minutes

The family

Fig. 2. Separation of the soluble peptides in a tryptic digest of the fl chain of Hb Doha by reverse-phase HPLC.

The proposita was a 24-year-old female with a significant anemia (Hb: 7.6-8.2 g / d l ) upon delivery of her fourth child. She was an otherwise healthy woman without abnormalities on clinical examination; her Hb levels varied between 11.2 and 11.9 g / d l prior to delivery. Examination of her Hb by cellulose acetate electrophoresis revealed the presence of a 'fast-moving' abnormal Hb (Fig. 1). The same abnormality was present in the blood of the patient's mother and one of her sisters. The three Hb Doha heterozygotes had a normal hematology (Table I), although the proposita was recovering from her anemia when these data were collected.

Hb X and Hb A was possible by cation-exchange H P L C and by DEAE-cellulose chromatography. Hb X eluted from the cation exchanger in front of the minor Hb A 1 zones, and was retained at the top of the DEAE-cellulose column, requiring an 0.06 M NaCl-glycine-KCN developer for its elution. Its quantity was 49.8% (including the minor H b X~ components) and that of Hb A was 48.9%. This quantitative value was similar to those obtained for the three Hb Doha heterozygotes by scanning of the cellulose acetate electrophoretic strips (Table I).

Hemoglobin analyses

Structural analyses

Studies conducted in Augusta confirmed the electrophoretic mobility of Hb X; at p H 6.1 with citrate agar gel electrophoresis it moved in the position of Hb F. Chromatographic separation of

A larger quantity of Hb X was isolated by preparative DEAE-cellulose chromatography, and its fix chain isolated on a column of CM-cellulose. After digestion with trypsin the resulting soluble peptides were separated by reverse phase HPLC. Fig. 2 illustrates one of the chromatograms that were obtained. All soluble peptides (T-l, T-2, T-3, T-4, T-5, T-6, T-7, T-8, T-9, T-13, T-14 and T-15) were recovered and had the expected retention times and amino acid compositions, except for "1"-1. This peptide was eluted at about 45 46 rain after the start of the experiment, in contrast with normal T-l, which elutes in about 32-34 min in this H P L C system. Its amino acid composition is listed in Table II; the data indicated the presence of one extra methionine residue and one additional glutamic acid residue and the loss of a valine residue, which suggested a possible Met, Glu, His N-terminus. Sequence analyses of the N-terminus of both the intact B x chain and the flx T-1 peptide by the manual ultramicroprocedure

i

Control AA Propositus Sister

CSFA Control AA Mother

-

t Start

ttttt C

+

S FA DOHA

Fig. 1. Demonstration of Hb Doha in lysates of red blood cells from three members of Family K using cellulose acetate electrophoresis.

259 TABLE I HEMATOLOGICAL AND HEMOGLOBIN COMPOSITION DATA HB, hemoglobin; PCV, packed cell volume; RBC, red blood cells; MCV, mean cellular volume; MCH, mean cellular hemoglobin; M C H C , mean cellular hemoglobin concentration. Subject

Sex/age

Diagnosis

Relation

Hb (g/dl)

PCV (1/1)

RBC (1012/1)

MCV (fl)

MCH (pg)

MCHC (g/dl)

Retic. (%)

A2a (%)

FAD b (%)

Doha ~ (%)

K.K. M.K. Ma.K. c R.K. A.K.

F/55 F/32 F/24 F/60 M/31

A-Doha A-Doha A-Doha AA AA

mother sister propositus aunt husband

12.2 11.9 9.6 12.7 16.0

0.372 0.377 0.314 0.410 0.498

3.77 4.58 4.17 4.82 5.62

99 82 75 84 89

32.3 25.9 23.1 26.2 28.5

32.7 31.5 30.7 31.0 32.2

3.7 1.1 1.5 1.4 2.1

2.4 2.5 2.2 2.9 2.9

0.7 0.7 0.8 0.9 0.7

49.4 53.2 53.1 0 0

a By cellulose acetate electrophoresis [1]. b By alkali denaturation [2]. c Recovering from iron deficiency anemia.

of Chang et al. [7,8] were unsuccessful, suggesting that the new N-terminus was blocked. This blocking group was removed by treatment of the flXT-1 peptide with 0.5 M HCL in methanol for 48-72 h at room temperature [11]. Unfortunately, this treatment is not specific, as it produces some random cleavages, notably N-terminal to threonine and serine residues [12]. As a consequence, some of the peptide was hydrolyzed at the Leu-Thr peptide bond, resulting in a mixture of three peptides, i.e. the intact ]3XT-1 (minus the blocking group), the N-terminal tetrapeptide (minus the blocking group), and the C-terminal pentapeptide (Fig. 3). The various amino acid residues could readily be placed in sequence, as in the first five steps two sets of residues and in the remaining four steps only one set of residues were identified. T A B L E 11 A M I N O ACID C O M P O S I T I O N OF PEPTIDE T-1 O F T H E fix C H A I N OF Hb D O H A A N D ITS T H E R M O L Y T I C FRAGMENTS Results expressed in mol/peptide. Amino acid

,SXT-1

Threonine Glutamic acid Proline Valine Methionine Leucine Histidine Lysine

0.95 3.15 0.92 0 0.77 0.96 1.07 1.01

/3XT-1A

,sx T-1 B

flAT-I

0.98

0.97 2.11 0.98

1 2 1 1 0 1 1 1

0.84 0.98 1.02 0.95

These results confirm a (blocked)-N-Met-Glu-HisLeu-Thr-Pro-Glu-Glu-Lys sequence for the Nterminus of the /3 x chain. Supporting data were obtained from the analyses of the thermolytic fragments of the/3XT-1 peptide. The amino acid compositions of these peptides are also listed in Table II. Sequence analyses failed to detect an N-terminal residue in the Met-Glu-His tripeptide (/3XT1A), which suggested that it was blocked, while a Leu-Thr- N-terminal sequence was readily established for peptide/3XT-1B. Since such an abnormality has not been described before, the Hb variant was named Hb Doha after the city in which the variant was first discovered. The nature of the blocking group Attempts were made to identify the blocking group by direct probe electron impact mass spectroscopy using the X-Met-Glu-His tripeptide as experimental sample and pyroglutamylhistidinylprolinamide ( = T R F or TSH releasing hormone) as control. The mass spectrum for T R F showed fragmentation patterns classical for this

1

2

3

4

5

6

7

8

9

MET-GLU-HI S-LEIJ-THR-PRO-GLU-GLU-LYS f

r

~"

/

/

/

r

/

f

f

MET-GLU-HI S-LEU-

THR-PRo-6LU-6Lu-LYs l

f

f

~

I

Fig. 3. Identification of the amino acid sequence of the Nterminus of the ~ chain of H b Doha. See text for further details.

260

peptide as well as a parent molecular ion. The spectrum of the X-Met-Glu-His probe, however, showed peaks at role 44 ( = CO 2) and at role 60 ( = C H 3 - C O O H ), but no significant ions at a higher molecular weight. This result may represent a unique property of this tripeptide, but does not allow an interpretation of the spectrum other than to be suggestive for the presence of an acetyl moiety. Alternate methods will be employed to analyze this peptide further.

Comments The presence of methionine in the N-terminal position of one of the chains of human Hb is surprising, as this residue, incorporated during the initiation process of the synthesis of the polypeptide chain, is enzymatically removed [13]. Cleavage of this residue, however, is greatly influenced by the next amino acid residue; if this residue is (relatively) small and uncharged, removal of the initiator is promoted [14,15]. A Val ~ G l u replacement in position fll(NA1) apparently prevents the removal of the methionyl N-terminal. Hb Doha is different from Hb Long Island [16,17], also known as Hb Marseille [18], which has an N-terminus extended by a methionine residue. The fl-chain of this Hb variant has an N-terminal sequence Met-Val-PRO-Leu-Thr-, resulting from a His--* Pro substitution in the second position (NA2), quite different from the (blocked)-Met-GLU-His-Leu-Thr N-terminus of the fl-Doha chain. The introduction of proline in position fl2(NA2) apparently results in an ineffective removal of the methionine residue; this has also been observed for other proteins [19-21]. The oxygen affinity of Hb Long Island-Marseille and Hb Doha can be expected to be normal although the binding of 2,3-diphosphoglycerate may be impaired. Functional studies of Hb Doha have not yet been completed, and these may be reported at a later time. It is of interest, however, that the Hb Doha heterozygotes show no effects of an apparent change in functional properties of nearly 50% of their circulating Hb.

Acknowledgements This research was supported in part by USPHS Research Grants HLB-05168 and HLB-15158. The

authors are indebted to Dr. L.B. Hendry and his staff (section of Metabolic and Endocrine Disease, Department of Medicine, Medical College of Georgia) for the initial mass spectroscopic data.

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