Single Amino Acid Substitutions in the Reactive Site of Antithrombin ...

Vol. 263, No. 12, Issue of April 25, pp. 5589-5593,1988 Printed in U.S.A.

THEJOURNAL OF BIOLOGICAL CHEMISTRY 0 1988 by The American Society for Biochemistry and Molecular Biology, Inc.

Single Amino AcidSubstitutions in the Reactive Site of Antithrombin Leading to Thrombosis CONGENITAL SUBSTITUTION OF ARGININE 393 TO CYSTEINE IN ANTITHROMBIN NORTHWICK PARK AND TOHISTIDINEINANTITHROMBIN GLASGOW* (Received for publication, October 22, 1987)

Hediye Erdjument, David A. Lane$, Maria Panico8, Vincenzo Di MarzoQ,and Howard R. Morris8 From the Department of Haematobgy, Charing Cross and Westminster Medical School, Hammersmith, London, W6 8RF and the $Department of Bwchemistry, Imperial College, South Kensington, London, SW7 2AZ. United Kingdom

family of related inhibitors now collectively known as “serpins” (6, 7). Reaction between antithrombin and serine proteinases is catalyzed by heparin which induces a conformational change in the inhibitor (8) and also provides a template for “approximation” of inhibitor and enzyme during its accelerated inhibition (9,lO). Appreciable evidence indicates that theaminoterminal region of antithrombin is involved in its interaction with heparin (11-13). Furthermore, it has been demonstrated that thrombin attacks specific a reactive bond of antithrombin near its COOH terminus during antithrombin-thrombincomplex formation. This bond has been identified as Arg393-Ser394 (14). Structural investigations of congenital variants of antithrombin exhibiting defective binding to heparin have established amino acid substitutions of Arg47to Cys in antithrombin Toyama (15), Arg47to His in antithrombin Rouen (16), and to Leu in antithrombin Base1(171, providing further support for an NH2-terminal heparin binding domain. In a study of another variant antithrombin, antithrombinDenver (18), characterized by impaired ability to inhibit thrombin, an amino acid substitution Ser394to Leu was identified. We have previously described some properties of two antithrombinvariants,antithrombin Northwick Park (19,20) and antithrombinGlasgow (21). Both of these variantsexhibit defective interaction with thrombin and somewhat increased binding affinity toward heparin-Sepharose (19, 21). Both are associated with familial thrombosis (21, 22). Plasma containing antithrombin Northwick Park is characterized by an additional fast moving anodal electrophoretic component on two-dimensional crossed immunoelectrophoresis (22). Ina Antithrombin is a single-chain plasma glycoprotein which recent communication, we have demonstrated that this abcan inactivate thrombin and most of the otherserine protein- normal component comprises an inactive, covalent disulfideases of the blood coagulation system (1).The amino acid linked variantantithrombin-albumin complex (20). Antisequence of antithrombin hasbeen established by protein and thrombin Glasgow exhibits normal electrophoretic behavior cDNA sequencing studies (2-5). It is composed of 432 amino (21). Using fast atombombardment mass spectrometry (FABacid residues and three disulfide bridges. Antithrombin is MS)’ of chemically and enzymatically fragmented isolated homologous with other serine proteinase inhibitors such as variant antithrombins, we have now established substitutions a,-proteinase inhibitor, a,-antichymotrypsin, a,-antiplasmin, of A r e to Cys in antithrombin Northwick Park and A r e heparin cofactor 11, C1 inhibitor, and protein C inhibitor, a to His in antithrombin Glasgow.

AntithrombinNorthwick Park andantithrombin Glasgow are functionally variant antithrombins with impaired abilities to interact with thrombin. Thrombosis is associated with their inheritance. Both of the purified, reduced, and S-carboxymethylated variant antithrombins were treated with cyanogen bromide and themajor pools of each containing theamino acid sequence Gly3s9-Met42Swere isolated. Following treatment of these pools with trypsin, fast atom bombardment mass spectrometryidentifiedtryptic peptides (found also in normal antithrombin treated in same the way)that corresponded to amino acid sequences Glyss9-Lyss70and Va1400-Met‘2S. The trypticpeptides, corresponding to amino acid sequences Ala37‘-Arg393 and SerSS4-ArgSg9 were present in both variant preparations in greatly reduced amounts compared to a normal antithrombin preparation. However, two novel tryptic peptides of molecular mass (M + H)+2976 and 2962 were identified in the digests of antithrombin Northwick Park and Glasgow, respectively. Further analyses of these novel tryptic peptides were carried out by V8 protease treatment and sequential Edman degradation coupled with mass spectrometric analysis of the shortened peptides. This established that these peptides comprised the amino acid sequence Alas7’ArgS”, but with singleamino acid substitutions at the reactive site, ArgSgs replaced by Cys (in antithrombin Northwick Park) and by His (in antithrombin Glasgow).

*This work was supported by a grant from the British Heart Foundation (to D.A. L.) and by a Medical Research Council Programme Grant (to H. R. M.). The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked “oduertisement” in accordance with 18 U.S.C. Section 1734 solely to indicate this fact. $ To whom correspondence should be addressed Dept. of Haematology, Charing Cross and Westminster Medical School, Hammersmith, London, W6 8RF, United Kingdom.

MATERIALSANDMETHODS

Antithrombin was isolated from patients and normal subjects by heparin-Sepharose affinity chromatography, with further purification using anion-exchange chromatography (FPLC, Pharmacia LKBBioThe abbreviations used are: FAB-MS, fast atom bombardment mass spectrometry; FPLC,fast protein liquid chromatography; HPLC, high performance liquid chromatography.

5589

5590

Substitutions inReactive Site of Antithrombin Lead to Thrombosis

technology Inc.) (19, 21, Antithrombin 23). from the plasma of the propositus with antithrombin Northwick Park isolated in this way comprised two distinct peaks, designated NWP peak 1 and peak 2 in order of elution from the anion-exchange column (19,20).NWP peak 2 was reduced, S-carboxymethylated, and chromatographed using reverse-phase HPLC (see below). Acetonitrile gradient elution resolved two peaks, designated NWP peaks 2.1 and 2.2 (20). We have previously shown that NWPpeak 2.1 is albumin (20).NWP peak 2.2 (variant antithrombin), denoted henceforth as antithrombin Northwick Park, was used for further study here. Following its initial isolation from plasma by heparin-Sepharose chromatography, antithrombin Glasgow was further purified to remove normal antithrombin (the patientis undoubtedly heterozygous) by passage through a thrombin-Sepharose column. This was prepared using human a-thrombin (agift from Dr. J. Fenton, New York State Department of Health, New Albany, NY) and CNBr-activated Sepharose 4B (Pharmacia). The column was equilibrated with 0.1 M NaHC03 containing 0.15 M NaCI, pH 8.3,and preliminary experiments established its capacity to bind to normal antithrombin. The variant antithrombin preparation was applied to the column in a small volume of the same buffer a t room temperature. After 20 min, unbound antithrombin was eluted and thiswas monitored at 280 nm. The variant antithrombin eluted in this way lacked the ability to inactivate thrombin and will henceforth be denoted antithrombin Glasgow. Functional analysis of the variant antithrombins was performed by a heparin cofactor assay (24),using thrombin as enzyme and the chromogenic substrate S2238 (Flow Laboratories). The activities of the variant antithrombin preparations were compared with that of a normal antithrombin preparation, designated as having 100% activity. Under the conditions of the assay, both Northwick Park peak 2 and antithrombin Glasgow eluted from thrombin-Sepharose column had no detectable functional activity. S-Carboxymethylation of the antithrombin preparations was performed as described (20,25).HPLC of reduced, S-carboxymethylated protein was carried out using a C8 ProRPC 5-pm HR 5/5 column with FPLC delivery system (Pharmacia), as described (20).For chemical fragmentation, 5'-carboxymethylated antithrombin preparations were dissolved in 70% formic acid in the presence of cyanogen bromide (CNBr) at a CNBr:protein ratio of 3:l (w/w) and incubated a t room temperature under nitrogen for 17 h. The mixtures were diluted 10-fold with distilled water and injected onto a 15-pm C18 Pep RPC column with FPLC delivery system (Pharmacia) (see also legend to Fig. 1). Trypsin (~-l-t0sylamido-2-phenylethyl chloromethyl ketone-treated, Sigma) digestion was carried out in 50 mM NH,HCOs, pH 8.4,at 37 "C for 4 h, at an enzyme:substrate ratio of

10%

150 (w/w). Staphylococcus aureus V8 protease (ICN) digestion was performed in 100 mM NH4HC03, pH 7.8, a t 40 "C for 6 h at an enzymembstrate ratio of 1:20 (w/w). Sequential Edman degradation (26) was carried outon digest mixtures (tryptic or V8 protease) using, at each stage, phenylisothiocyanate (Rathburn Chemicals Ltd, Walkerburn, Scotland) a t 45 'C for 1 h, followed by cleavage with trifluoroacetic acid (Rathburn Chemicals) for 10 min a t 45 "C. The exact molecular size (M H)+ of remaining and shortened peptides (after n cycles) was then determined by high field FAB-MS. FAB-MS was performed using a VG ZAB HF instrument equipped with an "Scan FAB gun operating a t 20 pA beam current at 8 keV (20, 27). Results of molecular mass determinations are expressed as (M + H)+.

+

RESULTS AND DISCUSSION

Peptides produced by CNBr fragmentation of reduced, Scarboxymethylated normal and variant antithrombins all exhibited broadly similar elution profiles upon reverse-phase HPLC; a typical elution profile of normal antithrombin is illustrated in Fig. 1. Each peak and pool designated in Fig. 1 was subsequently lyophilized and themolecular masses (M + H)+ of peptides produced by trypsin digestion were studied using FAB-MS, via the FAB mapping strategy (25, 27). Because nearly all trypticpeptides of normal antithrombin have unique mass (M + H)+ values, each peak and pool could be TABLEI Results of FAB mapping of tryptic digests of CNBr pool 4 of normal antithrombin, antithrombin Northwick Park, and antithrombin Ghgow Tryptic digests of CNBr pool 4 (see Fig. 1)were analyzed by FABMS and all of the (M + H)+ contained in the digests recorded (lefthand column). Each (M H)+ was then assigned to a tryptic peptide theoretically cleavable from the known amino acid sequence of antithrombin (see also Fig. 3). Confirmation of correct assignment was sought by Edman degradation of these tryptic digests followed by FAB-MS analyses. This produced positive identification of the NH, termini of all but seven of the peptides. Peptides of (M H)+ 2290* and 700* found in digests of normal antithrombin were assigned to peptides around Arge3, Ala371-Arg393,and Ser3''-Arg3=. These peptides were present ingreatly reduced amounts indigests of antithrombin Northwick Park and Glasgow. Signals at (M + H)+ 2976**and (M + H)' 2952" were novel and found only in the tryptic digests of CNBr pool 4 from antithrombin Northwick Park and antithrombin Glasgow, respectively (see text). ND, not detected.

+

+

Mass

(M H)' 522 839 1340 563 451 659 579 860 516 503 699 1036 418 2054 1104 1126 911 1309 2290* 700* 494 1232 1146 2976** 2952** +

M

%B

0

FIG. 1. Reverse-phase HPLC separation of CNBr fragments from reduced, S-carboxymethylated antithrombin. 1-2 mg of reduced S-carboxymethylated normal antithrombin was suspended in 70% formic acid and subjected to CNBrfragmentation under nitrogen a t CNBr:protein ratio of 3:1, for 17 h in the dark at room temperature. Reaction was stopped by dilution with distilled water and the reactants injected onto a 15-pm C18 Pep RPC column with FPLC delivery system. Separation was achieved using a 155-ml gradient of 0-55% acetonitrile with 0.1% trifluoroacetic acid, after initial equilibration with water acidified with 0.1% trifluoroacetic acid. Detection was at 214 nm, and a flow rate of 2 ml/min was used. Very similar elution profiles were observed for reduced, S-carboxymethylated antithrombin Northwick Park and Glasgow also treated with CNBr.

Amino acid sequence in antithrombin

(104-107) (108-114) (115-125) (126-129) (130-132) (140-145) (146-150) (170-176) (184-188) (223-226) (237-241) (282-290) (291-294) (298-314) (339-348) (349-359) (351-359) (360-370) (371-393) (394-399) (400-403) (404-413) (414-423)

Mass (M + H)' after 1 s t step Edman degradation

ND 692 1239 ND ND 546 466 747 445 446 570 937 ND 1925 1047 1039 ND 1194 2219 613 ND 1161 1017 ND 2881

Amino acid lost

ND Phe Thr ND ND Leu Leu Leu Ala G~Y Glu Val ND Glu Gly Ser ND ASP Ala Ser ND Ala Glu ND Ala

Substitutions in Reactive Site of Antithrombin Lead to Thrombosis assigned to theknown primary structure of antithrombin (3). Thus, CNBr fragmentGly339-Met423 of normal antithrombin was identified in CNBr pool 4 (see Fig. 1) on the basis of the (M H)' of its tryptic peptides (Table I, Figs. 2 and 3). Also contained in the same poolwere CNBr fragments G1u"Metzs1 and Val*a2-Met314.One step of Edman degradation followed by FAB-MS carried out on the pool from normal antithrombin treated with trypsin further substantiated the assignments made in theknown sequence of antithrombin for all but six of these peptides (Table I). The masses (M + H)' of tryptic peptides found in CNBr pool 4 from antithrombin Northwick Park and antithrombin Glasgow and their assigned amino acid sequences are also indicated in Table I. The masses (M + H)' 2290 and 700

+

418

519

451

I

5591

assigned to normal antithrombin amino acid sequences Ala371and Ser394-Arg399,respectively, could only be detected in small amounts in both of the variant preparations. One possible explanation of this finding which was entertained is that in both variant proteins the reactive bond Arg393-Ser3g4 is altered, preventing itscleavage by trypsin. For this conjecture to be correct, additional peptides would be present inthe variant protein digests, with (M + H)' values corresponding to the sum of those of the (modified) peptides contained within Ala371-Arg3m. Accordingly, the FAB maps obtained with the digests of antithrombins Northwick Park and Glasgow were examined in the mass region corresponding to (M + H)' exceeding 2290. Two novel tryptic peptides of masses (M H)' 2976 and 2952 were indeed identified in thetryptic digests of antithrombin Northwick Park and antithrombin Glasgow, respectively (see Table I and Fig. 4). Because of the high frequency of Arg to Cys and to His substitutions in another coagulation protein, fibrinogen (28),

+

A

I

I

I

2 952

C

I

2290

FIG. 2. FAB map of the peptides obtained by tryptic digestion of CNBr pool 4 of normal antithrombin showing the quasimolecular ions of component peptides. CNBr pool 4 from normal antithrombin (see Fig. 1) was lyophilized and dissolved in 50 mM NH,HCO, buffer, pH 8.5. Trypsin was added atanenzyme:substrate ratio of 1:50 (w/w), and the digest was incubated for 4 h at 37 'C. The incubation mixturewas freeze-dried twice, dissolved in 5%acetic acid, and the mixture of peptides analyzed by FAB-MS under the conditions described (20, 25). Quasimolecular ion signals are labeled, and other signals are due to anticipated satellites of -15 or +22. 339

FIG. 4. Comparison of the FAB mass spectra of tryptic digests of HPLC-purified CNBr pool 4 from normal antithrombin ( A ) ,antithrombin Northwick Park ( B ) ,and antithrombin Glasgow (C). Only regions of the spectra where differences were observed between the three digests are illustrated, i.e. the region corresponding to the quasimolecular ions m / z 700, 2290, 2976, and 2952. Note the presence of the signal of m/z 700, which is more abundant than the expected 13C isotope of that at m/z 699 in the normal antithrombin digest ( A ) .Its presence to a lesser extent inthe variantantithrombinpreparations ( B and C), together with the reduction in the signal at m/z 2290, confirms that these preparations contain relatively little normal antithrombin. FAB-MS analysis was performed as described (20,25). 348

359

9.3

360

370

Gly-Leu-Val- Asp.Leu-Phe Ser-Pro-Glu-Lys -Ser-Lys-Leu-Pro- Gly-lle-Val.Ala-Glu-Gly-Arg-Asp~Asp~Leu-Tyr-Val-Ser-Asp-Ala-PheHs-Lys.

FIG. 3. Amino acid sequence of antithrombin between GlyssgMet'". A fragment with this sequence is contained in CNBr pool 4 (see Fig. 1). Peptides theoretically cleavable by trypsin (see text), indicated by solid lines, have the masses (M H)' observed by FAB mapping. Subsequent V8 protease digestion (see text) has produced peptides with (M + H)' indicated by broken lines.

+

(M+H)+9ii L

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(M+H)+130'3

(M+H)+1126

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(M+H)%98

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(M+H)+913 371

-Ala-Phe-Leu-Glu-VaI-Asn-Glu-Glu-Gly-Ser-Glu-Ala-A~a-Ala-Ser~Thr-Ala-Val-Val-lle-Ala.Gly.A~~393 ~ ~

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