the journal of biological chemistry vol. no. pp. · 13848 . plasmic digest of fibrinogen kyoto i...

THE JOURNAL 0 1988 by The American Society for Biochemistry

OF BIOLOGICAL CHEMISTRY and Molecular Biology, Inc.

Vol. 263, No. 27, Issue of September 25, PP.

Characterization of an Apparently Lower Molecular Weight ?-Chain Variant in Fibrinogen Kyoto I THE REPLACEMENT OF -/-ASPARAGINE 308 BY LYSINE WHICH CAUSES ACCELERATED CLEAVAGE OF FRAGMENT Dl BY PLASMIN AND THE GENERATION OF A NEW PLASMIN CLEAVAGE SITE*

(Received for publication, February 26, 1988)

Nobuhiko YoshidaSQ, Shigeharu TerukinaS, Minoru Okumall, Masaaki MoroiII , Nobuo Aoki**, and Michio MatsudaS From the $Institute of Hematology and the 11 Department of Biochemistry 11, Jichi Medical School, Tochigi 329-04, The llFirst Division, Department of Internal Medicine, Faculty of Medicine, Kyoto University, Kyoto 606, and the **First Department of Internal Medicine, Tokyo Medical and Dental University, Tokyo 113, Japan

Congenitally abnormal fibrinogen Kyoto I with impaired fibrin monomer polymerization contains a normal y-chain and a y-chain variant (yKyota I) that has an apparently lower M, on sodium dodecyl sulfate-polyacrylamide gel electrophoresis in the Laemmli system (Laemmli, U. K. (1970) Nature 227, 680-685) but migrates with apparently normal M. in the Weber and Osborn system (Weber, K., and Osborn, M. (1969) J. Biol. Chem. 244, 4406-4412). Reverse-phase high performance liquid chromatographic analyses of the cyanogen bromide or lysyl endopeptidase cleavage fragments of the purified y-chains of fibrinogen Kyoto I showed the presence of peptides not seen from normal fibrinogen. Amino acid sequence analysis of these peptides indicated that yAsnSoB of the y-chain variant is replaced by lysine. Purified fragment Dl of fibrinogen Kyoto I also contains two types of Dl y-remnants: normal and apparently lower M, types. Abnormal fragment Dl is cleaved faster to fragments D2 and D3 by plasmin in the presence of [ethylenebis(oxyethylene- nitri1o)ltetraacetic acid (EGTA) than normal fragment Dl, as analyzed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis, followed by immunoblotting using anti-y-chain monoclonal antibody. Analysis of peptides released from fragment Dl by plasmin in the presence of EGTA demonstrated the cleavage of the yLys308-Gly308 bond. Fragment Dl of fibrinogen Kyoto I has normal calcium binding properties. The data suggest that a region or conformation containing yAsn308 affects the polymerization of fibrin monomers and that the yAsnSoS + Lys replacement causes a conformational change in the y-chain which results in the accelerated cleavage of yLy~~~’-Ala~’’ and yLys302- Phe303 bonds by plasmin and also results in the generation of a new plasmin cleavage site between Lys308 and GlySoe in the presence of EGTA. During these

* This work was supported in part by a Scientific Grant-in-Aid from the Ministry of Education of the Government of Japan, a Research Grant for Intractable Diseases from the Ministry of Health and Welfare of the Government of Japan, a grant-in-aid from the Japan Private School Promotion Foundation, and by the Shimabara Foundation. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked “advertisement” in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

Part of this work was presented at the XIth International Congress on Thrombosis and Haemostasis, Belgium, July 1987 (1).

§ To whom reprint requests should be addressed Inst. of Hema- tology, Jichi Medical School, Minamikawachi-Machi, Kawachi-Gun, Tochigi-Ken 329-04, Japan.

studies, we found that part of the yLys212-G1~213 bond in fragment Dl is cleaved by plasmin in the presence of EGTA.

Most of the congenitally abnormal fibrinogens have prolonged thrombin-clotting times and impaired polymerization of fibrin monomers, and some of them have been described to have additional abnormalities in their plasmic digestion profiles (2-8). Since calcium ion is able to protect fibrinogen fragment Dl against further attack by plasmin (9, lo), it has been possible to analyze the plasmin cleavage sites or calcium- binding sites in the COOH-terminal region of the normal y- chain (11-16). Abnormal fibrinogens Bern I (4) and Haifa (7) have been suggested to lack this ability to be protected by calcium. However, the relationship between abnormalities in the plasmic digestion patterns and structural abnormalities has not been fully studied for abnormal fibrinogens.

Fibrinogen Kyoto (17) (Kyoto I) was identified as a congenitally abnormal fibrinogen with defective fibrin monomer polymerization. This fibrinogen contains a y-chain variant with an apparent M , which is 2000 lower than the normal y- chain on SDS-PAGE’ by the Laemmli method (18). In our study, amino acid sequence analysis of the y-chain variant and investigations of the plasmic digestion of fragment Dl in the presence of EGTA were performed. The results indicate accelerated digestion of abnormal fragment Dl in fibrinogen Kyoto I, which is suggested to be closely related to a new type of single amino acid substitution, yAsn308 + Lys, and the generation of a new plasmin cleavage site between Lys308 and ~ 1 ~ 3 0 9 .

EXPERIMENTAL PROCEDURES

Materials-The reversed-phase HPLC columns were from the following sources: TSK gel TMS-250 from Toyo Soda (Tokyo, Japan), Cosmosil 5C18-P from Nakarai (Kyoto, Japan), and Biofine RPC- SC18 from Nihonbunko (Tokyo, Japan). DEAE-Sephacel was obtained from Pharmacia (Uppsala, Sweden), Ultrogel AcA 44 from LKB (Bromma, Sweden), and Chelex 100 from Bio-Rad. Human plasminogen was purified as described (19). Streptokinase was obtained from AB Kabi (Stockholm, Sweden), and contaminating albumin was removed with blue Sepharose CL-GB (Pharmacia). Plas- min was prepared by the activation of plasminogen with streptokinase (4000 units/mg of plasminogen) for 24 h at room temperature and

The abbreviations used are: SDS-PAGE, sodium dodecyl sulfate- polyacrylamide gel electrophoresis; EGTA, [ethylene bis(oxyethy1- enenitri1o)ltetraacetic acid; HPLC, high performance liquid chromatography.

13848

Plasmic Digest of Fibrinogen Kyoto I with yAsn30S + LYS 13849

was stored at -70 "C in 50% glycerol. CNBr, lysyl endopeptidase, and dithioerythritol were obtained from Wako Chemical Co. (Osaka, Japan). Affinity-purified goat anti-mouse IgG horseradish peroxidase conjugate and peroxidase substrate (4-chloro-1-naphthol) were obtained from Bio-Rad. "CaC12 (27.68 mCi/mg) and scintillation fluid (Biofluor) were from DuPont-New England Nuclear.

Purification of Fibrinogen-Fibrinogen was purified from citrated or acid citrate dextrose-plasma using lysine-Sepharose 4B chromatography, gelatin-Sepharose 4B chromatography, and fractionation by ammonium sulfate as previously described (17).

Purification of y-Chain-Reduced and carboxymethylated fibrinogen was prepared according to the method of Doolittle et al. (20) with the following modification, as previously described (21). 10 mg of fibrinogen was dialyzed against 0.2 M Tris-HC1, 6 M guanidine HCl, pH 8.2, before the addition of 3 mg of dithioerythritol, and the vessel was flushed with nitrogen before the reduction and carboxymethylation steps. HPLC separation of reduced and carboxymethylated fibrinogen chains (22) was performed using a TSK gel TMS-250 reverse-phase HPLC column. A 0.09% trifluoroacetic acid (solvent system A) and 0.09% trifluoroacetic acid in acetonitrile (solvent system B) gradient system was used as the eluant; a linear gradient of 30-60% solvent system B in 2 h with a flow rate of 0.5 ml/min was employed. The column effluent was monitored at 280 nm. Separation of the abnormal y-chain variant (yKyotoI) from the normal y-chain could not be achieved. The y-chain fractions were pooled, purified by rechromatography, and then lyophilized.

?-Chain Digestion with CNBr and Lysyl Endopeptidase-Purified y-chain dissolved in 70% formic acid was incubated with CNBr (1500 nmol/nmol of y-chain) in the dark for 24 h at room temperature after flushing with nitrogen. The CNBr cleavage products were lyophilized, dissolved in 6 M guanidine HC1, and fractionated on a Cosmosil5C18- P reversed-phase HPLC column. The solvent system used was the same as for TSK gel TMS-250, with a linear gradient of 10-50% solvent system B in 1 h; and the column effluent was monitored at 214 nm. Relevant CNBr fragments were lyophilized; dissolved in 50 mM Tris-HC1, 4 M urea, pH 9.0; and then further digested with lysyl endopeptidase (0.2 pg/nmol of original y-chain) at 37 "C for 18 h. Lysyl endopeptidase digests were also analyzed by HPLC with a linear gradient of 0-50% solvent system B for 100 min. Lysyl endopeptidase digestion of the y-chains (1 pg/nmol of y-chain) was also performed, and the digest was analyzed by HPLC with the following nonlinear gradient system: 0-5% solvent system B for 0-10 min, 5- 20% solvent system B for 10-20 min, and 20-40% solvent system B for 20-100 min.

Plasmic Digestion of Fibrinogen-Fibrinogen (2.5 mg/ml) in 50 mM Tris-HC1,0.135 M NaCI, 5 mM CaC12, pH 7.4, was incubated with 0.1 mg/ml human plasminogen and 3000 units/ml streptokinase for 6 h at 37 "C. The reaction was terminated by the addition of 500 kalli- krein-inactivating units/ml aprotinin as described (21). A 20-mg sample of the plasmic digest was dialyzed against 39 mM Tris-HC1, 5 mM CaC12, pH 8.6; passed through lysine-Sepharose 4B; and applied to a 40-1111 column of DEAE-Sephacel equilibrated with the above buffer and eluted with a 0-0.15 M NaCl linear gradient in 200 ml of the above buffer for fragment Dl and with 1 M NaCl in the above buffer for fragment E. Various kinds of gradient elution systems failed to separate abnormal fragment Dl with an apparently lower M , y-remnant (see "Results") from the normal one, although abnormal fragment Dl tended to be eluted slightly earlier than the normal fragment. Fractions with fragment Dl were further fractionated on an Ultrogel AcA 44 column (1.6 X 80 cm). After dialysis against 50 mM Tris-HC1, 0.135 M NaC1, pH 7.4, purified fragment Dl (0.2 mg/ ml) was treated with 0.02 CTA (Committee on Thrombolytic Agents) U/ml plasmin in the presence of 10 mM EGTA for various times at 37 "C. The reaction was terminated by the addition of 1% SDS and heating for 5 min at 100 'C, and then the digest was analyzed by SDS-PAGE. For the analysis of peptides cleaved by plasmin, 2 mg of fragment Dl was incubated with 0.18 mg of plasminogen and 6000 units of streptokinase in 10 ml of 50 mM Tris-HC1,0.135 M NaCl, 10 mM EGTA, pH 7.4, at 37 "C for 1 h; and the reaction was terminated by heating the mixture at 100 "C for 15 min. Cleaved peptides were obtained by centrifugation of the heat-denatured samples and fractionated on a Biofine RPC-SC18 reversed-phase HPLC column with a linear gradient of 0-40% solvent system B for 160 min. Relevant peaks were lyophilized; dissolved in 0.2 M Tris-HC1, 6 M guanidine HCl, pH 8.2; and incubated with dithioerythritol(1 mg/mg of original fragment Dl) for 1 h at 37 "C after flushing with nitrogen, followed by reaction with 15 mM iodoacetic acid for 1 h at room temperature. Dithioerythritol-treated peptides were also fractionated by HPLC

with the following gradient system: a linear gradient of 0-30% solvent system B for 30 min and then further elution with 30% solvent system B for the following 30 min.

Amino Acid Sequence Analysis-Amino acid sequence analysis was performed by automated Edman degradation (Applied Biosystems Model 470A Protein Sequencer to which a Model 120A phenylthio- hydantoin analyzer was connected).

SDS-PAGE and Zmmunoblotting-SDS-PAGE was performed according to the method of Laemmli (18) as described previously (see Ref. 23). Transfer of proteins from polyacrylamide gels onto nitro- cellulose (Western blotting) was performed as described (24, 25). Monoclonal antibody against the y-chain (21) was used for identifying the y-chain in the blots. The antigenic determinant for this antibody is within the sequence spanning ySerss to Lys302. SDS-PAGE was also performed according to the method of Weber and Osborn (26).

Two-dimensional SDS-PAGE was performed according to the method of Purves et al. (10) with minor modifications. The first dimension SDS-PAGE was performed on a 5 2 0 % gradient gel in the Laemmli system (18) under nonreducing conditions. After electrophoresis, the lane to be used was cut out and electrophoresed on the second dimension gel, a 5 2 0 % gradient gel in the Laemmli system, with a 1% agarose stacking gel containing 0.1 M dithiothreitol.

Calcium Binding to Fragment D-Equilibrium dialysis to charac- terize the Ca2+ binding properties of fragment Dl was performed essentially according to the method of Van Ruijven-Vermeer et a1. (27). The buffer used (50 mM Tris-HC1, 0.135 M NaCl, pH 7.6) was filtered through a Chelex 100 column, and the dialysis bags were pretreated according to the method of Marguerie et al. (28). Purified fragment Dl was added with 5 mM EGTA and dialyzed against the above buffer with 3 mM EGTA, followed by extensive dialysis against the above buffer without EGTA at 4 "C. 0.1 ml of EGTA-treated fragment Dl (1 mg/ml) was dialyzed against 20 ml of buffer containing Ca2+ at concentrations from 1 to 100 p~ at 25 "C for 48 h. Each vessel contained 2 pCi of 46Ca2'. After dialysis, 50-pl aliquots of the materials inside and outside the dialysis bag were mixed with 20 ml of scintillation fluid and counted in an LSC-700 liquid scintillation system (Aloka, Tokyo, Japan). Scatchard analysis was performed assuming M, of 95,000 for fragment Dl.

Other Methods-Protein concentration was assayed according to the method of Lowry et al. (29) using crystalline bovine serum albumin as the standard. Densitometric scanning of the immunoblots was done on a CS-930 dual-wavelength TLC scanner (Shimadzu, Kyoto, Japan) at 540 nm.

RESULTS

SDS-PAGE Analysis of Fibrinogen-Fibrinogen Kyoto I analyzed by SDS-PAGE in the Laemmli system (18) after reduction contained two types of y-chains: the y-chain variant yKyotol, with an apparent M, of 48,000, and the normal y- chain, with a M, of 50,000 (Ref. 17 and Fig. 1, lane K-I) . Both y-chains were positively stained by anti-y-chain monoclonal antibody (data not shown). The faster electrophoretic migration of y~~~~ I was not affected by a &fold increase of dithiothreitol for the reduction of fibrinogen (data not shown), nor by reduction and carboxymethylation of fibrinogen (Fig. 1, lane K-2), which exclude any possibility of insufficient cleavage of intra-y-chain disulfide bonds. However, SDS-PAGE following the method of Weber and Osborn (26) failed to differentiate y~~~~~ I in the reduced fibrinogen or reduced and carboxymethylated fibrinogen (Fig. 1, lanes K-3 and K-4) , which suggested that y~~~~ I actually has almost the same M, as the normal y-chain and that the apparently lower M , obtained on the Laemmli gels was caused by an amino acid substitution(s). Nonreduced fibrinogen could not be separated into two populations on Laemmli gels of various acrylamide concentrations (5, 7.5, and 10%) (data not shown).

CNBr and Lysyl Endopeptidase Cleavage of y-Chain-The HPLC elution pattern of the CNBr-cleaved y-chains of fibrinogen Kyoto I (Fig. 2 A ) revealed a decrease in one peak, (designated KB2) compared to the corresponding normal peak (designated NB1) and the appearance of a new peak (designated KB1). The sequences of the first 7 residues of NB1,

13850 Plasmic Digest of Fibrinogen Kyoto I with yAsn308 + Lys

A N

1 2 3 4 1 2 3 4

K N FIG. 1. SDS-PAGE of fibrinogen. The samples (2-4 pg of pro-

tein) were electrophoresed on 10% Laemmli gels (lanes 1 and 2) or 5% Weber and Osborn gels (lanes 3 and 4 ) and stained for protein. N, normal control; K, fibrinogen Kyoto I; lanes 1 and 3, reduced fibrinogen; lanes 2 and 4, reduced and carboxymethylated fibrinogen. The locations of the chains are indicated.

KB2, and KB1 were all Phe-Lys-Val-Gly-Pro-Glu-Ala, indicating that the peptides correspond to residues 265-310 of the y-chain (30) (data not shown). In another run, amino acid sequence analysis of KB1 was achieved up to residue 290 of the y-chain, with no difference from the sequence of KB2 being observed (data not shown). To analyze fully peak KB1, three peaks (NB1, KB1, and KB2) were further digested with lysyl endopeptidase and analyzed by HPLC (Fig. 2B). The NB1 digest profile (data not shown) was the same as that of the KB2 digest. In the KB1 digest (Fig. 2B, lowerpanel), peak KB2L2 in the KB2 digest, which had a retention time of 25.3 min (upper panel), was missing; instead, a new peak, (KBlL2) with a retention time of 22.8 min appeared. As shown in Table I, the amino acid sequence of KB2L2 corresponded to residues 303-310 and that of KBlL2 was Phe-Phe-Thr-Ser-His-Lys, which suggested that y A ~ n ~ ~ was replaced by lysine in abnormal y~~~~ I. The complete amino acid sequences of KB2L3 and KB1L3 were obtained, and they corresponded exactly to residues 274-302 (data not shown). The peptide corresponding to residues 309-310 in the KB1 digest was not recognized on this HPLC profile, like Phe-Lys (residues 265-266); it was probaby not retained by the column or was too small to be recognized as a peak. Thus, sequence analysis of abnormal peak KB1, which corresponds to residues 265-310, demonstrated that there was a normal sequence for residues 265- 307 and substitution of lysine for asparagine at residue 308, whereas residues 309-310 remained unknown. To resolve this question, purified y-chains were directly digested with lysyl endopeptidase, and the digests were analyzed by HPLC (Fig. 3). As expected, the lysyl endopeptidase digest of the y-chains of fibrinogen Kyoto I contained two new peaks (KL1-1 and KL1-2), and one peak (KL2) was decreased as compared to the corresponding normal peak NL1. The amino acid sequences of NL1 (not shown) and KL2 (Table I) indicated that they corresponded to residues 303-321. The amino acid sequence of KL1-1 was again Phe-Phe-Thr-Ser-His-Lys, corresponding to residues 303-308, and that of KL1-2 indicated that this peptide corresponded to residues 309-321 of the normal y-chain (Table I), which confirms the replacement of lysine for asparagine at residue 308 of y~~~~ I.

Plasmic Digestion of Fibrinogen and Fragment D1-The yAsn"* - Lys replacement in yKyoto I led us to investigate the

m

! o

r

z

60

Time (min)

B

KB 2 KB2L3

KB2L2

KB2Ll \

KB 1 KB1 L3

0 20 40 Time (min)

60 80

FIG. 2. HPLC analysis of CNBr peptides of 7-chains. A, separation of the CNBr peptides from the normal y-chain ( N ) and the y-chains of fibrinogen Kyoto I ( K ) was accomplished by reversed- phase HPLC as described under "Experimental Procedures." For these chromatograms, peptides from 0.75 mg of y-chain were injected. KB2 is the peak with the same elution time as NB1; KB1 is the new peak. B, KB2 and KB1 in A were further digested with lysyl endopeptidase and separated by HPLC. The very minor peaks KB2Ll and KBlLl were recognized as clear peaks if a steep gradient was used, and they correspond to residues 267-273 of the y-chain (21). The other peaks without designations proved to be minor contami- nants.

plasmic digestion of this fibrinogen. Purified fragment Dl was electrophoresed on 10% Laemmli gels. Fragment Dl of fibrinogen Kyoto I after reduction (Fig. 4, lane 2) had the same apparent M, remnants as the normal control, but an immu-

Plasmic Digest of Fibrinogen Kyoto I with yAsn30R + Lys 13851 TABLE I

Amino acid sesuence of Dentides derived from y-chuim I

KB2L2 KBlL2 KL2 KL1-1 KL1-2

N

I

- I

1 Phe 543 Phe 960 Phe 129 Phe 246 Gly 78 2 Phe 466 Phe 1004 Phe 115 Phe 230 Met 102 3 Thr 135 Thr 346 Thr 68 Thr 153 Gln 70 4 Ser -' Ser - Ser - Ser - Phe 85 5 His 27 His 217 His 15 His 25 Ser - 6 Asn 21 Lys 196 Asn 4 Lys 11 Thr 30 7 Gly 9 Gly 9 Trp 22 8 " Met 4 9

Asp 36 Gln 3 Asn 16

10 Phe 4 Asp 30 11 Ser - 12

"

"

303-310b 303-308 303-321 303-308 309-321 -, positions at which it was not possible to assign a residue or

Corresponding residues of the y-chain. to quantitate recovery.

!

al U c D L 0

a

.A D 4

i K

I I 1 I

0 30 60 9 0 Tine ( m i n )

FIG. 3. HPLC analysis of lysyl endopeptidase digests of 7- chains. Purified y-chains were directly digested with lysyl endopeptidase and separated by reversed-phase HPLC. For these chromatograms, peptides obtained from 0.125 mg of the y-chains were injected. KL1-1 and KL1-2 are new peptides; KL2 is the peak with the same elution time as NL1.

noblot (Fig. 5A, lune K - I ) demonstrated the presence of two types of fragment Dl y-remnants, consistent with the two types of fibrinogen y-chains: the normal fragment Dl y- remnant with an apparent M , of 42,000 and the abnormal fragment Dl y-remnant with an apparent M , of 40,000, which originated from 7 ~ ~ ~ ~ 1 . The abnormal fragment Dl y-remnant, fragment Dl ~ K ~ ~ ~ I remnant, showed almost the same electrophoretic migration as the @-remnant in the Laemmli system. Weber and Osborn gels could not differentiate the two types of y-remnants (data not shown). Although nonreduced fragment Dl of fibrinogen Kyoto I (Fig. 4, lunes 6 and

D3/7 - "-

1 2 3 4 5 6 7 8 9 101112 FIG. 4. SDS-PAGE and immunoblotting of fragment Dl and

its plasmic digest in presence of EGTA. Purified fragment Dl (lanes 1, 2, 5, 6, 9, and 10) and its plasmic digest (lanes 3, 4, 7, 8, 11, and 12) in the presence of EGTA for 24 h at 37 "C were electrophoresed on 10% Laemmli gels and stained for protien (lanes 1-8) or stained with anti-y-chain monoclonal antibody using immunoblotting (lanes 9-12) as described under "Experimental Procedures." Lunes 1, 3, 5, 7, 9, and 11, normal control; lanes 2, 4, 6, 8, 10, and 12, samples obtained from fibrinogen Kyoto I; Lunes 1-4, reduced samples; lanes 5-12, nonreduced samples. Amounts of proteins applied were: 1 pg for lanes 1, 2, 11, and 12; 2 pg for lanes 3, 4, 7, and 8; and 0.3 pg for lanes 5, 6, 9, and 10. The locations of the fragments are indicated. Dl/y and Da/y, y-remnants of fragments D1 and Da, respectively; /@, @-chain remnant; D6& D39, and D a fragments with apparent M, values of 62,000, 39,000, and 38,000, respectively. The a-chain remnant migrated at the dye front in lanes 1-4.

IO) migrated as an apparent single band at the same position as the normal fragment on 10% Laemmli gels, it could be separated into two species on 5% Laemmli gels (data not shown). Since DEAE-Sephacel chromatography, fibrin monomer-Sepharose chromatography, or chromatofocusing of fragment Dl (data not shown) failed to separate clearly abnormal fragment Dl from the normal fragment, analyses of fragment Dl of fibrinogen Kyoto I were performed with the whole fragment Dl fraction.

Fragment Dl was further digested with plasmin in the presence of EGTA and analyzed by SDS-PAGE, followed by immunoblotting. The apparent M , values of the normal fragment DP and D3 y-remnants were 34,000 and 30,000, respectively, as shown in Fig. 5A, lunes N-2-N-6. The COOH termini of these remnants are yLys3= and yLys302, respectively (14, 15). A minor band with slower mobility than the fragment DP y-remnant may represent the remnant with a COOH terminus yLys373. In the case of fibrinogen Kyoto I, an additional abnormal y-remnant with an apparent M, of 32,000 appeared in the early stage of plasmic digestion (Fig. 5A, lune K-2). This remnant would be the fragment DP y~~~~ I

remnant because of its early appearance and its apparent M,, which is 2,000 smaller than the normal fragment DZ y-remnant, corresponding to the apparent M , difference between the normal Dl y-remnant and the fragment Dl ~ K ~ ~ ~ I remnant. In addition, in the late stage of plasmic digestion, the M , = 32,000 remnant disappeared, and only one type of the normal fragment D3 y-remnant was detected, as would be expected inasmuch as the yAsn3"" + Lys exchange occurs beyond the COOH terminus of the y-remnant in fragment D3.

Interestingly, fragment Dl obtained from fibrinogen Kyoto I was digested faster to fragments DP and D3 by plasmin in the presence of EGTA than normal fragment Dl. The fragment Dl ~ K ~ ~ ~ I remnant was cleaved more rapidly than the normal fragment Dl y-remnant as demonstrated at the 60- min digestion point (Fig. 5A, lune K-4); and compared to the normal control (Fig. 5A, lune N-2), a considerable amount of

13852 Plasmic Digest of Fibrinogen Kyoto I with yAsn3'' + Lys

A the presence of EGTA for 1 h at 37 "C, and the cleaved peptides were analyzed by HPLC (Fig. 6A). For fibrinogen Kyoto I, three new peaks (KP1, KP2, and KP3) were detected.

Lys, corresponding to residues 303-308 of ~ K ~ ~ ~ ~ I (Table 11). Kyoto1 The NH2-terminal amino acid of both KP2 and KP3 was

1 ~ ~ 1 7 Kyoto I glycine. Sequencing of common peak NP4 in normal fibrino-

L amino acids, phenylalanine and threonine, at the NH, ter-

M r IkDaI The amino acid sequence of KP1 was Phe-Phe-Thr-Ser-His-

4 2 1 --"""-- --------- - 40 ~

34 - 32 ' 30'

- - - - -""a"- __ "" - -

" """"-e D21 Y

I D d 7 gen and peak KP4 in Kyoto I indicated the presence of two 1 2 3 4 5 6 1 2 3 4 5 6

I I I

N K A

B

100

h

D

Time (min)

FIG. 5. Time course of plasmic digestion of fragment Dl in presence of EGTA as analyzed by immunoblotting. A, purified fragment Dl was digested with plasmin in the presence of EGTA at 37 "C for various times (lane I, 0 min; lane 2, 15 min; lune 3, 30 min; lane 4, 60 min; lane 5, 2 h; lane 6, 24 h), electrophoresed on 10% Laemmli gels after reduction, and stained by anti-y-chain monoclonal antibody using immunoblotting. Amounts of proteins applied were 1 pg for lanes N-I-N-5 of the normal control (N), 1.6 pg for lane N-6, and 2 pg for samples from fragment D1 of fibrinogen Kyoto I (IO. The locations of the fragments and the corresponding apparent M, values are indicated. Dl/?, D2/y, and D3/y, y-remnants of fragments Dl, DP, and D3, respectively. B, immunoblots at various digestion times as described for A were scanned densitometrically, and the amount of the residual fragment Dl y-remnant was plotted as a function of time. The amount of the fragment D1 Y K ~ ~ ~ ~ remnant and the normal fragment Dl y-remnant in fragment Dl obtained from fibrinogen Kyoto I without plasmic digestion (0 min) was taken as 100%. M, normal fragment Dl y-remnant; 0---0, normal fragment Dl y-remnant of fibrinogen Kyoto I; 0- - -0, fragment Dl y~~~~ I remnant.

the fragment D3 y-remnant had already appeared after only 15 min of digestion (Fig. 5A, lane K-2). Densitometric scans of the immunoblots were performed, and the decay of the fragment Dl ~ K ~ ~ ~ I remnant was compared to that of the normal Dl y-remnant (Fig. 5B). Since the fragment Dl ~ K ~ ~ ~ I

remnant co-migrated with the P-remnant, it is not possible to determine accurately the amount of protein by densitometry. This was also the case with the use of radiolabeled anti-mouse IgG instead of goat anti-mouse IgG horseradish peroxidase conjugate (data not shown). However, the fragment Dl ~ K ~ ~ ~ ~ I

remnant was found to be cleaved about 2-fold faster than the fragment Dl y-remnant of the normal control. In contrast, the normal portion of the fragment Dl y-remnant in fibrinogen Kyoto I was cleaved slightly faster than or at almost the same rate as the normal control.

Peptides Cleaved by Plasmin from Fragment D-To investigate the possibility that the yLy~~~*-Gly~"$ bond in y~~~~ I is cleaved by plasmin in the presence of EGTA, purified fragment Dl was digested with plasminogen and streptokinase in

0 "50 75 100 125

Time (rnin)

B

KP 4 4c

I I I 1 I I 0 10 20 30 40 50

l ime (min)

FIG. 6. Peptides cleaved by plasmin in presence of ECTA from fragment Dl. A, peptides obtained from plasmic digestion of 0.1 mg of fragment Dl in the presence of EGTA were analyzed by reversed-phase HPLC as described under "Experimental Procedures." Chromatographic profiles between 0 and 50 min of elution time were omitted to condense the figure and because there were no obvious peaks. B, reduced KP4 and KP2 in A were analyzed by HPLC. For these chromatograms, KP4 and KP2 obtained from 1 mg of fragment Dl digest were reduced and injected.

Plasmic Digest of Fibrinogen Kyoto I wi th yAsn3" + Lys 13853

minus. Amino acid sequences of common peaks NP7 and KP7 were exactly the same as that of residues 357-373 of the normal y-chain (data not shown). Peaks KP2, KP3, KP4, and NP4 were treated with dithioerythritol, carboxymethylated, and again analyzed by HPLC. Dithioerythritol-treated KP4 (Fig. 6B, upper panel) was separated into three peaks (4A, 4B, and 4C), and the chromatographic profile of dithioerythritol- treated NP4 was the same as that of dithioerythritol-treated KP4 (data not shown). Dithioerythritol-treated KP2 (lower panel) was separated into two peaks (2A with the same elution time as 4A and 2C with an earlier elution time than 4C). Dithioerythritol-treated KP3 remained as one fraction (data not shown). Complete amino acid sequences of peaks 4A and 2A with Cys(Cm) as the NH2 terminus were obtained, and they were exactly the same as that of residues 339-356 of the normal y-chain (data not shown). In this peptide Lys3= was clearly detected, whereas Varadi and Scheraga (15) reported that it was missing. The sequence of the first 5 residues of peak 4B was Thr-Arg-Trp-Tyr-Ser, indicating that this peptide corresponded to residues 374-4061411 of the y-chain. The amino acid sequence of peak 4C indicated that the peptide corresponded to residues 303-338 of the normal y-chain and that of peak 2C to residues 309-338 (Table 11).

The amino acid sequence of the other new peak, KP3 (Table 11), indicates that this peptide corresponds to residues 309- 356 of the normal y-chain because it was not separated into two peaks by dithioerythritol treatment. This peak disappeared after prolonged digestion by plasmin (data not shown). In the plasmin cleavage of abnormal fragment Dl, a single- chain peptide with an intact y L y ~ ~ ~ - C y s ~ ~ ~ bond was present

TABLE I1 Amino acid sequence of peptides cleaved from fragment Dl

4C from KP4 KP1 2C from KP2 KP3

Residue pmol Residue pmol Residue pmol Residue pmol Cycle

1 Phe 298 Phe 240 Glv 223 Glv 2 Phe 268 Phe 3 Thr 175 Thr 4 Ser - Ser 5 His 17 His 6 Asn 34 Lys 7 Gly 38 8 Met 42 9 Gln 22

10 Phe 28 11 Ser 12 Thr 10 13 Trp 10 14 Asp 17 15 Asn 15 16 Asp 22 17 Asn 22 18 Asp 27 19 Lys 8 20 Phe 10 21 Glu 6 22 Gly 10 23 Asn 16 24 Cys(Cm) - 25 Ala 4 26 Glu 9 27 Gln 8

29 30

-

28 - -

226 Met 250 Met 47 Gln 158 Gln - Phe 186 Phe 55 Ser - 22 Thr 74 Thr

Ser

Trp 36 Trp ASP 41 Asp Asn 43 Asn ASP 51 Asp Asn 48 Asn ASP 49 Asp LYS 26 Lys Phe 30 Phe Glu 22 Glu GlY 22 Gly Asn 25 Asn Cys(Cm) - - Ala 19 Glu 19 Gln 14 ASP 13 GIY 17 Ser GlY

- 18

- Met 3 Asn 6 LYS 3

-

64 60 49 50

5 11 13 13 13 13 13 8 5 5 7 4

-

-

303-338b 303-308 309-338 309-356 -, positions at which it was not possible to assign a residue or

Corresponding residues of the y-chain. to quantitate recovery.

z OD

I I h I PNR - R

- f 6

D3/ y 4 l Y 1 2 - 1

FIG. 7. Two-dimensional SDS-PAGE of plasmic digest of fragment Dl in presence of EGTA. Plasmic digests of normal fragment Dl in the presence of EGTA (6 pg) were electrophoresed on a 5-20% gradient gel under nonreducing conditions ( N R ) as the first dimension, followed by second dimension SDS-PAGE after reduction ( R ) using a 5-20% gradient gel. Upper panel, stained for protein; lower panel, immunoblotting. SDS-PAGE pattern of the first dimension gel is shown above the upper panel. The one-dimension nonreduced and the reduced SDS-PAGE/immunostaining patterns of the sample are, respectively, shown above and to the left of the lower panel. fs, fragment with an apparent M, of 6,000; /&?, remnant with an apparent M, of 15,000, believed to be derived from the j3-remnant (/j3); /yla y-remnant with an apparent M, of 12,000. Note that the y-remnant of fragment DG2 which migrated to the position of /y12

may be difficult to see because it was weakly, but positively, stained by the antibody. However, we ran a number of gels on this sample, and this spot was consistently and reproducibly seen in all of them.

during the early stage of digestion, leading us to investigate whether such a single-chain peptide was also present during normal fragment Dl digestion. The very small peaks (NP5 and KP5) in Fig. 6A could not be separated into two fractions by reduction and carboxymethylation, and the sequence of their first four amino acids was Phe-Phe-Thr-Ser, corresponding to residues 303-356 of the normal y-chain. However, a peptide corresponding to residues 303-356 or 303-338 of yKyotoI in which the LyP-G1yIW bond is intact was not detected. On the other hand, the abnormal y-remnant with Lys308 as the COOH-terminal residue was not detected (Fig. 5A, lanes K-Z-K-5), which, if present, should migrate between the fragment D2 ~ K ~ ~ ~ I remnant and the fragment DI y- remnant.

Thus, normal fragment Dl is cleaved to fragment DI by plasmin in the presence of EGTA, where the cleavage of the yLys302-Phe303 bond precedes that of the yLy~~~-Cys"' bond; and abnormal fragment Dl with the fragment Dl y~~~~ I remnant is cleaved to fragment DI, where there is almost simul- taneous cleavage of the y L y ~ ~ " ~ - P h e ~ " ~ and y L y ~ ~ ~ - G l y ~ ~ bonds, which are followed by y L y ~ ~ ~ ' - C y s ~ ~ ' bond cleavage.

Amino acid sequence analyses of peaks NP6 and KP6 were also performed because the size of these peaks increased with prolonged plasmic digestion (data not shown). The sequence

13854 4

3

YI I 0 r x 2 0 \

1

a

Plasmic Digest of Fibrinogen Kyoto I with yAsn3'' + Lys

r

FIG. 8. Calcium binding to fragment Dl determined by Scat- chard analysis of equilibrium dialysis data. r, moles of Ca2+ bound per mole of fragment Dl; c, free Ca'+ concentration in moles/ liter. c-"., normal control; 0- - -0, fragment Dl of fibrinogen Kyoto I.

for the first nine amino acids of these peaks was Glu-Gly- Phe-Gly-His-Leu-Ser-Pro-Thr of the y-chain), suggesting the cleavage of the bond. If this bond had been cleaved, y-remnants with much smaller M, values than the fragment Ds y-remnant would have appeared. Immunoblots of reduced plasmic digests (Fig. 5A) failed to detect such smaller M , y-remnants, but those of the nonreduced samples demonstrated the presence of fragments smaller than fragment D3, as shown in lunes 11 and 12 of Fig. 4, although fragment D3 never disappeared even after 24 h of digestion. To estimate the M, values of the y-remnants of smaller M , fragments De*, D39, and D38, the plasmic digest of normal fragment Dl in the presence of EGTA was analyzed by two-dimensional SDS-PAGE using 5 4 0 % gradient gels (Fig. 7). Fragment D62 was composed of an a-remnant with an apparent M, of 12,000, a &remnant with an apparent M, of 40,000, and a y-remnant with an apparent M, of 12,000. Fragments DS9 and D38 were composed of a remnant with an apparent M, of 12,000 (probably the a-remnant), a remnant with an apparent M, of 15,000 (probably the @-remnant), and a y-remnant with an apparent M, of 12,000. A very small fragment with an apparent M , of 6,000 (fragment f6), which was detected by first dimension SDS-PAGE, was present even after reduction.

Calcium Binding to Fragment Dl-Equilibrium dialysis was performed to examine Ca2+ binding to fragment Dl because of the accelerated plasmin cleavage of fragment Dl obtained from fibrinogen Kyoto I in the presence of EGTA. A Scat- chard analysis of the data (Fig. 8) showed that one Ca2+- binding site was present in both the normal fragment Dl and fragment Dl of fibrinogen Kyoto I, with comparable dissociation constants of 2.8 and 3.2 pM, respectively. The rate of cleavage of fibrinogen Kyoto I to fragments Dl and E by plasmin in the presence of calcium was the same as that of normal fibrinogen (data not shown).

DISCUSSION

The hereditary congenital abnormal fibrinogen designated as fibrinogen Kyoto I is characterized by normal release of

fibrinopeptides A and B, defective polymerization of the fibrin monomer, and the presence of a y-chain variant ( 7 ~ ~ ~ I) with an apparently lower M, on SDS-PAGE in the Laemmli system (18). Amino acid sequence analysis of the y-chains demonstrated a new single amino acid substitution, yAsn308 + Lys, in y ~ ~ , , ~ I. As far as we know, Asn + Lys replacement has not been reported for abnormal coagulation or fibrinolytic factors. The amino acid sequence from residues 265 to 356 of y~~~~ I,

reconstructed solely from peptides obtained in CNBr or lysyl endopeptidase digestion of the y-chains and plasmic digests of fragment Dl, was the same as that of the normal y-chain except for the above-described single amino acid substitution. A substitution of lysine for asparagine at position y308 can arise from a point mutation involving a single nucleotide change in the codon (AAT) responsible for position y308 (30): the codon is most likely altered from AAT to AAA or AAG.

It has been shown that a fibrin y-chain polymerization site resides in the COOH-terminal portion (11, 12, 15) and that the native tertiary y-chain structure is necessary for the expression of the polymerization site (14, 16). These reports are supported by the discovery of fibrinogen Kyoto I as well as other recently reported abnormal fibrinogens with a single amino acid replacement in the COOH-terminal portion of the y-chain and with impaired polymerization of fibrin monomers

y~~~~ I was detected on Laemmli gels (18) but not on Weber and Osborn gels (26) (Fig. 1). The normal fragment Dl y- remnant migrates more slowly than the @-remnant in the Laemmli system (Ref. 21 and Fig. 4), but behaves conversely in the Weber and Osborn system (9). The unique electrophoretic nature of various proteins on Laemmli gels (34-37) has also been employed for the detection of fibrinogen Tochigi with a yArgZ?S 4 Cys replacement (21) and fibrinogen Osaka I11 with a yAr'$75 -+ His replacement.' SDS-PAGE in the Laemmli system may be useful as a screening method for the investigation of abnormal fibrinogen.

Since fibrinogen Kyoto I is a heterozygous abnormality of fibrinogen, three types of fibrinogen molecules are possible in this case, as was indicated for fibrinogen New York I (38). In a previous study (17), we could not separate fibrinogen Kyoto I into two or three main populations using fibrin monomer- Sepharose chromatography, DEAE-Sephacel chromatography, immunoelectrophoresis, or differences in clottability. However, using a low concentration of thrombin or batroxo- bin, we obtained unclottable material from fibrinogen Kyoto I in which abnormal ~ K ~ ~ ~ I appeared to be the predominant form, with the clot being enriched with normal molecules (data not shown). This suggests that fibrinogen Kyoto I also contains normal molecules and abnormal homodimer molecules, although the possibility of the presence of heterodimer molecules could not be ruled out.

The yAsn308 + Lys replacement led us to investigate the plasmic digestion of fibrinogen Kyoto I. Plasmic digestion of fibrinogen Kyoto I in the presence of calcium yielded fragments Dl and E. The cleavage of the y-chains of Kyoto I to the fragment Dl y-remnant occurred at the same rate as this cleavage in normal fibrinogen, and fragment Dl contained two types of y-remnants with the same M, on Weber and Osborn gels. Residue 308 of the y-chain is in (15) or near (13) the calcium-binding site on the y-chain, but calcium binding to purified fragment Dl obtained from fibrinogen Kyoto I was normal (Fig. 8). The dissociation constant obtained (2.8-3.2 p ~ ) is the same as that (3 p ~ ) for rat fragment Dl (271, but at variance with that (8.9 p ~ ) determined by Nieuwenhuizen

N. Yoshida, Y. Morikami, M. Imaoka, S. Asakura, K. Yamazumi,

(21, 31-33).

S. Terukina, and M. Matsuda, manuscript in preparation.

Plasmic Digest of Fibrinogen Kyoto I with -yAsn3" -P Lys 13855

et al. (39) for human fragment Dl. The dissociation constant (9 p ~ ) for human fibrinogen determined by Nieuwenhuizen et al. (39) is different from those (2-3.7 pM) for human (13), rat (27), or bovine (28) fibrinogen. Calcium binding studies in abnormal fibrinogens have been previously reported only for fibrinogen Haifa, although the actual data were not shown (40). Fibrinogen Haifa with a yArg275 -+ His replacement (31) was reported to have normal calcium binding properties (40), whereas plasmic digestion of this fibrinogen in the presence of calcium resulted in the formation of fragments Dl, D3, and E (7). However, fibrinogen Osaka I11 with the same amino acid substitution as fibrinogen Haifa did not yield fragment D3 when it was digested with plasmin in the presence of calcium.'

Compared to normal fragment Dl, the fragment Dl T K y o t a I

remnant was digested more rapidly by plasmin to the fragment DP and D3 y-remnants in the presence of EGTA (Fig. 5 ) . Analyses of peptides released by plasmin (Fig. 6) demonstrated the cleavage of the yLys308-Gly309 bond in ~ K ~ ~ + , , , I ,

which occurs very slightly after cleavage of the L y ~ ~ ~ ' - - P h e ~ ~ ~ bond. However, this does not explain the accelerated cleavage of fragment Dl y K y o t o ~ . The distribution of hydropathy (41) over the sequence of ?Kyoto I and preliminary measurement of the circular dichroism spectra of purified fragment Dl a t room temperature showed little or no difference from the normal control (data not shown), as expected from the substitution of lysine for asparagine. The local secondary structure of -)'Kyoto1 as analyzed according to the method of Chou and Fasman (42) predicted the incorporation of yLys30s into a helix (data not shown). Fragment Dl of fibrinogen Kyoto I could not be separated into two populations on plasminogen- Sepharose (data not shown). Thus, the precise mechanism of the accelerated cleavage of fragment Dl YKyotoI awaits future analysis, although the substitution of lysine for asparagine at position 7308 in T K y o t o I may cause a conformational change that does not affect calcium binding or the protective effect of calcium on fragment Dl against further attack by plasmin. It may be possible that such a conformational change results, after the removal of calcium from fragment Dl, in the Lys356- Ala357 and Lys302-Phe303 bonds being relocated to a region closer to the outer surface of the molecule or in the increased affinity of the molecule for plasmin, thereby increasing the susceptibility of these bonds to attack by plasmin in the presence of EGTA. We cannot tell if the cleavage of the newly generated plasmin site, Lys308-Gly309, affects the cleavage rate at the L y ~ ~ ~ ' - - P h e ~ ~ ~ bond.

Analysis of peptides released from fragment Dl by plasmin in the presence of EGTA showed the presence of a peptide corresponding to the Phe303 to Lys356 segment of the normal y-chain (NP5 and KP5 in Fig. 6A) or to the Gly309 to Lys356 segment of the abnormal y-chain (KP3 in Fig. 6A) in which the Lys338-Cys339 bond is not cleaved. However, such a peptide could only be detected in the early stage of plasmic digestion as a small peak, and the Lys338-Cys339 bond in the corresponding main peaks (NP4, KP4, and KP2 in Fig. 6A) is cleaved, as shown by VAradi and Scheraga (15). Nevertheless, these data strongly suggest that the cleavage of the Lys338-Cys339 bond is preceded by that of the Lys302-Phe303 bond.

Analysis of peptides released from fragment Dl by plasmin in the presence of EGTA also suggested the cleavage of the

bond of the y-chain (NP6 and KP6 in Fig. 6A), which corresponds to the appearance of fragments smaller than fragment D3 (Figs. 4 and 7). The presence of such smaller fragments has already been reported (9, 10, 43-46), although these reports used fibrinogen and not purified fragment Dl. Fragment D62 with an apparent M, of 62,000 (Fig. 4, lanes 11

and 12) under nonreducing conditions is comparable to the M, = 62,000 fragment reported by Gaffney and Brasher (44) or to fragment D5 reported by Purves et al. (lo), and its y- chain remnant with an apparent M, of 12,000 (Fig. 7) is also comparable to a remnant slightly larger than the a-chain remnant (10). Fragments D39 and DS8, with apparent M, values of 39,000 and 38,000, respectively, are comparable to a fragment reported by Harverkate and Timan (9), which was smaller than fragment E. The presence of such low M , fragments cannot be interpreted without further degradation of the remnants. From the plasmic digests of denatured fragment D, Furlan et al. (46) obtained a y-chain fragment (&) with a M, of 6,000 under nonreducing conditions and glutamic acid as the NHB-terminal amino acid. Its amino acid composition showed 89% homology to residues Glu'13 to of the y- chain, with the M, of this stretch of amino acids being around 6,000. Although we did not attempt to find a peptide with yVa1267 as the NH' terminus, there may be a possibility that the bond in some of the molecules is also cleaved by plasmin, like the L y ~ ' ~ ~ - G l u ' ~ ~ bond. The origin of the very small fragment f6 with an apparent M, of 6,000 (Fig. 7) is unknown, but the fragment from G W 3 to LysZM might be comparable to fragment f6. In the same way, the y-chain remnant with an apparent M, of 12,000 (Fig. 7) might be the other half of the fragment D3 y-remnant derived from the cleavage of the bond. Fragments D39 and Dsa with an M, = 12,000 a-remnant, an M , = 15,000 @-remnant, and an M, = 12,000 y-remnant (Fig. 7) cannot be produced without further degradation of the @-remnant with an apparent M , of 40,000. Such degradation of the @-remnant has also been reported (43, 46). A reduced amount of the &remnant with a M , of 43,000 as well as the fragment D3 y-remnant after prolonged plasmic digestion was shown by Southan et al. (14), which would imply the possibility of further degradation of the p- and y-remnants. Preservation of the fragment D3 y-remnant as the main terminal product despite long-term plasmic digestion suggests that only a part of fragment Dl is susceptible to further cleavage by plasmin in the presence of EGTA. This may reflect in vitro heterogeneity of fragment Dl as discussed by Harverkate and Timan (9). The patho- physiological importance of fragments Dz and D3 or smaller fragments is unknown because these would not be generated in the blood.

In summary, fibrinogen Kyoto I with the yAsn308 -+ Lys replacement provides insights into the structure-function relationship in plasmic digestion of fibrinogen in the presence of EGTA.

Acknowledgments-We are grateful to Dr. Yoshihiko Uratani for the measurement of circular dichroism spectra, to Dr. Hajime Hirata for helpful discussions during the course of this work, and to Dr. Stephanie M. Jung for comments on the manuscript. We also wish to thank Yuko Muto for excellent technical assistance.

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