Biochimica et Biophysica Acta, 439 (1976) 47-50
© Elsevier ScientificPublishing Company, Amsterdam-- Printed in The Netherlands BBA 37392 INVESTIGATIONS ON THE PRIMARY STRUCTURE OF HUMAN PLASMINOGEN FURTHER EVIDENCE FOR SEQUENCE HOMOLOGY EGON E. RICKLI", WILLIAMLERGIERb and DIETER GILLESSENb "Institute of Biochemistry and Theodor Kocher Institute, University of Berne, Postfach 99, CH-"000 Berne 9, and bDiagnostie Research Department F. Hoffmann-La Roche & Co. Ltd. CH-4002 Basel (Switzerland)
(Received January 13th, 1976)
SUMMARY NHz-Terminal sequences were determined by the automated Edman method in four fragments which were isolated from a mixture of fragments obtained by CNBr cleavage of human plasminogen. One of the fragments whose sequence was determined over the first 31 residues shows sequence homologies with the fragment that forms the linkage between the plasmin chains and also with the non-thrombin part of prothrombin.
Human plasminogen contains a single polypeptide chain and has a molecular weight of 92 000 [1]. Glutamic acid [2, 3] and asparagine [4] are in the NH2- and COOH-terminal positions, respectively. The activation of the single chain proenzyme with urokinase proceeds in a two-step mechanism and yields the active two-chain plasmin molecule [5, 6, 7]. It has been shown by Wiman [8] that CNBr cleavage of human plasminogen which contains 8 methionine residues [9] results in the liberation of the NH2-terminal CNBr fragment, the adjacent undecapeptide and of a tripeptide comprising the COOH-terminus [10]. The remaining fragments which are linked to each other by disulfide bridges form a high molecular weight fraction which is easily separated from the small fragments by gel filtration. Recently, Wiman and Wall6n [I 1] were the first to publish the sequence of a fragment (Ca) containing the Arg-Val bond (positions 98-99) which is cleaved in the second step of the plasminogen activation. The present work describes some of our results on the primary structure of human plasminogen. Plasminogen was isolated from human plasma by affinity chromatography with lysine-Biogel [3]. The protein was dialyzed against 10-3 M HC1 and lyophilized and cleaved with CNBr. In a typical experiment 200 mg protein, dissolved in 20 ml 70 ~ formic acid, were treated with 200 mg CNBr for 24 h at 25 °C in the dark under nitrogen. The fragmented protein, recovered from the reaction mixture by lyophilization, was then chromatographed on a column (2.5 × 90 cm) of Sephadex G-75 in 1 M acetic acid. In later experiments the chromatograms were also developed with
48 equal success with 0.13 M formic acid. This resulted in the separation of the high molecular weight fraction of disulfide bridged fragments from the smaller, noncrosslinked peptides. The high molecular weight fraction was reduced in 0.13 M Tris/ 6 MLguanidinium chloride/1 mg per ml EDTA (pH 7.6) with a 100-fold molar excess of 2-mercaptoethanol (vs. protein) for 15 h at 25 °C under nitrogen. The sulfhydryl groups were blocked with a 1.2-fold molar excess of iodoacetamide (vs. total -SH) at a pH of 8.9-9.0 for 30 min. The reaction mixture was desalted on a Sephadex G-25 column in 1 M acetic or 0.13 M formic acid. The recovered peptide material was then separated by gel filtration on a column (7 x 90 cm) of Sephadex G-75 in 1 M acetic or 0.13 M formic acid. The fragment mixture was resolved into 5 different fractions (I, II, III, IV, V) which were analyzed by sodium dodecyl sulfate gel electrophoresis [12] for purity and for the estimation of the molecular weight of individual components. ! cEO'61
50.4
I[
IE
~a2 0 ------,,- • 0
1400 1600 1800 2000
2400 2800 3200 Elution volume (ml)
Fig. 1. Elution diagram of reduced and carboxamidomethylated CNBr fragments. 230 mg of a fragment mixture were applied to a 7 x 90 cm Sephadex G-75 column. Elution was performed with 0.13 M formic acid at a constant flow rate of 73 ml'h -1. Fraction I gave at least five different bands in the molecular weight range between 20 000 and 40 000. Fraction II, after rechromatography on Sephadex G-75 was obtained in an electrophoretically pure form and it had a molecular weight of 12 500. This value was later confirmed by gel filtration parameters. The elution volume in a Sephadex G-75 column of fraction II was identical with that of horse heart cytochrome c. Fractions III and IV were only partially resolved and had to be purified by ion exchange chromatography. The molecular weight of these two fractions was between 9000 and 10 000. Fraction V was a low molecular weight fragment which could not be stained after sodium dodecyl sulfate electrophoresis. Fractions III and IV were separated from each other by ion exchange chromatography on DEAESephadex A-25 with 0.1 M triethylamine-acetic acid, pH 7.9, as the starting buffer. The chromatogram was developed with a linear gradient to 0.8 M in triethylamine/ acetic acid, pH 7.9. Fragment V was purified in a similar way on CM-Sephadex C-25 using a linear gradient of ammonium acetate from 0.1 to 1.0 M at pH 8.1. The sequence analyses were performed by automated Edman-degradation with the use of a Beckman sequencer (model 890). The phenylthiohydantoin amino acids were identified by means of thin-layer and gas-liquid chromatography according
49 TABLEI NH~-TERMINAL Fragment
II :
SEQUENCES
OF CNBr-FRAGMENTS
Phe - Gly -Asn
II-V OF HUMAN
PLASMINOGEN
- Gly - Lys - Gly - Tyr -
Arg - Gly - Lys - Arg - Ala - Thr - Thr Val - Thr - Gly - Thr - Pro - Cys - Gln Asp - Trp - Ala - Ala - Gin - Glu - Pro His - Arg -
III:
Fragment
Ser
- Lys - Thr - Lys -Asn
- Gly - Ile
-
Thr - Cys - Gin - Lys - Ala - Pro Fragment
IV:
Ash - Tyr - Cys - Arg -Asn
- Pro - Asp -
Ala - Asp - Lys - Gly - Pro - Trp - Cys Phe - Thr - Thr - Asp - Pro - Val - Val Arg - Trp - Glu - Tyr - Cys -Asn
- Leu -
Lys - Lys - Cys Fragment
V:
Arg - Asp - Val - Val - Leu - Phe - Glu Lys - Lys - Val -
TABLE
II
SEQUENCE AND
HOMOLOGIES
PROTHROMBIN
Number
BETWEEN
IV
AND
IV
Ca O F
PLASM1NOGEN
PART)
of necessary base-changes indicating degree of homology.
l e t t e r s y m b o l s ( E u r . J. B i o c h e m . Fragment
FRAGMENTS
(NON-THROMBIN
Amino acids are expressed as one-
5, 1 5 1 - 1 5 3 ( 1 9 6 8 ) ) .
1-31
(Plasminogen)
Fragment
C a [11]
44-78
L E K
Y C R N
P D G
V G
Y
N
R K L Y D
D V
P
(Plasminogen)
Minimum
No. of base
Iololololololo111ol21 Iolololo111olo111ol2121112111olo111112111ol
No. of base
10101010111olol 21o12111o111ol21ol11112121o'1
changes
Minimum changes
Prothrombin
Fragment
IV
[14] 1 2 4 - 1 4 4
11-31
F
VV
W
KK
(Plasminogen) Prothrombin
Minimum changes
[14] 2 2 9 - 2 4 9
No. of base
~~
o
rlzlYicl~lLl~ YW
IIIIIIIIIIIIIIIIIIIIII O i O O 1 2 1 0 1 2 1 2 2 O O O O O 1 2 0
50 to the standard procedures and the results obtained are shown in Table I. On comparing these results with the NH2-terminal sequences of CNBr fragments of human plasminogen as reported by Wiman and Wall6n [10], it is obvious that the fragments lI, III, IV and V correspond in this order to their fragments Ca, Db, Da and Ea resp. The D and E fragments are located in the heavy chain and the C fragment forms the overlap between the heavy and light chain of plasmin [10]. We also sequenced the NH2-terminal CNBr fragment and identified the adjacent undecapeptide (to be published elsewhere). Wiman and Wall6n [11] have already shown internal sequence homologies within the plasmin heavy chain which can be attributed to gene duplication during the evolution of the plasminogen molecule. In addition homologous parts within the plasmin heavy chain and the non-thrombin part of prothrombin were also observed [11, 13]. Our studies on the sequence of fragment IV confirm and extend these findings (Tabel II). Furthermore these data indicate that the homology of this plasminogen fragment with the non-thrombin part of prothrombin is not restricted to the NH2terminal end but also exists in other regions suggesting a close genetic relationship between these two proteins. ACKNOWLEDGEMENTS We are indebted to the Central Laboratory of the Blood Transfusion Service of the Swiss Red Cross for the generous supply of plasma. The excellent technical assistance of Miss E. Burri and Miss M. Pedrocca is gratefully acknowledged. We are grateful to Mr. R. Rueher, Physical Research Dept., F. Hoffmann-La Roche & Co. Ltd. for performing the identification of the phenylthiohydantoin amino acids by gas-liquid chromatography. This investigation was supported by Grants Nos. 3.525.71 and 3.272.74 of the Swiss National Science Foundation, granted to E.E.R. REFERENCES 1 Sj6holm, I., Wiman, B. and Wall6n, P. (1973) Eur. J. Biochem. 39, 471--479 2 Wall6n, P. and Wiman, B. (1970) Biochim. Biophys. Acta 221, 20-30 3 Rickli, E. E. and Cuendet, P. A. (1972) Biochim. Biophys. Acta 250, 447-451 4 Robbins, K. C., Summaria, L., Hsieh, B. and Shah, R. J. (1967) J. Biol. Chem. 242, 2333-2342 5 Rickli, E. E. and Otavsky, W. I. (1973) Biochim. Biophys. Acta 295, 381-384 6 Wiman, B. and Wall6n, P. (1973) Eur. J. Biochem. 36, 25-31 7 Walther, P. J., Steinmann, H. M., Hill, R. L. and McKee, P. A. (1974) J. Biol. Chem. 249, 1173118l 8 Wiman, B. (1972) Thromb. Res. 1, 89-94 9 Wall6n, P. and Wiman, B. (1972) Biochim. Biophys. Acta 257, 122-134 10 Wiman, B. and Wail6n, P. (9175)'Eur. J. Biochem. 57, 387-394 I1 Wiman, B. and Wall6n, P. (1975)*Eur. J Biochem. 58, 539-547 12 Weber, K. and Osborn, M. (1969) J. Biol. Chem. 244, 4406-4412 13 Sottrup-Jensen, L.,rZaidel, M., Claeys, H , Petersen ,T. and Magnusson, S. (1975) Proc. Natl. Acad. Sci. U.S. 72, 2577-2581 14 Magnusson, S., Petersen, T., Sottrup-Jensen, L. and Claeys, H. (1975) Cold Spring Harbor Syrup. Quant. Biol., in the press.
© Elsevier ScientificPublishing Company, Amsterdam-- Printed in The Netherlands BBA 37392 INVESTIGATIONS ON THE PRIMARY STRUCTURE OF HUMAN PLASMINOGEN FURTHER EVIDENCE FOR SEQUENCE HOMOLOGY EGON E. RICKLI", WILLIAMLERGIERb and DIETER GILLESSENb "Institute of Biochemistry and Theodor Kocher Institute, University of Berne, Postfach 99, CH-"000 Berne 9, and bDiagnostie Research Department F. Hoffmann-La Roche & Co. Ltd. CH-4002 Basel (Switzerland)
(Received January 13th, 1976)
SUMMARY NHz-Terminal sequences were determined by the automated Edman method in four fragments which were isolated from a mixture of fragments obtained by CNBr cleavage of human plasminogen. One of the fragments whose sequence was determined over the first 31 residues shows sequence homologies with the fragment that forms the linkage between the plasmin chains and also with the non-thrombin part of prothrombin.
Human plasminogen contains a single polypeptide chain and has a molecular weight of 92 000 [1]. Glutamic acid [2, 3] and asparagine [4] are in the NH2- and COOH-terminal positions, respectively. The activation of the single chain proenzyme with urokinase proceeds in a two-step mechanism and yields the active two-chain plasmin molecule [5, 6, 7]. It has been shown by Wiman [8] that CNBr cleavage of human plasminogen which contains 8 methionine residues [9] results in the liberation of the NH2-terminal CNBr fragment, the adjacent undecapeptide and of a tripeptide comprising the COOH-terminus [10]. The remaining fragments which are linked to each other by disulfide bridges form a high molecular weight fraction which is easily separated from the small fragments by gel filtration. Recently, Wiman and Wall6n [I 1] were the first to publish the sequence of a fragment (Ca) containing the Arg-Val bond (positions 98-99) which is cleaved in the second step of the plasminogen activation. The present work describes some of our results on the primary structure of human plasminogen. Plasminogen was isolated from human plasma by affinity chromatography with lysine-Biogel [3]. The protein was dialyzed against 10-3 M HC1 and lyophilized and cleaved with CNBr. In a typical experiment 200 mg protein, dissolved in 20 ml 70 ~ formic acid, were treated with 200 mg CNBr for 24 h at 25 °C in the dark under nitrogen. The fragmented protein, recovered from the reaction mixture by lyophilization, was then chromatographed on a column (2.5 × 90 cm) of Sephadex G-75 in 1 M acetic acid. In later experiments the chromatograms were also developed with
48 equal success with 0.13 M formic acid. This resulted in the separation of the high molecular weight fraction of disulfide bridged fragments from the smaller, noncrosslinked peptides. The high molecular weight fraction was reduced in 0.13 M Tris/ 6 MLguanidinium chloride/1 mg per ml EDTA (pH 7.6) with a 100-fold molar excess of 2-mercaptoethanol (vs. protein) for 15 h at 25 °C under nitrogen. The sulfhydryl groups were blocked with a 1.2-fold molar excess of iodoacetamide (vs. total -SH) at a pH of 8.9-9.0 for 30 min. The reaction mixture was desalted on a Sephadex G-25 column in 1 M acetic or 0.13 M formic acid. The recovered peptide material was then separated by gel filtration on a column (7 x 90 cm) of Sephadex G-75 in 1 M acetic or 0.13 M formic acid. The fragment mixture was resolved into 5 different fractions (I, II, III, IV, V) which were analyzed by sodium dodecyl sulfate gel electrophoresis [12] for purity and for the estimation of the molecular weight of individual components. ! cEO'61
50.4
I[
IE
~a2 0 ------,,- • 0
1400 1600 1800 2000
2400 2800 3200 Elution volume (ml)
Fig. 1. Elution diagram of reduced and carboxamidomethylated CNBr fragments. 230 mg of a fragment mixture were applied to a 7 x 90 cm Sephadex G-75 column. Elution was performed with 0.13 M formic acid at a constant flow rate of 73 ml'h -1. Fraction I gave at least five different bands in the molecular weight range between 20 000 and 40 000. Fraction II, after rechromatography on Sephadex G-75 was obtained in an electrophoretically pure form and it had a molecular weight of 12 500. This value was later confirmed by gel filtration parameters. The elution volume in a Sephadex G-75 column of fraction II was identical with that of horse heart cytochrome c. Fractions III and IV were only partially resolved and had to be purified by ion exchange chromatography. The molecular weight of these two fractions was between 9000 and 10 000. Fraction V was a low molecular weight fragment which could not be stained after sodium dodecyl sulfate electrophoresis. Fractions III and IV were separated from each other by ion exchange chromatography on DEAESephadex A-25 with 0.1 M triethylamine-acetic acid, pH 7.9, as the starting buffer. The chromatogram was developed with a linear gradient to 0.8 M in triethylamine/ acetic acid, pH 7.9. Fragment V was purified in a similar way on CM-Sephadex C-25 using a linear gradient of ammonium acetate from 0.1 to 1.0 M at pH 8.1. The sequence analyses were performed by automated Edman-degradation with the use of a Beckman sequencer (model 890). The phenylthiohydantoin amino acids were identified by means of thin-layer and gas-liquid chromatography according
49 TABLEI NH~-TERMINAL Fragment
II :
SEQUENCES
OF CNBr-FRAGMENTS
Phe - Gly -Asn
II-V OF HUMAN
PLASMINOGEN
- Gly - Lys - Gly - Tyr -
Arg - Gly - Lys - Arg - Ala - Thr - Thr Val - Thr - Gly - Thr - Pro - Cys - Gln Asp - Trp - Ala - Ala - Gin - Glu - Pro His - Arg -
III:
Fragment
Ser
- Lys - Thr - Lys -Asn
- Gly - Ile
-
Thr - Cys - Gin - Lys - Ala - Pro Fragment
IV:
Ash - Tyr - Cys - Arg -Asn
- Pro - Asp -
Ala - Asp - Lys - Gly - Pro - Trp - Cys Phe - Thr - Thr - Asp - Pro - Val - Val Arg - Trp - Glu - Tyr - Cys -Asn
- Leu -
Lys - Lys - Cys Fragment
V:
Arg - Asp - Val - Val - Leu - Phe - Glu Lys - Lys - Val -
TABLE
II
SEQUENCE AND
HOMOLOGIES
PROTHROMBIN
Number
BETWEEN
IV
AND
IV
Ca O F
PLASM1NOGEN
PART)
of necessary base-changes indicating degree of homology.
l e t t e r s y m b o l s ( E u r . J. B i o c h e m . Fragment
FRAGMENTS
(NON-THROMBIN
Amino acids are expressed as one-
5, 1 5 1 - 1 5 3 ( 1 9 6 8 ) ) .
1-31
(Plasminogen)
Fragment
C a [11]
44-78
L E K
Y C R N
P D G
V G
Y
N
R K L Y D
D V
P
(Plasminogen)
Minimum
No. of base
Iololololololo111ol21 Iolololo111olo111ol2121112111olo111112111ol
No. of base
10101010111olol 21o12111o111ol21ol11112121o'1
changes
Minimum changes
Prothrombin
Fragment
IV
[14] 1 2 4 - 1 4 4
11-31
F
VV
W
KK
(Plasminogen) Prothrombin
Minimum changes
[14] 2 2 9 - 2 4 9
No. of base
~~
o
rlzlYicl~lLl~ YW
IIIIIIIIIIIIIIIIIIIIII O i O O 1 2 1 0 1 2 1 2 2 O O O O O 1 2 0
50 to the standard procedures and the results obtained are shown in Table I. On comparing these results with the NH2-terminal sequences of CNBr fragments of human plasminogen as reported by Wiman and Wall6n [10], it is obvious that the fragments lI, III, IV and V correspond in this order to their fragments Ca, Db, Da and Ea resp. The D and E fragments are located in the heavy chain and the C fragment forms the overlap between the heavy and light chain of plasmin [10]. We also sequenced the NH2-terminal CNBr fragment and identified the adjacent undecapeptide (to be published elsewhere). Wiman and Wall6n [11] have already shown internal sequence homologies within the plasmin heavy chain which can be attributed to gene duplication during the evolution of the plasminogen molecule. In addition homologous parts within the plasmin heavy chain and the non-thrombin part of prothrombin were also observed [11, 13]. Our studies on the sequence of fragment IV confirm and extend these findings (Tabel II). Furthermore these data indicate that the homology of this plasminogen fragment with the non-thrombin part of prothrombin is not restricted to the NH2terminal end but also exists in other regions suggesting a close genetic relationship between these two proteins. ACKNOWLEDGEMENTS We are indebted to the Central Laboratory of the Blood Transfusion Service of the Swiss Red Cross for the generous supply of plasma. The excellent technical assistance of Miss E. Burri and Miss M. Pedrocca is gratefully acknowledged. We are grateful to Mr. R. Rueher, Physical Research Dept., F. Hoffmann-La Roche & Co. Ltd. for performing the identification of the phenylthiohydantoin amino acids by gas-liquid chromatography. This investigation was supported by Grants Nos. 3.525.71 and 3.272.74 of the Swiss National Science Foundation, granted to E.E.R. REFERENCES 1 Sj6holm, I., Wiman, B. and Wall6n, P. (1973) Eur. J. Biochem. 39, 471--479 2 Wall6n, P. and Wiman, B. (1970) Biochim. Biophys. Acta 221, 20-30 3 Rickli, E. E. and Cuendet, P. A. (1972) Biochim. Biophys. Acta 250, 447-451 4 Robbins, K. C., Summaria, L., Hsieh, B. and Shah, R. J. (1967) J. Biol. Chem. 242, 2333-2342 5 Rickli, E. E. and Otavsky, W. I. (1973) Biochim. Biophys. Acta 295, 381-384 6 Wiman, B. and Wall6n, P. (1973) Eur. J. Biochem. 36, 25-31 7 Walther, P. J., Steinmann, H. M., Hill, R. L. and McKee, P. A. (1974) J. Biol. Chem. 249, 1173118l 8 Wiman, B. (1972) Thromb. Res. 1, 89-94 9 Wall6n, P. and Wiman, B. (1972) Biochim. Biophys. Acta 257, 122-134 10 Wiman, B. and Wail6n, P. (9175)'Eur. J. Biochem. 57, 387-394 I1 Wiman, B. and Wall6n, P. (1975)*Eur. J Biochem. 58, 539-547 12 Weber, K. and Osborn, M. (1969) J. Biol. Chem. 244, 4406-4412 13 Sottrup-Jensen, L.,rZaidel, M., Claeys, H , Petersen ,T. and Magnusson, S. (1975) Proc. Natl. Acad. Sci. U.S. 72, 2577-2581 14 Magnusson, S., Petersen, T., Sottrup-Jensen, L. and Claeys, H. (1975) Cold Spring Harbor Syrup. Quant. Biol., in the press.
