CN1117154C - Urokinase zymogen mutant - Google Patents
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- CN1117154C CN1117154C CN 00109829 CN00109829A CN1117154C CN 1117154 C CN1117154 C CN 1117154C CN 00109829 CN00109829 CN 00109829 CN 00109829 A CN00109829 A CN 00109829A CN 1117154 C CN1117154 C CN 1117154C
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Abstract
The present invention relates to a prourokinase mutant which has thrombolytic activity. The prourokinase mutant is composed of the amino acid sequence of natural prourokinase with the site-directed mutagenesis of proline in No. 309 site. The site-directed mutagenesis causes the prourokinase mutant to have intrinsic activity (namely single chain activity) 2.5 to 20 times lower than that of the natural prourokinase including lowered fibrinogen dissolving activity and lowered non-specific fibrin dissolving zymogen activation. Compared with the natural prourokinase, the fibrin dissolving zymogen activation of the present invention can be promoted by both of the E segment and the D segment of fibrin degradation.
Description
The novel uPA that the present invention relates to use in thrombolytic treatment is particularly had a urokinase zymogen mutant of thrombolysis activity.
UPA is that a kind of single chain serine protease is former, through the activation of fibrinoclase (Plasmin) and form double chain urokinase (UK).It is fibrinoclase that uPA and urokinase all can make plasminogen (Plasminogen) activate, fibrinoclase then can be with the degraded of the scleroproein in the thrombus and other plasma proteins, so uPA and urokinase can be used for the treatment of thromboembolism.
Compare with other serine stretch protein proenzyme, a distinguishing feature of uPA is that it has very high intrinsic activity (Intrinsic Activity), shows as to the hydrolysis ability of small molecules substrate and to the activation of plasminogen.Although above-mentioned activity is low about 250 times than urokinase, its intrinsic activity is still high by 10 than other serine stretch protein proenzyme (as trypsinogen or chymotrypsinogen)
4.0～4.3Doubly.There is long delay in uPA to the activation of plasminogen, when having the E fragment of fibrin degradation in the reactive system, above-mentioned activation is significantly strengthened, and scleroproein and degradation fragment D thereof have no significant effect above-mentioned reaction.
The intrinsic activity that uPA is higher relatively makes it can produce some bad side effects when clinical use.The same with urokinase, uPA also can cause nonspecific plasminogen activation, thereby causes being partly dissolved of fibrinogenic degraded and thrombocyte and vessel wall, produces systematic fibrinolytic state, causes complication such as hemorrhage or apoplexy.When low dosage used, uPA had more selectivity than urokinase, and this is to free or all very strong with the activation of scleroproein bonded plasminogen because of urokinase.And the intrinsic activity of uPA only is 0.2%～0.5% of a urokinase, and it is then very weak to the activation of free plasminogen to combining fibrinous plasminogen priority activation when lower concentration.But, when using high dosage in order to ensure thromboclastic curative effect, the specificity when uPA has just been lost its low dosage.
Purpose of the present invention is exactly in order to address the above problem, and proposes a kind of urokinase zymogen mutant with good thrombolytic effect.
We show the thrombolytic Study on mechanism result of uPA, uPA or urokinase are two committed steps of thrombolysis process to the activation near the uPA thrombus of the activation of the plasminogen on thrombus surface and fibrinoclase.Therefore, compare with the wild-type uPA, the pro urokinase mutant with good efficacy should possess: 1. lower intrinsic activity, to guarantee the security of clinical use; 2. to having higher activation with thrombus bonded plasminogen, and very weak to the activation capacity of free plasminogen, this can realize by the Substratspezifitaet that changes uPA; 3. this variant should be suitable with the wild-type uPA by fibrinoclase activated speed, and the ability of its corresponding double chain urokinase activation fiber protein dissolution proenzyme should be similar to the wild-type urokinase
The present invention is based on following discovery: Lys in the natural urokinase original molecule
300With Asp
355Between electrostatic interaction (sat linkage) be the prerequisite of keeping its high intrinsic activity, therefore, the amino acid mutation of the above-mentioned electrostatic interaction of any weakening all may make the intrinsic activity of pro urokinase mutant descend.Work as Pro
309After being sported other amino acid, can change Lys
300The locus, thereby influence Lys
300And Asp
355Between electrostatic interaction, and the intrinsic activity of mutant is reduced.In addition, this site mutation not only can influence the activity of mutant double chain urokinase, and can change the Substratspezifitaet of (strand) mutant, its activation to wandering fibre protein dissolution proenzyme is descended, and the activation capability with scleroproein or its degradation fragment bonded plasminogen is risen.
Compare with natural urokinase is former, some urokinase zymogen mutants in this site are good thrombolysis medicaments, because they are subjected to the promoted degree of scleroproein former above natural urokinase to the activation of plasminogen, thereby, these urokinase zymogen mutants pair show than the former stronger specificity of natural urokinase with the activation of scleroproein bonded plasminogen, in addition because its low intrinsic activity, so when using these pro urokinase mutants to the patient, higher, the more productive dosage of the former use of comparable natural urokinase.
Therefore the invention is characterized in a kind of active urokinase zymogen mutant of thrombus that has, it has the former aminoacid sequence of natural urokinase, but its proline(Pro) of the 309th is suddenlyd change, this sudden change can make its intrinsic activity former lower more than 2.5 times than natural urokinase, and its activation to plasminogen can be promoted by scleroproein and degradation fragment thereof.
When using to the patient, this sudden change can induce than former lower fibrinogenolysis of natural urokinase and non-specific plasminogen activation it, and makes it show higher fibrin-specific.
Term used herein " natural " is meant the protein of a kind of natural existence or wild-type form, perhaps refers to a kind of natural existence in the naturally occurring protein or the amino acid of wild-type form.A kind of " newly " amino acid is meant the common indefinite amino acid that is positioned on the given site in the natural protein, specifically, and at the 309th other amino acid except that proline(Pro) of uPA.
Feature of the present invention also is a kind of dna molecular, for example, and any mutant uPA that a kind of recombinant DNA molecules, this molecule encoding are described herein.The present invention also comprises the dna molecular cell transformed that a kind of usefulness is above-mentioned, for example a kind of Mammals, bacterium or yeast cell.Feature of the present invention also is the method for the mutant uPA that a kind of preparation is described herein, this method comprises that the dna molecular with a kind of mutant uPA of coding transforms a kind of cell, cultivate this cell, and express this mutant, and separate this mutant (albumen).
Embodiment: the preparation of urokinase zymogen mutant
A kind of method that is applicable to the urokinase zymogen mutant that preparation is described herein is the oligonucleotide rite-directed mutagenesis, this method can make the former nucleotides sequence of natural urokinase be listed in specific site and change, and can cause its amino acid sequence coded to change into other amino acid in corresponding site.Coding natural urokinase former gene order identified fully, and can (Primm, Milano Italy) locate to obtain from for example Dr.David Dichek (NIH) and Dr.Paolo Sarmientos.Preserving number at ATCC is DNA 57329 or bacterium/phage 57328.Can in NIH Computer Database Protein Identity Resource, retrieve its nucleic acid and aminoacid sequence with the UKHU title.The former aminoacid sequence of natural urokinase is seen Fig. 1.
The technology that is used to carry out the Nucleotide rite-directed mutagenesis at present is very ripe, this paper with the sudden change test kit of Stratagene (the reagent catalog number (Cat.No.):
#200518) be example, illustrate that the uPA gene is corresponding to Pro
309The rite-directed mutagenesis at codon place is seen Fig. 2.
With comprising uPA (cDNA) gene and having the plasmid of modifying that methylates is template, with the oligonucleotide that contains mutant nucleotide sequence is primer, after sex change and anneal, make the pairing of Oligonucleolide primers and plasmid template, and with have pfu archaeal dna polymerase that height duplicates fidelity of reproduction make Oligonucleolide primers extend with synthetic contain the uPA mutator gene and with the DNA of plasmid template complementary pairing.This reaction can be finished on the PCR instrument, and behind about about the 15 times sex change-annealing-extension of process, products therefrom enough is used to the competence bacterium of transduceing.
Get above-mentioned synthetic product 2 μ l, add the DpnI enzyme, this enzyme can optionally be degraded and be contained methylated DNA chain, and the plasmid DNA that contains wild-type uPA gene as template is degraded, and lose the ability of transduction competence bacterium, then methylate and can not be degraded at the DNA that external synthetic contains the uPA rite-directed mutagenesis with the Pfu archaeal dna polymerase because of not existing, but and complementary pairing for containing the double-stranded DNA of jagged (nick).This double-stranded DNA can be transduceed to competent escherichia coli cell effectively.Select positive colony and the uPA gene order in its plasmid DNA is identified, thereby obtain to contain the uPA gene of rite-directed mutagenesis.
We are with Pro
309Random mutation is other 19 seed amino acid, with the intrinsic activity of each mutant of comparison and to the plasminogen activated spy property led.We design following Oligonucleolide primers and are used for uPA Pro
309The random mutation of codon: primer 1.ACAG TCA TTT TCA GCT GCT C
NN NAT AGA GAT AGT
CGG TAG AAT TC primer 2 .GAA TTC TAC CGA CTA TCT CTA T
NN NGA GCA GCT
GAA AAT GAC TGT wherein N represents A, T, C, G
The expression of urokinase zymogen mutant DNA:
According to the host cell type that is used for protein expression, the uPA gene clone that will contain sudden change is in suitable expression vector, to contain uPA expression carrier transduction (for example chemistry transduction or electroporation transduction etc.) then to host cell (for example bacterium, yeast or mammalian cell), so that express urokinase zymogen mutant.If adopt secreted expression carrier, urokinase zymogen mutant albumen can reclaim from substratum, if do not adopt secreted expression carrier, uPA albumen then can reclaim from host cell.If host cell is an eukaryotic cell, then can directly carry out separation and purification to it.If host cell is a prokaryotic cell prokaryocyte, then must carry out activation treatment to its expression product (existing) earlier with the inclusion body form.Urokinase zymogen mutant albumen after separation and purification (purification process comprises ion-exchange, affinity chromatography and gel-filtration etc.) can identify its biochemical characteristic.
The mutation analysis of uPA:
The recombinaton urokinase of producing with intestinal bacteria was reference originally, and research is the biochemical property of urokinase zymogen mutant relatively.Detect index and comprise that mainly intrinsic activity, natural urokinase or the mutant urokinase of urokinase zymogen mutant are to the former activation of (Glu-type) plasmin, fibrinoclase is to the activation of urokinase zymogen mutant, scleroproein and degraded product E fragment thereof and D fragment activate the promoter action of (Glu-type) plasminogen to uPA or its mutant, and in the blood plasma environment to fibrinogenic residual quantity in the dissolution rate of fibrinogen grumeleuse and the blood plasma.
Intrinsic activity detects:
According to mutant uPA and (corresponding) urokinase thereof hydrolytic action, the intrinsic activity of coming each urokinase zymogen mutant of comparison to the chromogenic substrate S2444 of chemosynthesis.At 25 ℃ and determination of activity damping fluid (0.05M Tris-HCl, 0.1M NaCl, 0.1%BSA and 0.01% tween 80, pH7.4) in, uPA or urokinase zymogen mutant and finite concentration scope (0.03 with 2.5 μ M, 0.06,0.12,0.18,0.24,0.3,0.6,1.2,1.8 and 2.4mM) S2444 add in the microtiter plate (96 orifice plate), on enzyme mark determinator, do to calculate the reaction power mathematic(al) constant according to what measure in 410nm (410/490nm) wavelength place's optical density(OD) (Δ OD) value increase of (t) in time (Δ OD/t) with reference to wavelength with 490nm.The result shows, urokinase zymogen mutant Pro
309→ Ala, Pro
309→ Gly, Pro
309→ Leu, Pro
309→ Ser, Pro
309→ Thr, Pro
309The intrinsic activity of → Val (to S2444) be respectively recombinaton urokinase former 10.5 ± 2.0%, 10.8 ± 2.4%, 8.5 ± 1.8%, 9.6 ± 2.2%, 11.2 ± 2.5%, 18.4 ± 3.6%.
Urokinase and urokinase mutant detect the activity of S2444: in the determination of activity damping fluid, the uPA of 1 μ M or urokinase zymogen mutant mixed with the fibrinoclase of 20nM put 37 ℃ of reactions 1 hour, and confirm that according to reduced form SDS-gel electrophoresis uPA or urokinase zymogen mutant are converted into corresponding urokinase fully, get urokinase or the mutant urokinase of 10nM and measure its activity as stated above S2444, the result shows: urokinase mutant, Pro
309→ Ala, Pro
309→ Gly, Pro
309→ Leu, Pro
309→ Ser, Pro
309→ Thr, Pro
309→ Val is respectively 75 ± 2.8%, 38 ± 2.4%, 24 ± 1.5%, 89 ± 3.6%, 92 ± 4.8%, 68 ± 2.7% of recombinaton urokinase to the activity of S2444.
Urokinase mutant is to the former activation of Glu-type plasmin: in 25 ℃ and determination of activity damping fluid, urokinase or urokinase mutant and finite concentration scope (1.0 with 0.2nM, 1.5,2.5,3.5,4.5,5.5,7.5 Glu-fiber type protein dissolution proenzyme and 10 μ M) and the S2251 of 1.5mM add in the microtiter plate, do with reference to wavelength with 490nm with enzyme mark determinator, according to measure at 410nm (410/490nm) wavelength place's optical density(OD) (Δ OD) value square (t in time
2) increase (Δ OD/t
2) and calculate the reaction power mathematic(al) constant.The result shows, urokinase mutant Pro
309→ Ala, Pro
309→ Gly, Pro
309→ Leu, Pro
309→ Ser, Pro
309→ Thr, Pro
309→ Val is a recombinaton urokinase to the activity of plasminogen: 72 ± 4.2%, 36 ± 2.5%, 18 ± 1.5%, 96 ± 6.8%, 75 ± 5.4%, 23 ± 1.6%.
Fibrinoclase (Plasmin) is to the activation of urokinase zymogen mutant: in 25 ℃ and determination of activity damping fluid, fibrinoclase and finite concentration scope (0 with 0.1nM, 0.1,0.2,0.4,0.6,0.8,1.0,1.2,1.4,2.5,3.5 uPA and 5.0 μ M) or the S2444 of urokinase zymogen mutant and 1.2mM add in the microtiter plate, do with reference to wavelength with 490nm with enzyme mark determinator, according to 410nm wavelength place optical density(OD) (Δ OD) value of measuring square (t in time
2) increase (Δ OD/t
2) and calculate the reaction power mathematic(al) constant, the result shows: fibrinoclase is close to the activation capability of wild-type uPA and urokinase zymogen mutant, and fibrinoclase activates urokinase zymogen mutant Pro
309→ Ala, Pro
309→ Gly, Pro
309→ Leu, Pro
309→ Ser, Pro
309→ Thr, Pro
309The speed of → Val is respectively 106 ± 5%, 87 ± 6%, 84 ± 8%, 104 ± 3%, 109 ± 8%, 85 ± 7% of the former speed of its activation recombinaton urokinase.
Scleroproein and degraded product E fragment thereof and D fragment are to the promoter action of urokinase zymogen mutant activation fiber protein dissolution proenzyme:
In 25 ℃ and determination of activity damping fluid, the S2251 of the Glu-fiber type protein dissolution proenzyme of the uPA of 0.5nM or urokinase zymogen mutant and 2.0 μ M and 1.5nM mixed place microtiter plate, do not add or add soluble fibrin (0.2 μ M) with enzyme mark determinator mensuration, when fibrin degradation D fragment (0.4 μ M) and fibrin degradation E fragment (5 μ M), reaction mixture is at 410nm wavelength place optical density(OD) (Δ OD) changing value of (t) (Δ OD/t) in time, and is worth (Δ OD/t) assessment urokinase zymogen mutant is subjected to scleroproein and degradation fragment thereof to the plasminogen activation influence with this.The result shows: fibrin degradation product (FDP) E fragment has similar promoter action to reorganization uPA and urokinase zymogen mutant activation fiber protein dissolution proenzyme.Scleroproein and degraded product D fragment thereof are to the not influence of reorganization uPA activation fiber protein dissolution proenzyme, and scleroproein (0.2 μ M) is to urokinase zymogen mutant Pro
309→ Ala, Pro
309→ Gly, Pro
309→ Leu, Pro
309→ Ser, Pro
309→ Thr, Pro
309The promoter action of → Val activation fiber protein dissolution proenzyme is respectively: 3.8 times, and 8.4 times, 9.2 times, 4.4 times, 4.7 times, 5.4 times.The promoter action of fibrin degradation product (FDP) D fragment (0.4 μ M) is respectively: 5.4 times, and 10.6 times, 12.4 times, 5.9 times, 6.5 times, 6.8 times.
The plasma fibrinogen lytic activity detects:
UPA (0～10 μ g/ml) or urokinase zymogen mutant (0～100 μ g/ml) and 1ml blood plasma is mixed and put 37 ℃ of insulations after 6,16 or 24 hours, Trypsin inhibitor,Trasylol (the aprotinin that adds 0.2ml, 10,000 KIU/ml), adopt zymoplasm-grumeleuse method to measure the plasma fibrin that after preset time, stays.Wherein former to make the fibrinogen degradation 50% required consumption of 9 μ M in 6 hours be 1.0 μ g/ml to recombinaton urokinase, and when causing the fibrinogen degradation of same degree, urokinase zymogen mutant Pro
309→ Ala, Pro
309→ Gly, Pro
309→ Leu, Pro
309→ Ser, Pro
309→ Thr, Pro
309The consumption of → Val is respectively: 6 μ g/ml, 9.8 μ g/ml, 12 μ g/ml, 8.4 μ g/ml, 6.9 μ g/ml, 15.5 μ g/ml.
The fibrinogen clot dissolution is analyzed:
According to people J.Clin Invest such as Gurewich; The description of 73:1731 (1980) prepares from 0.3ml blood plasma
125The grumeleuse of I mark.In 3ml blood plasma, add the former or urokinase zymogen mutant (1,1.25,1.5,2.0,3.0,5.0,7.5,10.0,20.0 μ g/ml) of certain density recombinaton urokinase and carry out the fibrin clot solubility test.The radioactive activity that discharges according to specified time carries out quantitatively (before accounting for the fiber clot dissolution with the radioactive activity that has discharged in the specified time the fibrinolysis reaction
125The active percentage ratio of the gross activity of I is represented).After the fibrin clot in the blood plasma disappears, take out 0.5ml blood plasma immediately and add 0.1ml 10, (with the blood plasma that does not add uPA is contrast with residual Fibrinogen percentage composition in the mensuration blood plasma for the Trypsin inhibitor,Trasylol of 000KIU/ml and the zymoplasm of 5IU/ml, setting its Fibrinogen percentage composition is 100%), the result shows: made plasma clot dissolve required recombinaton urokinase consumption former or the mutant uPA fully in 4 hours and be respectively: 2 μ g/ml, fibrinogenic residual quantity is 72% in its blood plasma.Pro
309→ Ala urokinase zymogen mutant: 1.5 μ g/ml, fibrinogenic residual quantity is 80% in its blood plasma; Pro
309→ Gly mutant: 10 μ g/ml, fibrinogenic residual quantity is 89% in its blood plasma; Pro
309→ Leu mutant: 20 μ g/ml, the Fibrinogen residual quantity is 95% in its blood plasma; Pro
309→ Ser mutant: 1.25 μ g/ml, the Fibrinogen residual quantity in its blood plasma is 76%; Pro
309→ Thr mutant: 1.5 μ g/ml, the Fibrinogen residual quantity is 78% in its blood plasma; Pro
309→ Val mutant: 7.5 μ g/ml, fibrinogenic residual quantity is 92% in its blood plasma.
Claims (10)
1, a kind of urokinase zymogen mutant with thrombolysis activity, it is the mutant that the 309th proline(Pro) that natural urokinase is former suddenlys change and form, and wherein said sudden change is with the 309th proline(Pro) of aminoacid replacement that one of is selected from Serine, Threonine, L-Ala, glycine, Xie Ansuan or the leucine.
2, urokinase zymogen mutant according to claim 1, it is characterized in that not only can be by the E fragment of fibrin degradation to the activation of plasminogen, and can be promoted by the D fragment of scleroproein and degraded thereof.
3, urokinase zymogen mutant according to claim 1 is characterized in that in the natural urokinase original acid sequence that the 309th proline(Pro) is replaced by Serine.
4, urokinase zymogen mutant according to claim 1 is characterized in that in the natural urokinase original acid sequence that the 309th proline(Pro) is replaced by Threonine.
5, urokinase zymogen mutant according to claim 1 is characterized in that in the natural urokinase original acid sequence that the 309th proline(Pro) is replaced by L-Ala.
6, urokinase zymogen mutant according to claim 1 is characterized in that in the natural urokinase original acid sequence that the 309th proline(Pro) is replaced by glycine.
7, urokinase zymogen mutant according to claim 1 is characterized in that in the natural urokinase original acid sequence that the 309th proline(Pro) is replaced by Xie Ansuan.
8, urokinase zymogen mutant according to claim 1, the proline(Pro) that it is characterized in that in the natural urokinase original acid sequence the 309th is by leucine in place.
9, a kind of dna molecular of each the described urokinase zymogen mutant of claim 1-8 of encoding.
10, with the described recombinant DNA molecules cell transformed of claim 9.
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