PLASMINOGEN-ACTIVATOR INHIBITOR TYPE-2 (PAI-2) IS A SPONTANEOUSLY POLYMERIZING SERPIN - BIOCHEMICAL-CHARACTERIZATION OF THE RECOMBINANT INTRACELLULAR AND EXTRACELLULAR FORMS

被引:77
作者
MIKUS, P [1 ]
URANO, T [1 ]
LILJESTROM, P [1 ]
NY, T [1 ]
机构
[1] UMEA UNIV,DEPT CELL & MOLEC BIOL,S-90187 UMEA,SWEDEN
来源
EUROPEAN JOURNAL OF BIOCHEMISTRY | 1993年 / 218卷 / 03期
关键词
D O I
10.1111/j.1432-1033.1993.tb18467.x
中图分类号
Q5 [生物化学]; Q7 [分子生物学];
学科分类号
071010 ; 081704 ;
摘要
Plasminogen-activator inhibitor type 2 (PAI-2) is a specific inhibitor of plasminogen activators (PA) that exists in an intracellular, low-molecular-mass form and a secreted, high-molecular-mass form that varies with respect to glycosylation. Here we have developed expression systems for both forms of PAI-2 and biochemically characterised the purified proteins. In order to obtain efficient secretion, we constructed an artificial signal sequence and fused it to the coding region of PAI-2. With this construct, more than 90% of PAI-2 was secreted as a glycosylated, 60-kDa molecular-mass form, but the level of expression was low and unstable. To obtain higher expression of secreted PAI-2, a novel expression vector based on the Semliki-forest-virus replicon was used. Secreted PAI-2 was purified to homogeneity and N-terminal sequence analysis showed that the artificial signal peptide was correctly removed. The intracellular, non-glycosylated form of PAI-2 was expressed in Escherichia coli and purified to homogeneity. Both the secreted and the intracellular forms of PAI-2 were found to inhibit plasminogen activators by forming SDS-resistant complexes and the second-order rate constants were similar for both forms, ranging over 2.4-2.7 X 10(6) M-1 s-1 for urokinase-type PA, 2.5-2.7 x 10(5) M-1 s-1 for two-chain tissue-type PA and 0.8-1.2 X 10(4) M-1 s-1 for single-chain tissue-type PA. None of the purified PAI-2 forms bound to vitronectin. Circular-dichroism spectral analysis revealed that PAI-2 has a CD spectrum that resembles oval-bumin more than PA-inhibitor type 1, confirming the greater similarity between these two members of the serine-protease inhibitor family. Similar to what has been described for the Z-form of alpha1-antitrypsin, purified PAI-2 was found to spontaneuosly form polymers during incubation at room temperature. Attempts to convert PAI-2 to a stable locked conformation resembling the conformation of latent PAI-1 by treatment with diluted guanidinium chloride were unsuccessful. Instead, this treatment enhanced the formation of PAI-2 polymers, possibly by the loop-sheet polymerisation mechanism described for alpha1-antitrypsin.
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页码:1071 / 1082
页数:12
相关论文
共 65 条
[11]   MOBILE REACTIVE CENTER OF SERPINS AND THE CONTROL OF THROMBOSIS [J].
CARRELL, RW ;
EVANS, DL ;
STEIN, PE .
NATURE, 1991, 353 (6344) :576-578
[12]   ALPHA-1-ANTITRYPSIN - MOLECULAR PATHOLOGY, LEUKOCYTES, AND TISSUE-DAMAGE [J].
CARRELL, RW .
JOURNAL OF CLINICAL INVESTIGATION, 1986, 78 (06) :1427-1431
[13]   ANALYSIS OF GENE-CONTROL SIGNALS BY DNA-FUSION AND CLONING IN ESCHERICHIA-COLI [J].
CASADABAN, MJ ;
COHEN, SN .
JOURNAL OF MOLECULAR BIOLOGY, 1980, 138 (02) :179-207
[14]   MACROPHAGE FIBRINOLYTIC-ACTIVITY - IDENTIFICATION OF 2 PATHWAYS OF PLASMIN FORMATION BY INTACT-CELLS AND OF A PLASMINOGEN-ACTIVATOR INHIBITOR [J].
CHAPMAN, HA ;
VAVRIN, Z ;
HIBBS, JB .
CELL, 1982, 28 (03) :653-662
[15]   HIGH-EFFICIENCY TRANSFORMATION OF MAMMALIAN-CELLS BY PLASMID DNA [J].
CHEN, C ;
OKAYAMA, H .
MOLECULAR AND CELLULAR BIOLOGY, 1987, 7 (08) :2745-2752
[16]   KINETICS OF THE INHIBITION OF PLASMINOGEN ACTIVATORS BY THE PLASMINOGEN-ACTIVATOR INHIBITOR - EVIDENCE FOR 2ND SITE INTERACTIONS [J].
CHMIELEWSKA, J ;
RANBY, M ;
WIMAN, B .
BIOCHEMICAL JOURNAL, 1988, 251 (02) :327-332
[17]   PLASMINOGEN ACTIVATORS, TISSUE DEGRADATION, AND CANCER [J].
DANO, K ;
ANDREASEN, PA ;
GRONDAHLHANSEN, J ;
KRISTENSEN, P ;
NIELSEN, LS ;
SKRIVER, L .
ADVANCES IN CANCER RESEARCH, 1985, 44 :139-266
[18]  
DECLERCK PJ, 1988, J BIOL CHEM, V263, P15454
[19]  
EHRLICH HJ, 1990, J BIOL CHEM, V265, P13029
[20]  
EVANS DLI, 1991, THESIS U CAMBRIDGE