Recombinant Plasminogen Kringle Research Articles

Anti-tumor effect of recombinant plasminogen kringle 1–3 in KB cells: Histological observation and immunoprecipitation-based high performance liquid chromatography

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Direct binding of recombinant plasminogen kringle 1–3 to angiogenin inhibits angiogenin-induced angiogenesis in the chick embryo CAM

Angiogenin is one of the most potent angiogenesis-inducing proteins. Angiostatin is one of the most potent angiogenesis inhibitors, and it contains the first four kringle domains of plasminogen (K1–4). Recombinant human plasminogen kringle 1–3 (rK1–3) was expressed in Escherichia coli and purified to homogeneity. The binding of t-4-aminomethylcyclohexanecarboxylic acid with the purified kringle 1–3 was determined by changes in intrinsic fluorescence. rK1–3 exhibits comparable ligand-binding properties as native human plasminogen kringle 1–3. The purified rK1–3 inhibits neovascularization in the chick embryo chorioallantoic membrane (CAM) assay. Interaction of angiogenin with rK1–3 was examined by immunological binding assay and surface plasmon resonance kinetic analysis, and the equilibrium dissociation constants for the complex, K d, are 0.89 and 0.18 μM, respectively. rK1–3 inhibits angiogenin-induced angiogenesis in the chick embryo CAM in a concentration-dependent manner. These results indicate that rK1–3 directly binds to angiogenin and thus rK1–3 inhibits the angiogenic activity of angiogenin.

Expression of Recombinant Plasminogen Kringle 1–1 in Escherichia coli, and Its Interaction with Angiogenin In Vivo

Expression of Recombinant Plasminogen Kringle 1–1 in <i>Escherichia coli</i>, and Its Interaction with Angiogenin <i>In Vivo</i>

Tetranectin-binding site on plasminogen kringle 4 involves the lysine-binding pocket and at least one additional amino acid residue.

Kringle domains are found in a number of proteins where they govern protein-protein interactions. These interactions are often sensitive to lysine and lysine analogues, and the kringle-lysine interaction has been used as a model system for investigating kringle-protein interactions. In this study, we analyze the interaction of wild-type and six single-residue mutants of recombinant plasminogen kringle 4 expressed in Escherichia coli with the recombinant C-type lectin domain of tetranectin and trans-aminomethyl-cyclohexanoic acid (t-AMCHA) using isothermal titration calorimetry. We find that all amino acid residues of plasminogen kringle 4 found to be involved in t-AMCHA binding are also involved in binding tetranectin. Notably, one amino acid residue of plasminogen kringle 4, Arg 32, not involved in binding t-AMCHA, is critical for binding tetranectin. We also find that Asp 57 and Asp 55 of plasminogen kringle 4, which both were found to interact with the low molecular weight ligand with an almost identical geometry in the crystal of the complex, are not of equal functional importance in t-AMCHA binding. Mutating Asp 57 to an Asn totally eliminates binding, whereas the Asp 55 to Asn, like the Arg 71 to Gln mutation, was found only to decrease affinity.

The inhibitory effects of recombinant plasminogen kringle 1-3 on the neovascularization of rabbit cornea induced by angiogenin, bFGF, and VEGF.

Angiostatin is a potent angiogenesis inhibitor that is composed of the first four kringles of plasminogen fragment. Angiostatin with one less kringle molecule (kringle 1 to 3) was recently demonstrated to be an effective angiogenic inhibitor. To determine whether recombinant plasminogen kringle 1-3 (rPK1-3) can inhibit the corneal neovascularization induced by potent angiogenic factors; angiogenin, bFGF, or VEGF, hydron polymer discs each containing 2.0 microg of angiogenin, 500 ng of bFGF, or 500 ng of VEGF respectively were implanted into the corneal stroma of 138 rabbit eyes, and then discs each containing 10 microg, 12.5 microg, 20 microg or 30 microg of rPK1-3 were implanted randomly. Discs containing phosphate buffered saline were also implanted as a control. The angiogenesis score on number and length of newly formed vessels on the each of the rabbit's cornea were recorded daily by two observers (blinded). The treated corneas were also examined histologically. Recombinant PK1-3 treated corneas showed less neovascularization induced by all angiogenic factors (p < 0.05). and the extent of inhibition of neovascularization was proportional to the concentration of rPK1-3 (p < 0.05). Histologic examination showed leukocyte infiltration into the corneal stroma on the PBS treated eyes whereas rPK1-3 treated eyes showed only traces of leukocytes. These results of the effective rPK1-3 inhibition of corneal neovascularization induced by angiogenin, bFGF, or VEGF suggest that this angiostatin related fragment, rPK1-3, may be useful in the treatment of various neovascular diseases.

The structure of recombinant plasminogen kringle 1 and the fibrin binding site.

The structure of recombinant (Hoover et al. Biochemistry, 1993; 32:10936-10944) plasminogen (PG) kringle 1 (K1) has been determined and refined at 2.48 A resolution to a crystallographic R value of 0.159. In addition, 71 water molecules and two chloride ions have been located. The folding of PGK1 is very similar to that of PGK4. The lysine/fibrin binding site, however, differs from that of both PGK4 and tissue-type PG activator (t-PA) K2 at the cationic centre. Although PGK1 can potentially have a doubly charged cationic centre utilizing Arg34 and Arg71, the side chain of Arg34 is outside of Arg71 in a solvent region and its guanidino group is flexibly disordered. Moreover, site specific mutagenesis studies show unequivocally that Arg34 can be changed to glutamine without affecting the binding ability of PGK1. Thus, PGK1 only has Arg71 at the cationic site, PGK4 has Lys35/Arg71 and t-PAK2 has only Lys33. The cationic site differences may result in subtle responses in the binding affinities of the kringles. The two chloride ions are located in the lysine binding site and effectively compensate the positive charges of the region. They also appear to be involved intermolecularly in a complex way in the crystal structure. Such intermolecular anionic interactions are also found in PGK4 and t-PAK2.

Protein-ligand interactions in the lysine-binding site of plasminogen kringle 4 are different in crystal and solution. Electrostatic interactions studied by site-directed mutagenesis exclude Lys35 as an important acceptor in solution.

Three amino acid residues previously reported to establish the interactions between lysine-like derivatives and plasminogen kringle 4 have been replaced by other residue types using the methods of site-directed mutagenesis. The effect of these modifications on the binding constant have been measured. The residues are Lys35, Asp57, and Arg71, according to the sequence numbering scheme adapted from the plasminogen kringle 5 domain. The plasminogen kringle 4 derivatives where Lys35 of the native molecule is replaced with isoleucine and methionine residues, respectively, were seen to bind the ligands, respectively, with association constants similar to those of the unmodified recombinant kringle 4 domain. The modification of Asp57 to asparagine was shown to eliminate the ability to bind to the lysine affinity column used to purify the protein. Similarly the site-directed mutagenesis for Arg71 to glutamine resulted in a 12-19-fold decrease in binding of each of the two ligands. In addition, the effect of ionic strength on the binding of 6-aminohexanoic acid to the recombinant plasminogen kringle 4 and the three single substituted derivatives was examined. For the unmodified kringle domain as well as for the two derivatives modified only at the position of Lys35, an ionic strength of 0.5 M reduced the binding constant by a factor of 3 to 0.12 x 10(5) M-1. The derivative modified at the position of Arg71 was not effected by the ionic strength and maintained a rather low binding constant of 0.02 x 10(5) M-1. The observations suggest that the carboxylate of Asp57 and the guanidino group of Arg71 provide the electrostatic interaction in the binding site for the epsilon-amino group and the alpha-carboxylate of a C-terminal lysine residue.(ABSTRACT TRUNCATED AT 250 WORDS)