1H-NMR spectroscopic manifestations of ligand binding to the kringle 4 domain of human plasminogen

Structural aspects of the binding of the linear ligands N alpha-acetyl-L-lysine (AcLys) and epsilon-aminocaproic acid (epsilon ACA) and of the cyclic analogs trans-(aminomethyl)-cyclohexanecarboxylic acid (AMCHA) and p-benzylaminesulfonic acid (BASA) to the intact plasminogen kringle 4 domain have been investigated by 1H-NMR spectroscopy at 300 and 600 MHz. Ligand binding results in consistent shifts of the His-II (His31), Trp-I (Trp25?), Trp-II (Trp62?), Trp-III (Trp72), Tyr-II (Tyr50), and Phe64 ring signals. BASA tends to induce larger shifts than elicited by the aliphatic ligands, most noticeably on Trp-II and on Trp72, suggesting that the ligand aromatic ring interacts with the two indole groups. Trp-II and, to lesser extent, Trp-I interact with an acidic side chain group, in a manner that is blocked by BASA. BASA binding also perturbs Tyr-II (Tyr50), Tyr-III (Tyr41), and Tyr-IV (Tyr74) over a wide pH range and lowers the pKa* of His31 from approximately 4.8 to approximately 4.6. His-III (His33) responds to BASA and AMCHA but is relatively insensitive to the linear ligands. His33 carries a sterically shielded side chain which, in conjunction with Leu46, Trp-I, Tyr50, and Tyr74, participates in structuring the kringle hydrophobic core, contiguous to the binding site. Pronounced shifts are observed for aliphatic resonances stemming from the kringle-bound molecules of AMCHA, AcLys, and epsilon ACA. It is proposed that the lysine-binding site is mostly supported by the loop that extends from Cys51 through Cys71 and that aromatic residues, which include Trp-II, Trp72, and Phe64, play a major role in interacting with the nonpolar segment of the ligand molecule. The binding site also encompasses Tyr50, Tyr74, His31, and His33 although it is not clear the extent to which these residues interact directly with the ligand.     

Isolation, purification and 1H-NMR characterization of a kringle 5 domain fragment from human plasminogen

A scheme is proposed for generating the intact Val-448-Phe-545 polypeptide of human plasminogen which contains the fifth kringle domain of the plasmin heavy chain. The procedure is based on a pepsin fragmentation of miniplasminogen and involves the purification of the kringle 5-containing fragment by gel filtration and ion-exchange chromatography. The final product is characterized by amino acid analysis, N- and C-terminal analyses, and high-resolution 1H-NMR spectroscopy at both 300 MHz and 611 MHz. We detect a (40:60%) Asp/Asn heterogeneity at site 452 of the Glu-plasminogen molecule. In the conventional kringle numbering system, the kringle 5 domain extends from Cys-1 to Cys-80, which corresponds to Cys-461 to Cys-540 in plasminogen. A preliminary 1H-NMR characterization of kringle 5 focuses on the global conformational features of the polypeptide. Assignments are given for a number of resonances, including the Tyr-72, the His imidazoles' and the Trp indoles' spin systems. Comparison with human plasminogen kringles 1 and 4 shows that the kringle 5 conformation is highly structured and very similar to that of the homologous domains. This conservancy is particularly striking in the environment surrounding Leu-46 and in the overall features of the aromatic spectrum. There are some differences, particularly in the buried His-33 imidazole group, whose H2 resonance is shifted to 9.67 ppm. A preliminary study of benzamidine-binding shows that the ligand interacts weakly (Ka approximately equal to 1.7 mM -1) mainly through the amidino functional group. Trp-62 and Tyr-72 are significantly perturbed by benzamidine, suggesting that these residues are part of the ligand-binding site.     

Analysis of the aromatic 1H-NMR spectrum of the kringle 5 domain from human plasminogen. Evidence for a conserved kringle fold

A kringle 5 domain fragment from human plasminogen has been investigated by 1H-NMR spectroscopy at 300 MHz and 620 MHz. The study focuses on the kringle 5 aromatic spectrum as aromatic side chains appear to mediate the binding of benzamidine. Spin-echo experiments and acid/base-titration studies in conjunction with two-dimensional double-quantum and chemical-shift-correlated spectroscopies were used to identify individual spin systems. Sequence-specific assignments of aromatic resonances are derived from direct comparison of the kringle 5 spectrum with spectra of the homologous kringle 1 and kringle 4 domains of plasminogen. As previously observed for kringles 1 and 4, the pattern we detect for Tyr9 in kringle 5 reflects a slow conformational exchange between two states in equilibrium, one in which the Tyr9 ring is freely mobile and one in which its flip dynamics are constrained. Proton Overhauser experiments in 1H2O and in 2H2O have been used to probe aromatic ring interactions and to identify residues which are part of the hydrophobic core centered at the Leu46 side chain. Overall, the data indicate a strong structural homology among the three plasminogen kringles.     

Solution structure of the kringle 4 domain from human plasminogen by 1H nuclear magnetic resonance spectroscopy and distance geometry

Kringle 4 is an autonomous structural and folding domain within the proenzyme plasminogen. Homologous domains are found throughout the blood clotting and fibrinolytic proteins. In this paper, we present the almost complete assignment of the 1H nuclear magnetic resonance (n.m.r.) spectrum of the kringle 4 domain of human plasminogen. A detailed structural analysis has been completed. The sequential pattern of nuclear Overhauser enhancements indicated little regular secondary structure but rather a series of turns and loops connecting beta-strands. A small stretch of antiparallel beta-sheet was identified between the residues 61 to 63 and 71 to 73 and the close proximity of other strands was determined from two-dimensional nuclear Overhauser enhancement spectra. Slowly exchanging amide (NH) resonances were found to be associated with residues of the beta-sheet and neighbouring strands that support the hydrophobic core of the domain. A total of 526 interproton distance constraints and two hydrogen bonds were specified as input to the distance geometry program DISGEO. Tertiary structures were produced that were consistent with the n.m.r. data. The structures were compared with that of our earlier model based on n.m.r. studies and with that of prothrombin fragment 1 determined crystallographically.     

Two-dimensional 1H-NMR studies of the solution structure of plasminogen kringle 4

Native kringle 4 from human plasminogen has been studied by two-dimensional 1H-NMR methods in order to obtain new structural information about the kringle fold. Two-dimensional scalar correlated spectroscopy (COSY), two-dimensional dipolar correlated spectroscopy (NOESY) and two-dimensional relayed coherance transfer spectroscopy (RCT) experiments were recorded, allowing most resonances arising from the aromatic and methyl-containing residues to be assigned in the spectrum. From an analysis of NOE data, a small segment of double-stranded beta-sheet has been identified near residues Phe63 and Thr64. Further analysis of the NOESY spectrum has allowed detailed study of the conformation of sidechains located in regions near Leu45 and Val69. A model has been constructed of the polypeptide segment comprising residues 40-49 which accounts for the observed NOE interactions.     

Ligand interactions with the kringle 5 domain of plasminogen. A study by 1H NMR spectroscopy

The binding of small molecules to the kringle 5 domain fragment of human plasminogen has been investigated by 1H NMR spectroscopy at 300 MHz. The compounds tested as potential ligands include L-arginine, L-lysine, and a number of aliphatic and aromatic analogs of similar size but different ionic charge configurations. Ligand/kringle 5 association constant (Ka) values were obtained from ligand titration experiments at 22 degrees C, pH 7.2. Neither L-arginine nor N alpha-acetyl-L-arginine and N alpha-acetyl-L-arginine methyl ester bind measurably to kringle 5 (Ka approximately less than 0.05 mM-1). In contrast, binding of hexylamine or epsilon-aminocaproic acid (epsilon ACA) is favored (Ka approximately 2.9 and 10.5 mM-1, respectively). Benzamidine and p-benzylaminesulfonic acid associate with kringle 5 with similar affinities (Ka approximately 3.4 and 2.2 mM-1, respectively) while benzylamine binds about twice as tightly (Ka approximately 6.3 mM-1). The higher affinities toward both benzylamine and epsilon ACA indicate that a free carboxylate group is not, by itself, a main determinant of ligand-binding to kringle 5. The experiments also reveal a definite affinity for L-arginine methyl ester, L-lysine, and N alpha-acetyl-L-lysine methyl ester. It is suggested that, although weak (0.1 approximately less than Ka approximately less than 0.6 mM-1), these interactions could be of physiological relevance in the context of plasminogen binding to the fibrin clot. Ligand-induced shifts of kringle 5 proton resonances indicate that the Trp25, His33, Tyr50, Trp62, and Tyr72 (kringle numbering convention) side chains form or neighbor the kringle 5-binding site. Benzamidine-kringle 5 magnetization transfer (Overhauser) experiments verify a close proximity of the bound ligand to these aromatic groups. A model of the binding site is proposed in which the above residues interact closely with each other and define a lipophilic surface which is accessible to the free ligand.     

Proton magnetic resonance study of kringle 1 from human plasminogen. Insights into the domain structure

The aromatic H NMR spectrum of the kringle 1 domain from human plasminogen has been investigated by proton Overhauser experiments, acid-base titration, and two-dimensional chemical shift correlated spectroscopy. Spin-echo and pH response experiments lead to the identification of the N-terminal Tyr-3 phenol ring signals. The connectivities among the tryptophanyl aromatic protons have been established and sets of singlet-doublet-triplet resonances stemming from each of the two indole groups sorted according to their common side chain origin. Similarly, the four histidyl singlets have been identified and paired per imidazole group. From their pH responses, it is indicated that a histidyl (His31) and a tryptophanyl (Trp-II) residue are placed in the neighborhood of carboxyl groups. The high-field chemical shifts observed for proton resonances of the ligand epsilon-aminocaproic acid upon binding to kringle 1 indicate that the ligand-binding site is rich in aromatic components. Overhauser experiments reveal that Leu46 is surrounded by a cluster of interacting aromatic side chains, which includes Trp25, Phe36, His41, Trp62, and Tyr64, and define a hydrophobic region contiguous to the kringle lysine-binding site. Relative internuclear distances have been estimated for aromatic H-atoms in the vicinity of Leu46 by reference to one of the latter's CH3 sigma, sigma' groups. Some of the connectives have previously been found for Leu46 in kringle 4 which further supports the idea of a common structure for the homologous domains.     

The effects of ligand binding on the backbone dynamics of the kringle 1 domain of human plasminogen

The internal motions of the backbone nitrogen atoms of the kringle 1 domain of human plasminogen (K1(Pg)) were examined in the absence and presence of the ligand, epsilon-aminocaproic acid. These dynamic properties were determined from (15)N NMR relaxation data in terms of the extended model-free parameters. The model of isotropic reorientation was found sufficient to account for overall molecular tumbling for both apo and EACA-bound K1(Pg). The global rotational correlation time (tau(m)) for apo-K1(Pg) was 5.87(+/-0.01) ns, while the tau(m) for ligand-bound K1(Pg) was 5.20(+/-0.01) ns, suggesting that perhaps some small degree of aggregation occurred in the apo form of the kringle module. Complexation of K1(Pg) with ligand mainly reduced those internal motions that occurred on a 100 ps to 5 ns time-scale. The magnitude of the chemical exchange was also attenuated upon ligand binding. These data are consistent with studies employing other approaches that suggest that the binding pocket is preformed in K1(Pg).     

Examination of the secondary structure of the kringle 4 domain of human plasminogen

The structure of a small region of human plasminogen (F4), consisting of amino acid residues Val354-Ala439 and containing its kringle 4 (K4) domain (residues Cys357-Cys434), has been predicted from Chou-Fasman calculations and hydropathy profiles, and compared to circular dichroism (CD) measurements on the isolated fragment. Calculations, by the Chou-Fasman method, of the probabilities of various types of secondary structures that exist in this region reveal that no helical structures are present. Of the total of 86 amino acid residues present in this K4-containing peptide region, 37% can adopt conformations of beta-pleated sheets, 48% of the amino acids can exist in beta-turns, and 15% of the residues can be present as coils. The structure of F4 in dilute aqueous solution has been experimentally evaluated by CD measurements. At pH = 7.4, in dilute salt solutions, a total of 64% beta-structures, 30% beta-turns, and 6% coiled structures is estimated to be present in this peptide region. Consideration of the marginal stability of many of the conformational regions of F4, as predicted by Chou-Fasman calculations, suggests that secondary structural flexibility is present in this fragment, which could result in ready adoption of new conformations. The hydropathy profile of F4 has been determined and suggests that this polypeptide is highly hydrophilic, especially in the regions of residues His387-Tyr396 and Cys406-Lys413. Thus, it appears as though a large portion of the surface of F4 can be exposed to solvent in its native conformation.     

Analysis of ligand-binding to the kringle 4 fragment from human plasminogen

The interaction of the isolated human plasminogen kringle 4 with the four -amino acid ligands -aminocaproic acid (ACA), N-acetyl-l-lysine (AcLys), trans-aminomethyl(cyclohexane)carboxylic acid (AMCHA) and p-benzylaminesulfonic acid (BASA) has been further characterized by ¹H-NMR spectroscopy at 300 and 600 MHz. Pronounced high-field shifts, reaching 3 ppm, are observed for AMCHA resonances upon binding to kringle 4, which underscores the relevance of ligand lipophilic interactions with aromatic side chains at the binding site. Ligand titration curves for the nine His and Trp singlets found in the kringle 4 aromatic spectrum reveal a striking uniformity in the kringle response to the various ligands. The average binding curves exhibit a clear Langmuir absorption isotherm saturation profile and the data were analyzed under the assumption of one (high affinity) binding site per kringle. Equilibrium association constants (K _a ) and first order dissociation rate constants (k _off) were derived from linearized expressions of the Langmuir isotherm and of the spectral line-shapes, respectively. The results for the four ligands, at 295 K, pH^* 7.2, indicate that: (a) AMCHA exhibits the strongest binding (K _a =159 mM ^-1) and ACA the weakest (K _a =21 mM ^–1) with AcLys and BASA falling in between; (b) ACA dissociates readily (k _off = 5.3 × 10³ s^–1) and AMCHA associates the fastest (k _off = 2.0 × 10⁸ M ^–1 s^–1) while the kinetics for BASA exchange is relatively slow (k _off = 0.8 × 10³ s^–1, k _on = 0.6 × 10⁸ M ^–1s^–1); (c) the ligand-binding kinetics is close to diffussion-controlled.Abbreviations ACA -aminocaproic acid - AcLys N-acetyl-l-lysine - AMCHA t-aminomethyl(cyclohexane)carboxylic acid - BASA p-benzylaminesulfonic acid - K4 kringle 4 - NOE nuclear Overhauser effect - ppm parts-per-million - pH^* glass electrode pH reading uncorrected for deuterium isotope effects - K _a ligand-kringle 4 equilibrium association constant - k _off ligand-kringle 4 dissociation rate constant - k _on ligand-kringle 4 association rate constant     

Analysis of the aliphatic 1H-NMR spectrum of plasminogen kringle 4. A comparative study of human, porcine, bovine and chicken homologs

The aliphatic 1H-NMR spectrum of the kringle 4 domain of human plasminogen has been studied via two-dimensional chemical shift correlated (COSY) and nuclear Overhauser correlated (NOESY) experiments at 300 MHz and 620 MHz. A number of aliphatic proton spin systems have been identified and several definite assignments have been made. This was mainly achieved by comparison of the human kringle 4 spectrum with spectra of the porcine, bovine and chicken homologs and also with that of the kringle 1 from human plasminogen on which we have reported previously. The three valyl and two leucyl residues of human kringle 4 have been assigned. The eleven threonyl spin systems have been identified via a RELAYED-COSY experiment and Thr17 has been assigned. The three alanyl spin systems have been identified and assigned. Six seryl spin systems have been identified and the signals from the seven glycyl residues of human kringle 4 have been located with Gly45 assigned. Furthermore, 24 AMX spin systems have been mapped in the COSY spectrum of human kringle 4 and H alpha-H beta,beta' spin systems of Tyr2, Tyr41, Tyr50, Tyr74, Trp25 and Trp62 have been assigned. From the spectrum of a deglycosylated chicken homolog, the epsilon-methyl singlets of Met28 and Met48 have been assigned. Finally, ligand effects on selected aliphatic resonances were observed which could be analyzed in terms of residues likely to neighbor the kringle lysine-binding site.     

A 1H-NMR study of isolated domains from human plasminogen. Structural homology between kringles 1 and 4

Kringles 1 and 4 from human plasminogen are polypeptide domains of Mr approximately equal to 10000 each of which can be isolated by proteolysis of the zymogen. They have been studied by 1H-NMR spectroscopy at 300 MHz and 600 MHz. The spectra, characteristic of globular structures, show striking analogies that point to a close conformational relatedness among the two kringles, consistent with their high degree of amino acid conservancy and homology. The interaction of both kringles with p-benzylaminesulfonic acid (BASA), an antifibrinolytic drug that binds to a lysine-binding site, results in better resolved, narrower lines for both spectra. Aromatic and methyl-region spectra of BASA complexes of kringles 1 and 4 were compared and the latter was studied by two-dimensional NMR spectroscopy. Analysis of the CH3 multiplets in terms of their resonance patterns, and the amino acid compositions and sequences of the two kringles, leads to the identification of most signals and to some assignments. In particular, a doublet at -1 ppm, exhibited by both kringles and also found in reported proton spectra of homologous bovine prothrombin fragments, has been assigned to Leu46, a residue that is conserved in all of the kringles studied to date by 1H-NMR. Since this resonance is somewhat more sensitive to BASA than other methyl signals, it is likely that Leu46 is proximal to the lysine-binding site. Nuclear Overhauser experiments reveal that Leu46 is surrounded by a cluster of closely interacting hydrophobic and aromatic side chains. Kringle 4 was also compared with a derivative chemically modified at Trp72 with dimethyl(2-hydroxy-5-nitrobenzyl)sulfonium bromide. As judged from the proton spectra, the modified kringle 4 retains globularity and is perturbed mainly in the aromatic region, in analogy to that which is observed for the unmodified kringle upon BASA binding. Furthermore, although previous studies have indicated no retention of the modified kringle by lysine-Sepharose, the NMR studies point to a definite interaction between BASA and the kringle derivative. The spectroscopic data also suggest that the His31 imidazole is not significantly affected by the ligand and that the lysine-binding site is structured mostly by hydrophobic side chains, including Trp72 in the case of kringle 4, and probably Tyr72 in kringle 1.     

A study on human urine in a high-selenium area of China by 1H-NMR spectroscopy

In this study, high-resolution 600-MHz ¹H-NMR (nuclear magnetic resonance) spectroscopies were used to compare the urinary metabolic profiles of healthy humans and humans in a high-selenium area of China. NMR biomarkers for renal and liver lesions were observed by comparing the urine ¹H-NMR spectra. In urinary excretion, the concentrations in human urine samples of formate, lactate, acetate, hippurate, and alanine in overexposure to selenium were increased, whereas citrate, creatine, and TMAO excretion were decreased compared with that of the healthy human—some of them even disappeared. An interesting result was the appearance of formate in urine, which has previously been shown to lead to acidosis and chronic renal failure and interfere with the lumen and proximal tubular cells. The level of creatine was associated with the seminal activity. The changes of acetate and citrate may explain the disorder of the cellular energy metabolism caused by selenium, and the changes of other amino acids were a result of the reuptake of these compounds that had been blocked in the glomerulus and proximal tubule. The results elucidate the renal/liver lesion in humans in high-selenium area by ¹H-NMR spectroscopy and offer the molecular basic of selenium toxicity.     

Proton magnetic resonance study of lysine-binding to the kringle 4 domain of human plasminogen. The structure of the binding site

The binding of L-Lys, D-Lys and epsilon-aminocaproic acid (epsilon ACA) to the kringle 4 domain of human plasminogen has been investigated via one and two-dimensional 1H-nuclear magnetic resonance spectroscopy at 300 and 600 MHz. Ligand-kringle association constants (Ka) were determined assuming single site binding. At 295 K, pH 7.2, D-Lys binds to kringle 4 much more weakly (Ka = 1.2 mM-1) than does L-Lys (Ka = 24.4 mM-1). L-Lys binding to kringle 4 causes the appearance of ring current-shifted high-field resonances within the -1 approximately less than delta approximately less than 0 parts per million range. The ligand origin of these signals has been confirmed by examining the spectra of kringle 4 titrated with deuterated L-Lys. A systematic analysis of ligand-induced shifts on the aromatic resonances of kringle 4 has been carried out on the basis of 300 MHz two-dimensional chemical shift correlated (COSY) and double quantum correlated spectroscopies. Significant differences in the effect of L-Lys and D-Lys binding to kringle 4 have been observed in the aromatic COSY spectrum. In particular, the His31 H4 and Trp72 H2 singlets and the Phe64 multiplets appear to be the most sensitive to the particular enantiomers, indicating that these residues are in proximity to the ligand C alpha center. In contrast, the rest of the indole spectrum of Trp72 and the aromatic resonances of Trp62 and Tyr74, which are affected by ligand presence, are insensitive to the optical nature of the ligand isomer. These results, together with two-dimensional proton Overhauser studies and ligand-kringle saturation transfer experiments reported previously, enabled us to generate a model of the kringle 4 ligand-binding site from the crystallographic co-ordinates of the prothrombin kringle 1. The latter, although lacking recognizable lysine-binding capability, is otherwise structurally homologous to the plasminogen kringles.     

1H NMR studies of aliphatic ligand binding to human plasminogen kringle 4

A detailed 1H NMR analysis of ligand binding to the human plasminogen kringle 4 domain has been carried out at 300 MHz. The ligands that were investigated are N alpha-acetyl-L-lysine, L-lysine methyl ester, N alpha-acetyl-L-lysine methyl ester, L-lysine hydroxamic acid, trans-(aminomethyl)cyclohexanecarboxylic acid (AMCHA), and 4-(aminomethyl)bicyclo[2.2.2]octane-1-carboxylic acid (AMBOC). Specific ligand-binding effects were detected via two-dimensional COSY experiments. The side chains that are the most perturbed by ligand presence are those from Trp62, Phe64, and Trp72. Ligand-kringle saturation transfer (Overhauser) experiments show that the aromatic rings from these three residues, especially Trp72, are in direct contact with the ligand. These results add support to a previously reported model of the kringle 4 lysine-binding site [Ramesh, V., Petros, A. M., Llinás, M., Tulinsky, A., & Park, C. H. (1987) J. Mol. Biol. 198, 481-498] by which these aromatic groups are assigned a key role in establishing hydrophobic interactions with the ligand molecule. Equilibrium association constants (Ka) and kinetic rate constants (kon, koff) were determined for the binding of the various linear and cyclic ligands to kringle 4. We find that those ligands whose carboxylate function is blocked bind significantly weaker (Ka approximately less than 2 mM-1) than the corresponding analogues where the anionic center is present (Ka approximately greater than 20 mM-1), which underscores the relevance of the polar group in stabilizing the interaction with the kringle 4 binding site.(ABSTRACT TRUNCATED AT 250 WORDS)     

Fluorescence spectroscopic analysis of ligand binding to kringle 1 + 2 + 3 and kringle 1 fragments from human plasminogen

The ligand binding of kringle 1 + 2 + 3 and kringle 1 from human plasminogen has been investigated by fluorescence spectroscopy. Analysis of fluorescence titration of kringle 1 + 2 + 3 with 6-aminohexanoic acid shows that this fragment, besides the high-affinity lysine-binding site with Kd = 2.9 microM, contains two additional lysine-binding sites which differ in binding strength (Kd = 28 microM and Kd = 220 microM). This strongly suggests the existence of a lysine-binding site in each of the first three kringles. 6-Aminohexanoic acid, pentylamine, pentanoic acid and arginine were used for investigation of the ligand specificity of isolated kringle 1 prepared by pepsin hydrolysis of kringle 1 + 2 + 3. It has been established that kringle 1 has high affinity to 6-aminohexanoicacid, pentylamine and arginine (Kd values are 3.2 microM, 4.8 microM and 4.3 microM, respectively). At the same time pentanoic acid did not bind with kringle 1. These facts indicate, firstly, a broad ligand specificity of kringle 1 and, secondly, the paramount importance of the positively charged group of the ligand for its interaction with lysine-binding site of this kringle.     

Expression, purification, and characterization of the recombinant kringle 1 domain from tissue-type plasminogen activator.

A novel fusion protein expression plasmid that allows ready purification and subsequent facile release of the target molecule has been constructed and employed to express in Escherichia coli and purify the tissue plasminogen activator kringle 1 domain ([K1tPA] residues C92-C173). The resulting plasmid encodes the tight lysine-binding kringle (K)1 domain of human plasminogen ([K1HPg]) followed by a peptide (PfXa) containing a factor Xa-sensitive bond, downstream of which [K1tPA] was inserted. The recombinant (r) [K1HPg]PfXa[K1tPA] fusion polypeptide was purified from various cell fractions in one step by Sepharose-lysine affinity chromatography. After cleavage with fXa, the mixture was repassaged over Sepharose-lysine, whereupon the r-[K1tPA]-containing polypeptide passed unretarded through the column. A homogeneous preparation of this material was then obtained after a simple step employing fast protein liquid chromatography. The purified r-[K1tPA], which contained the amino acid sequence SNAS[K1tPA]S, provided an amino-terminal amino acid sequence, through at least 20 amino acid residues, that was identical to that predicted from the cDNA sequence. The molecular mass of r-SNAS[K1tPA]S, determined by electrospray mass spectrometry, was 9621.9 +/- 4.0 (expected molecular mass, 9623.65). 1H-NMR spectroscopy and thermal stability studies of r-SNAS[K1tPA]S revealed that the purified material was properly folded and similar to other isolated kringle domains. Additionally, employment of this methodology revealed that only a very weak interaction between epsilon-aminocaproic acid and the isolated r-[K1tPA] domain occurred.     

Purification and characterization of a novel, oligomeric, plasminogen kringle 4 binding protein from human plasma: tetranectin

Purification of alpha 2-plasmin inhibitor (alpha 2PI) from human plasma by affinity chromatography on plasminogen-Sepharose resulted in copurification of a contaminating protein with Mr 17,000 as judged by sodium dodecyl sulphate/polyacrylamide gel electrophoresis. This contaminating protein could not be removed from the purified alpha 2-PI preparation by several types of gel chromatography applied. The use of the kringle 1-3 part of plasminogen, K(1 + 2 + 3), bound to Sepharose for affinity chromatography, instead of plasminogen-Sepharose, resulted in an alpha 2PI preparation without this contaminant. The contaminating protein was found to interact specifically with the kringle 4 part of plasminogen (K4) and not with K(1 + 2 + 3) or miniplasminogen. The K4-binding protein was purified by ammonium sulphate precipitation, affinity chromatography on K4-Sepharose, ion-exchange chromatography and gel filtration on AcA 34. The relative molecular mass of the protein (Mr 68 000) was estimated by gel filtration. This suggests a tetrameric protein composed of four subunits (Mr 17,000), that are dissociated by 1% sodium dodecyl sulphate. Dissociation into subunits was also demonstrated by gel filtration in the presence of 6 M guanidine hydrochloride. A specific antibody was raised in rabbits against the purified protein and this antibody was shown not to react with any known fibrinolytic components. The pI of the K4-binding protein was found to be 5.8. The first three N-terminal amino acids were determined to be Glu-Pro-Pro. The concentration of the protein in plasma was estimated to be 0.20 +/- 0.03 microM (15 +/- 2 mg/l). The electrophoretic mobility of the K4-binding protein was shown by crossed immunoelectrophoresis to be influenced by the presence of Ca2+, EDTA and heparin. The protein was found to enhance plasminogen activation catalyzed by tissue-type plasminogen activator (t-PA) in the presence of poly(D-lysine). The protein appeared to be a novel plasma protein tentatively called 'tetranectin'.     

Structure of the plasminogen kringle 4 binding calcium-free form of the C-type lectin-like domain of tetranectin

Tetranectin is a homotrimeric protein containing a C-type lectin-like domain. This domain (TN3) can bind calcium, but in the absence of calcium, the domain binds a number of kringle-type protein ligands. Two of the calcium-coordinating residues are also critical for binding plasminogen kringle 4 (K4). The structure of the calcium free-form of TN3 (apoTN3) has been determined by NMR. Compared to the structure of the calcium-bound form of TN3 (holoTN3), the core region of secondary structural elements is conserved, while large displacements occur in the loops involved in calcium or K4 binding. A conserved proline, which was found to be in the cis conformation in holoTN3, is in apoTN3 predominantly in the trans conformation. Backbone dynamics indicate that, in apoTN3 especially, two of the three calcium-binding loops and two of the three K4-binding residues exhibit increased flexibility, whereas no such flexibility is observed in holoTN3. In the 20 best nuclear magnetic resonance structures of apoTN3, the residues critical for K4 binding span a large conformational space. Together with the relaxation data, this indicates that the K4-ligand-binding site in apoTN3 is not preformed.     

DGDA, a local sequence of the kringle 2 domain, is a functional motif of the tissue-type plasminogen activator’s antiangiogenic kringle domain

Antiangiogenic activity can be elicited by the kringle domains 1 and 2 of tissue-type plasminogen activator (TK1-2), or the kringle 2 domain alone. In a previous report, we showed that the anti-migratory effect of TK1-2 is mediated in part by its interference with integrin α2β1. Since integrin α2β1 interacts with collagen type I through the DGEA (Asp-Gly-Glu-Ala) amino acid sequence, and a similar sequence, DGDA (Asp-Gly-Asp-Ala), exists in the kringle 2 domain, we investigated whether the DGDA sequence has a role in antiangiogenic activity of TK1-2. In an adhesion assay, the DGDA peptide inhibited adhesion of human umbilical vein endothelial cells (HUVECs) to immobilized TK1-2. Pretreatment of the DGDA peptide also blocked anti-migratory activity of TK1-2. When the DGDA peptide alone was tested for antiangiogenic activity, it effectively inhibited VEGF-induced migration of HUVECs and tube formation on Matrigel. In addition, the DGDA peptide decreased differentiation of endothelial progenitor cells on collagen type I matrix. These data suggest that the DGDA sequence presents a functional epitope of TK1-2 and that it can be used as a potential novel antiangiogenic peptide.