Limited proteolysis of tumor cells increases their plasmin-binding ability

Mild proteolytic treatment of SW1116 tumor cells with trypsin or plasmin increases their plasmin-binding ability considerably by increasing the number of binding sites without altering their affinity. This mechanism may be operative for increasing the concentration of active plasmin at the surface of tumor cells. C-terminal lysine residues are involved in plasmin binding to cells, since treatment of cells with carboxypeptidase B decreases this binding by 50%.     

Alternative processing pathways for preprovasoactive intestinal peptide in the enteric nervous system of the rat

In order to study biosynthetic processing of preprovasoactive intestinal peptide (prepro VIP) we have raised antisera to sequences that flank the biologically active peptides VIP and PHI (peptide with N-terminal His and C-terminal Ile). We have used these antisera in radioimmunoassays to identify the N-terminal flanking peptide (NFP) and C-terminal flanking peptide (CFP)-like immunoreactivities in rat brain and gastrointestinal tract. Concentrations of NFP-LI were similar to those of VIP in brain and throughout the gut. Concentrations of CFP-LI were 10-20% those of VIP-LI but could be increased 5-fold by digestion with carboxypeptidase B, suggesting that the C-terminal lysine residue of prepro VIP is not normally removed during processing. In rat stomach the NFP-LI was of higher molecular weight and greater hydrophobicity than the intestinal component. The data are consistent with alternative processing pathways for prepro VIP in enteric nerves of rat stomach and intestine.     

Plasmin(ogen)-binding alpha-enolase from Streptococcus pneumoniae: crystal structure and evaluation of plasmin(ogen)-binding sites

Alpha-enolases are ubiquitous cytoplasmic, glycolytic enzymes. In pathogenic bacteria, alpha-enolase doubles as a surface-displayed plasmin(ogen)-binder supporting virulence. The plasmin(ogen)-binding site was initially traced to the two C-terminal lysine residues. More recently, an internal nine-amino acid motif comprising residues 248 to 256 was identified with this function. We report the crystal structure of alpha-enolase from Streptococcus pneumoniae at 2.0A resolution, the first structure both of a plasminogen-binding and of an octameric alpha-enolase. While the dimer is structurally similar to other alpha-enolases, the octamer places the C-terminal lysine residues in an inaccessible, inter-dimer groove restricting the C-terminal lysine residues to a role in folding and oligomerization. The nine residue plasminogen-binding motif, by contrast, is exposed on the octamer surface revealing this as the primary site of interaction between alpha-enolase and plasminogen.     

Contribution of conserved lysine residues in the alpha2-antiplasmin C terminus to plasmin binding and inhibition

α(2)-Antiplasmin is the physiological inhibitor of plasmin and is unique in the serpin family due to N- and C-terminal extensions beyond its core domain. The C-terminal extension comprises 55 amino acids from Asn-410 to Lys-464, and the lysine residues (Lys-418, Lys-427, Lys-434, Lys-441, Lys-448, and Lys-464) within this region are important in mediating the initial interaction with kringle domains of plasmin. To understand the role of lysine residues within the C terminus of α(2)-antiplasmin, we systematically and sequentially mutated the C-terminal lysines, studied the effects on the rate of plasmin inhibition, and measured the binding affinity for plasmin via surface plasmon resonance. We determined that the C-terminal lysine (Lys-464) is individually most important in initiating binding to plasmin. Using two independent methods, we also showed that the conserved internal lysine residues play a major role mediating binding of the C terminus of α(2)-antiplasmin to kringle domains of plasmin and in accelerating the rate of interaction between α(2)-antiplasmin and plasmin. When the C terminus of α(2)-antiplasmin was removed, the binding affinity for active site-blocked plasmin remained high, suggesting additional exosite interactions between the serpin core and plasmin.     

Thrombin-activable fibrinolysis inhibitor zymogen does not play a significant role in the attenuation of fibrinolysis

Activated thrombin-activable fibrinolysis inhibitor (TAFIa) plays a significant role in the prolongation of fibrinolysis. During fibrinolysis, plasminogen is activated to plasmin, which lyses a clot by cleaving fibrin after selected arginine and lysine residues. TAFIa attenuates fibrinolysis by removing the exposed C-terminal lysine residues. It was recently reported that TAFI zymogen possesses sufficient carboxypeptidase activity to attenuate fibrinolysis through a mechanism similar to TAFIa. Here, we show with a recently developed TAFIa assay that when thrombin is used to clot TAFI-deficient plasma supplemented with TAFI, there is some TAFI activation. The extent of activation was dependent upon the concentration of zymogen present in the plasma, and lysis times were prolonged by TAFIa in a concentration-dependent manner. Potato tuber carboxypeptidase inhibitor, an inhibitor of TAFIa but not TAFI, abolished the prolongation of lysis in TAFI-deficient plasma supplemented with TAFI zymogen. In addition, TAFIa but not TAFI catalyzed release of plasminogen bound to soluble fibrin degradation products. The data presented confirm that TAFI zymogen is effective in cleaving a small substrate but does not play a role in the attenuation of fibrinolysis because of its inability to cleave plasmin-modified fibrin degradation products.     

The heparin binding site of human extracellular-superoxide dismutase.

Extracellular-superoxide dismutase (EC-SOD) is a secretory glycoprotein that is major SOD isozyme in extracellular fluids. We revealed the possible structure of the carbohydrate chain of serum EC-SOD with the serial lectin affinity technique. The structure is a biantennary complex type with an internal fucose residue attached to asparagine-linked N-acetyl-D-glucosamine and with terminal sialic acid linked to N-acetyllactosamine. EC-SOD in plasma is heterogeneous with regard to heparin affinity and can be divided into three fractions: A, without affinity; B, with intermediate affinity; and C, with high affinity. It appeared that this heterogeneity is not dependent on the carbohydrate structure upon comparison of EC-SOD A, B, and C. No effect of the glycopeptidase F treatment of EC-SOD C on its heparin affinity supported the results. A previous report showed that both lysine and arginine residues probably at the C-terminal end, contribute to heparin binding. Recombinant EC-SOD C treated with trypsin or endoproteinase Lys C, which lost three lysine residues (Lys-211, Lys-212, and Lys-220) or one lysine residue (Lys-220) at the C-terminal end, had no or weak affinity for the heparin HPLC column, respectively. The proteinase-treated r-EC-SOD C also lost triple arginine residues which are adjacent to double lysine residues. These results suggest that the heparin-binding site may occur on a "cluster" of basic amino acids at the C-terminal end of EC-SOD C. EC-SOD is speculated to be primarily synthesized as type C, and types A and B are probably the result of secondary modifications. It appeared that the proteolytic cleavage of the exteriorized lysine- and arginine-rich C-terminal end in vivo is a more important contributory factor to the formation of EC-SOD B and/or EC-SOD A.     

Human ''creatine kinase conversion factor'' identified as a carboxypeptidase.

The effect of partially purified 'creatine kinase conversion factor' on rabbit muscle creatine kinase is shown to be that of a carboxypeptidase, removing the C-terminal lysine residue from both subunits. These changes fully explain the three-banded electrophoretic patterns of the partially and the fully modified rabbit and human enzymes. The factor also produces a similar electrophoretic pattern with haemoglobin A; comparison with the effects of carboxypeptidases A and B permits the inference that the C-terminal residues of both alpha- and beta-subunits are removed. Small synthetic peptides are poor or non-substrates. A low activity with hippuryl-L-lysine may be due to contamination of the preparation with carboxypeptidase N. The possibility has been excluded that the action of conversion factor on creatine kinase involves modification of the protein thiol groups. Mr, substrate-specificity, pH-activity profile and the effects of metal ions distinguish creatine kinase conversion factor from carboxypeptidases A, B and N. On the basis of this evidence it is proposed to give the conversion factor the provisional name of carboxypeptidase K.     

Subunit structure of D-galactose dehydrogenase from Pseudomonas saccharophila.

1. D-Galactose dehydrogenase from Pseudomonas saccharophila (molecular weight 102 000) dissociates in 8 M urea into its subunits (molecular weight 25 000) which migrate in polyacrylamide gels, containing 8 M urea, as a single band. 2. The N-terminal residue determination by the dansyl method revealed only serine. 3. The C-terminal group determination with carboxypeptidase A and B indicated the sequence -Tyr-His-Leu. Leucine as the single C-terminal amino acid was confirmed by the tritiation method and by tritiation and subsequent degradation with carboxypeptidases. 4. The fragmentation of D-galactose dehydrogenase (24 mol methionine per mol enzyme) by CNBr resulted in six peptides, as detected in disc electrophoresis and substantiated by end group determination, indicating the identity of the subunits. 5. The treatment of D-galactose dehydrogenase (24 mol lysine and 52 mol arginine per mol enzyme) with trypsin and subsequent peptide mapping showed 21, perhaps 22 peptides, indicating a structure comprising four identical subunits.     

Replacement of lysine by arginine, phenylalanine and tryptophan in the reactive site of the bovine trypsin-kallikrein inhibitor (Kunitz) and change of the inhibitory properties.

The reactive-site sequence of a proteinase inhibitor can be written as . . . -P3-P2-P1-P'1-P'2-P'3- . . . , where-P1-P'1-denotes the reactive site. Three semisynthetic homologues have been synthesized of the bovine trypsin-kallikrein inhibitor (Kunitz) with either arginine, phenylalanine or tryptophan in place of the reactive-site residue P1, lysine-15. These homologues correspond to gene products after mutation of the lysine 15 DNA codon to an arginine, phenylalanine or tryptophan DNA codon. Starting from native (virgin) inhibitor, reactive-site hydrolyzed, still active (modified) inhibitor was prepared by chemical and enzymic reactions. Modified inhibitor was then converted into inactive des-Lys15-inhibitor by reaction with carboxypeptidase B. Inactive des-Lys15-inhibitor was reactivated by enzymic replacement of the P1 residue according to Leary and Laskowski, Jr. The introduction of arginine was catalyzed by an inverse reaction with carboxypeptidase B, while phenylalanine or tryptophan were replaced by carboxypeptidase A. The reactivated semisynthetic inhibitors were trapped by complex formation with either trypsin or chymotrypsin. The enzyme - inhibitor complexes were subjected to kinetic-control dissociation, and the semisynthetic virgin inhibitors were isolated. The inhibitory properties of the semisynthetic inhibitors have been investigated against bovine trypsin and chymotrypsin and against porcine pancreatic kallikrein and plasmin. The homologues with either lysine or arginine in the P1 position are equally good inhibitors of trypsin, plasmin and kallikrein. The Arg-15-homologue is a slightly more effective kallikrein inhibitor than the Lys15-inhibitor. The semisynthetic phenylalanine and tryptophan homologues, however, are weak inhibitors of trypsin and still weaker inhibitors of kallikrein, but are excellent inhibitors of chymotrypsin. Their association constant with chymotrypsin is at least ten times higher than that of native Lys-15-inhibitor. A dramatic specificity change is observed with the phenylalanine and tryptophan homologues, which in contrast to the native inhibitor do not at all inhibit porcine plasmin. Thus, the nature of the P1 residue strongly influences the primary inhibitory specificity of the bovine inhibitor (Kunitz).     

Characterization of recombinant human alpha 2-antiplasmin and of mutants obtained by site-directed mutagenesis of the reactive site

Human alpha 2-antiplasmin (alpha 2AP) has been expressed in Chinese hamster ovary cells and purified from conditioned media. The recombinant protein (r alpha 2AP) is immunologically identical with natural alpha 2AP and indistinguishable with respect to plasmin(ogen) binding properties. Second-order rate constants (k1) for the interaction of alpha 2AP and r alpha 2AP with plasmin are both (1-2) X 10(7) M-1 s-1. In order to examine the effects of alterations within the reactive site of alpha 2AP, deletions of the P1 residue Arg-364 (r alpha 2AP-delta Arg364) or the P'1 residue Met-365 (r alpha 2AP-delta Met365) were introduced by in vitro site-directed mutagenesis. r alpha 2AP-delta Met365 completely retains its ability to inhibit both plasmin and trypsin, indicating that alpha 2AP has no absolute requirement for Met in the P'1 position. Unexpectedly, no increase in antithrombin activity was observed. r alpha 2AP-delta Arg364 has lost the ability to inhibit plasmin, trypsin, and thrombin, but unlike the wild-type protein, this variant is an effective elastase inhibitor (k1 = 1.5 X 10(5) M-1 s-1).     

alpha-Enolase binds to human plasminogen on the surface of Bacillus anthracis

alpha-enolase of Bacillus anthracis has recently been classified as an immunodominant antigen and a potent virulence factor determinant. alpha-enolase (2-phospho-d-glycerate hydrolase (EC 4.2.1.11), a key glycolytic metalloenzyme catalyzes the dehydration of d-(+)-2-phosphoglyceric acid to phosphoenolpyruvate. Interaction of surface bound alpha-enolase with plasminogen has been incriminated in tissue invasion for pathogenesis. B. anthracis alpha-enolase was expressed in Escherichia coli and the recombinant enzyme was purified to homogeneity that exhibited a K(m) of 3.3 mM for phosphoenolpyruvate and a V(max) of 0.506 microM min(- 1) mg(-1). B. anthracis whole cells and membrane vesicles probed with anti-enolase antibodies confirmed the surface localization of alpha-enolase. The specific interaction of alpha-enolase with human plasminogen (but not plasmin) evident from ELISA and the retardation in the native gel reinforced its role in plasminogen binding. Putative plasminogen receptors in B. anthracis other than enolase were also observed. This binding was found to be carboxypeptidase sensitive implicating the role of C-terminal lysine residues. The recombinant enolase displayed in vitro laminin binding, an important mammalian extracellular matrix protein. Plasminogen interaction conferred B. anthracis with a potential to in vitro degrade fibronectin and exhibit fibrinolytic phenotype. Therefore, by virtue of its interaction to host plasminogen and extracellular matrix proteins, alpha-enolase may contribute in augmenting the invasive potential of B. anthracis.     

Identification of the C-terminal portion of a protein by comparative peptide mapping

A method is presented for the simple identification of C-terminal fragment of proteins. The method consists of (i) C-terminal processing of a protein by carboxypeptidase and (ii) comparative peptide mapping of the intact and carboxypeptidase-excised protein after fragmentation by endoproteinase or by chemical cleavage. The peptide mapping was performed by means of high-performance reversed-phase chromatography, where the C-terminal fragment was identified as a peptide peak that was lost or decreased in the carboxypeptidase-excised protein. The C-terminal sequence of the protein could be then determined by sequential Edman degradation of the C-terminal fragment collected from the peptide mapping chromatography. The sensitivity of the method depends solely on the peptide detection and subsequent Edman degradation, currently available techniques of which require a nanomole to subnanomole quantity of protein. The present method can be coupled with conventional carboxypeptidase technology because it utilizes a protein portion remaining after carboxypeptidase digestion while released amino acids are needed in the conventional technique. The method would be particularly valuable in finding a gene probe site for a RNA message coding for the C-terminal portion of a molecule.     

C-terminal lysine variants in fully human monoclonal antibodies: investigation of test methods and possible causes

The C-terminal lysine variation is commonly observed in biopharmaceutical monoclonal antibodies. This modification can be important since it is found to be sensitive to the production process. The methods commonly used to probe this charge variation, including IEF, cIEF, ion-exchange chromatography, and LC-MS, were evaluated for their ability to effectively approximate relative percentages of lysine variants. A monoclonal antibody produced in a B cell hybridoma versus a CHO cell transfectoma was examined and it was determined that the relative amount of incorporated C-terminal lysine can vary greatly between these two production schemes. Another case study is shown whereby a different monoclonal antibody is subject to some minor process changes and the extent of lysine variation also exhibits a significant difference. During these studies the different methods for determining the extent of variation were evaluated and it was determined that LC-MS after trypsin digestion provides reproducible relative percentage information and has significant advantages over other methods. The final section of this work investigates the possible origins of this modification and evidence is shown that carboxypeptidase B or another basic carboxypeptidase causes this variation.     

Role of zinc ions in activation and inactivation of thrombin-activatable fibrinolysis inhibitor.

Thrombin-activatable fibrinolysis inhibitor (TAFI) circulates as an inactive proenzyme of a carboxypeptidase B-like enzyme (TAFIa). It functions by removing C-terminal lysine residues from partially degraded fibrin that are important in tissue-type plasminogen activator mediated plasmin formation. TAFI was classified as a metallocarboxypeptidase, which contains a Zn(2+), since its amino acid sequence shows approximately 40% identity with pancreatic carboxypeptidases, the Zn(2+) pocket is conserved, and the Zn(2+) chelator o-phenanthroline inhibited TAFIa activity. In this study we showed that TAFI contained Zn(2+) in a 1:1 molar ratio. o-Phenanthroline inhibited TAFIa activity and increased the susceptibility of TAFI to trypsin digestion. TAFIa is spontaneously inactivated (TAFIai) by a temperature-dependent intrinsic mechanism. The lysine analogue epsilon-ACA, which stabilizes TAFIa, delayed the o-phenanthroline mediated inhibition of TAFIa. We investigated if inactivation of TAFIa involves the release of Zn(2+). However, the zinc ion was still incorporated in TAFIai, indicating that inactivation is not caused by Zn(2+) release. After TAFIa was converted to TAFIai, it was more susceptible to proteolytic degradation by thrombin, which cleaved TAFIai at Arg(302). Proteolysis may make the process of inactivation by a conformational change irreversible. Although epsilon-ACA stabilizes TAFIa, it was unable to reverse inactivation of TAFIa or R302Q-rTAFIa, in which Arg(302) was changed into a glutamine residue and could therefore not be inactivated by proteolysis, suggesting that conversion to TAFIai is irreversible.     

Hepatitis induced by an IgM monoclonal antibody against procarboxypeptidase R

Procarboxypeptidase R (proCPR), also known as thrombin-activatable fibrinolysis inhibitor (TAFI), is present in plasma and can be activated to carboxypeptidase R (CPR) by trypsin-like enzymes such as thrombin and plasmin. CPR has the carboxypeptidase B-like activity that can inactivate the inflammatory peptides such as C5a by removing the C-terminal arginine and can interfere with fibrinolysis by removing C-terminal lysine residue of fibrin. In the present study, we conducted to produce monoclonal antibodies (mAbs) by using spleen cells from proCPR-deficient mice immunized by partially purified mouse proCPR. The mAbs obtained were IgM isotype and reacted with proCPR and interfered with activation of proCPR to CPR by thrombin-thrombomodulin complex. Some BALB/c mice implanted with the hybridoma died in 7 days, and intravenous injection of the mAb to BALB/c mice induced transient elevation of GOT and GPT in plasma although injection to the deficient mice did not. Furthermore, the histological features showed the focally lesions in liver tissue of BALB/c mice injected with the mAb. Since liver is the major site of proCPR synthesis, IgM mAb to proCPR should have induced local inflammation at the side resulting in induction of hepatitis.     

Role of cell-surface lysines in plasminogen binding to cells: identification of alpha-enolase as a candidate plasminogen receptor.

Plasminogen binding to cell surfaces results in enhanced plasminogen activation, localization of the proteolytic activity of plasmin on cell surfaces, and protection of plasmin from alpha 2-antiplasmin. We sought to characterize candidate plasminogen binding sites on nucleated cells, using the U937 monocytoid cell as a model, specifically focusing on the role of cell-surface proteins with appropriately placed lysine residues as candidate plasminogen receptors. Lysine derivatives with free alpha-carboxyl groups and peptides with carboxy-terminal lysyl residues were effective inhibitors of plasminogen binding to the cells. One of the peptides, representing the carboxy-terminal 19 amino acids of alpha 2-antiplasmin, was approximately 5-fold more effective than others with carboxy-terminal lysines. Thus, in addition to a carboxy-terminal lysyl residue, other structural features of the cell-surface proteins may influence their affinity for plasminogen. Affinity chromatography has been used to isolate candidate plasminogen receptors from U937 cells. A major protein of Mr 54,000 was recovered and identified as alpha-enolase by immunochemical and functional criteria. alpha-Enolase was present on the cell surface and was capable of binding plasminogen in ligand blotting analyses. Plasminogen binding activity of a molecular weight similar to alpha-enolase also was present in a variety of other cell types. Carboxypeptidase B treatment of alpha-enolase abolished its ability to bind plasminogen, consistent with the presence of a C-terminal lysyl residue. Thus, cell-surface proteins with carboxy-terminal lysyl residues appear to function as plasminogen binding sites, and alpha-enolase has been identified as a prominent representative of this class of receptors.     

Mutations in the substrate binding site of thrombin-activatable fibrinolysis inhibitor (TAFI) alter its substrate specificity

Thrombin-activable fibrinolysis inhibitor (TAFI) is a zymogen that inhibits the amplification of plasmin production when converted to its active form (TAFIa). TAFI is structurally very similar to pancreatic procarboxypeptidase B. TAFI also shares high homology in zinc binding and catalytic sites with the second basic carboxypeptidase present in plasma, carboxypeptidase N. We investigated the effects of altering residues involved in substrate specificity to understand how they contribute to the enzymatic differences between TAFI and carboxypeptidase N. We expressed wild type TAFI and binding site mutants in 293 cells. Recombinant proteins were purified and characterized for their activation and enzymatic activity as well as functional activity. Although the thrombin/thrombomodulin complex activated all the mutants, carboxypeptidase B activity of the activated mutants against hippuryl-arginine was reduced. Potato carboxypeptidase inhibitor inhibited the residual activity of the mutants. The functional activity of the mutants in a plasma clot lysis assay correlated with their chromogenic activity. The effect of the mutations on other substrates depended on the particular mutation, with some of the mutants possessing more activity against hippuryl-His-leucine than wild type TAFIa. Thus mutations in residues around the substrate binding site of TAFI resulted in altered C-terminal substrate specificity.     

The p11 subunit of annexin II heterotetramer is regulated by basic carboxypeptidase

The Ca(2+)-dependent phospholipid-binding protein annexin II heterotetramer (AIIt) is composed of two copies of annexin II and a p11 dimer. The interaction of the carboxyl-terminal lysine residues of the p11 subunit of AIIt with the lysine-binding kringle domains of plasminogen is believed to play a key role in plasminogen binding and stimulation of the tPA-catalyzed cleavage of plasminogen to plasmin. In the current report, we show that AIIt-stimulated plasminogen activation is regulated by basic carboxypeptidases, in vitro. The incubation of AIIt with a 1/400 molar ratio of carboxypeptidase B for periods as short as 2 min resulted in a significant loss in AIIt-stimulated plasminogen activation. Carboxypeptidase B (CpB) as well as thrombin-activated fibrinolysis inhibitor (TAFIa) and carboxypeptidase N (CpN) rapidly reduced AIIt-stimulated plasminogen activation by 80%. The molar ratio of carboxypeptidase/AIIt for half-maximal inhibition of AIIt was 1/4700, 1/700, and 1/500 for CpB, TAFIa, and CpN, respectively. Treatment of AIIt with carboxypeptidase resulted in loss of both carboxyl-terminal lysine residues from the p11 subunit, which correlated with a decrease in the k(cat) and an increase in the K(m) for plasminogen activation. The data reveal a novel mechanism for the regulation of AIIt-stimulated plasminogen activation.     

Biosynthesis of peptide neurotransmitters: studies on the formation of peptide amides

A high proportion of peptide transmitters and peptide hormones terminate their peptide chain in a C-terminal amide group which is essential for their biological activity. The specificity of an enzyme that catalyses the formation of the amide was investigated with the aid of synthetic peptide substrates. With peptides containing l-amino acids the enzyme exhibited an essential requirement for glycine in the C-terminal position; amidation did not take place with peptides that had leucine, alanine, glutamic acid, lysine or N-methylglycine at the C-terminus and a peptide extended by the attachment of lysine to the C-terminal glycine did not act as a substrate. Amidation did occur with a peptide containing C-terminal D-alanine but no reaction was detected with peptides having C-terminal, D-serine or D-leucine. In tripeptides with a neutral amino acid in the penultimate position, amidation, took place readily but the reaction was slower when this position was occupied by an acidic or a basic residue. A series of overlapping peptides with C-terminal glycine, based on partial sequences of calcitonin, underwent amidation at similar rates, indicating that the amidating enzyme recognizes only a limited sequence at the C-terminus of its substrates. The results provide evidence that the amidating enzyme has a highly compact substrate binding site.