Structure-function analysis of tissue-type plasminogen activator by linker-insertion, point and deletion mutagenesis

We undertook a structure–function analysis of human tissueplasminogen activator (tPA) using linker-scanning and deletionmutagenesis. Synthetic oligonucleotide linkers were introducedinto the tPA cDNA at pre-existing restriction enzyme sites.This generated a series of tPA variants which contained smallprimary sequence alterations consisting of point mutations,deletions or insertions. The majority of the linker-insertionvariants demonstrate a significant reduction in amidolytic andfibrinolytic activity in comparison to wild-type tPA. The exceptionsare the variants with linker-inserts placed at the BglII(115)and StyI(277) sites of the tPA cDNA (4SLEG5 and 57LEA58 respectively),which encode insertions at the boundaries of the finger domain.The variants with linker-inserts in the light chain (proteasedomain) of tPA are the lowest in enzymatic activity. Particularlysensitive to mutation are highly conserved amino acids. Heavychain deletion variants were constructed from point mutantsat the domain boundaries of tPA. Deletion of the kringle domainslowers the fibrinolytic activity to a greater extent than deletionof the finger or growth factor domains. We conclude that alterationsin any domain of the tPA molecule, and particularly in the highlyconserved residues within these domains, can affect fibrinolyticactivity.     

Variants of human tissue-type plasminogen activator substituted at the protease cleavage site and glycosylation sites, and truncated at the N- and C-termini

Mutations were directed to specific regions of the human tissue-typeplasminogen activator (t-PA) gene in an effort to better definestructure–function relationships of the enzyme. Threetypes of modifications were effected by in vitro mutagenesis:elimination of glycosylation sites; substitutions of amino acidsat the cleavage site for conversion of single-chain t-PA totwo-chain t-PA; and truncations of the N- and C-termini. Thirteenvariants were purified from permanent CHO cell lines and analyzedfor specific activity, fibrin stimulation, fibrin binding, inhibitionby plasminogen activator inhibitor-2 (PAI-2) and half-life.The results of these analyses are: (i) variants with carbohydrate–depletedkringle domains possessed higher specific activities than wild-typet-PA; (ii) a cleavage site variant substituted at Arg275 withGly had greatly reduced specific activity; (iii) two variantssubstituted at Lys277 exhibited altered interactions with PAI-2;(iv) the variant with a truncated C-terminus had reduced activityin the absence of fibrin; and (v) no variants had significantlyaltered half–lives. In order to test the effects of combiningmutations, four additional variants were produced. Each combinationvariant retained at least one of the altered properties observedin the original variants, and in three of the variants the diverseproperties were additive.     

The position of the structurally autonomous kringle 2 domain influences the functional features of tissue-type plasminogen activator

Tissue-type plasminogen activator (t-PA) is composed of structurallyautonomous domains. From the N-terminus of t-PA, a finger-likedomain (F), an epidermal growth factor-like domain (G), twokringle domains (Kl and K2) and a serine protease domain (P)can be discerned. The K2 domain of t-PA is known to be involvedin lysine binding, fibrin binding and fibrin-dependent plasminogenactivation. To study the functional autonomy of the K2 domainin t-PA we constructed, with the aid of a cassette t-PA gene[Rehberg et al. (1989) Protein Engng, 2,371–377], mutantt-PA genes coding for four molecules (FGK1K2P, FGK2K1P, GK1K2Pand GK2K1P) in which the K2 domain was placed in two differentpositions in t-PA. The DNAs of wild-type t-PA and the t-PA variantswere expressed in Chinese hamster ovary cells and the recombinantproteins were purified by affinity chromatography.All moleculeswere expressed in their single-chain form and could be convertedto their two-chain form. With these molecules, lysine binding,fibrin binding and fibrin-dependent plasminogen activation werestudied. All variants showed affinity for lysyl-Sepharose andaminohexyl-Sepharose. Reversal of the K domains (FGK2K1P versusFGK2K1P and GK1K2P versus GK2K1P) resulted in a 23–47%weaker interaction to both lysyl-Sepharose and aminohexyl-Sepharose.Deleting the F domain (FGK1K2P versus GK1K2P and FGK2K1P versusGK2K1P) resulted in a 20–70% improvement of the interactionslysyl-Sepharose and aminohexyl-Sepharose. All variants boundto a forming fibrin clot. Reversal of the K domains (FGK1K2Pversus FGK2K1P) reduced fibrin binding. In the presence of thelysine analogue -amino caproic acid, only FGK1K2P bound to fibrin.All variants activated plasminogen. In the absence of fibrinogenCNBr fragments (mimic of fibrin), the reversal of the K domain(FGK2K1P) resulted in a 2-fold improved plasminogen activation.In the presence of a fibrin mimic, the plasminogen activationsof the F domain deletion analogues GK1K2P and GK2K1P were foundto be decreased 2- to 4-fold. From these results we concludedthat the function of t-PA in lysine binding, fibrin bindingand fibrin-dependent plasminogen activation is dependent onthe correct spatial orientation of the K2 domain within thet-PA molecule     

Biochemical properties of the kringle 2 and protease domains are maintained in the refolded t-PA deletion variant BM 06.022

BM 06.022 is a t-PA deletion variant which comprises the kringle2 and the protease domain. Production of BM 06.022 in Escherichiacoli leads to the formation of inactive inclusion bodies, whichhave to be refolded by an in vitro refolding process to achieveactivity and proper structure of the domains. We analysed thebiochemical properties of BM 06.022 to obtain some informationabout the structure of kringle 2 and the protease as comparedwith the structure of these domains in the intact t-PA molecule.The kinetic analysis of the amidolytic activity of BM 06.022and CHO-t-PA yielded similar values for k_cat (13.9 s^-1and 11.4s^-1for the single chain forms and 33.9 s^-1and 27.1 s^-1for thetwo chain forms of BM 06.022 and CHO-t-PA, respectively) andfor k_m, (2.5 mM and 2.1 mM for the single chain forms and 0.5mM and 0.3 mM for the two chain forms of BM 06.022 and CHO-t-PA,respectively). BM 06.022 and CHO-t-PA have the same plasminogenolyticactivity in the absence of CNBr fragments of fibrinogen. However,BM 06.022 has a lower plasminogenolytic activity in the presenceof CNBr fragments of fibrinogen and a lower affinity to fibrinas compared with CHO-t-PA. The affinity of BM 06.022 for fibrinis completely suppressed by 0.3 mM eaminocaproic acid, whilethe intact t-PA has a residual affinity of 30%. The dissociationconstants for the interaction with the lysine analogue e-aminocaprokacid are 0.10 mM and 0.09 mM for BM 06.022 and the intact t-PA,respectively. Furthermore, BM 06.022 and CHO-t-PA are inhibitedby PAI-1 in a similar manner     

Construction of a tissue plasminogen activator gene having unique restriction sites to facilitate domain manipulation: a model for the analysis of multi-domain proteins

We have designed and constructed a DNA sequence encoding humantissue plasminogen activator (tPA) with convenient restrictionsites that flank each of the domains of the heavy chain. Toaccomplish this, the first 1095 bases of the gene coding forthe mature protein were synthesized with unique restrictionsites engineered into the interdomainal regions. This syntheticconstruction was then ligated to a cDNA fragment of the tPAgene that encoded the active site, thus generating a full-lengthtPA gene. The gene products produced by Chinese hamster ovary(CHO) cells transfected with either the tPA cassette gene orthe tPA cDNA gene were then compared with the tPA produced byBowes melanoma cells to determine whether or not synthetic interdomainalamino acid changes had an effect on the biochemical characteristicsof the molecule. Specifically, molecular weight, specific activity,enhancement by fibrinogen fragments and kinetic constants wereanalysed. None of the properties examined were significantlydifferent from those of the native melanoma tPA. Therefore,the cassette gene described herein should provide considerableversatility and precision in the construction of tPA mutantsby facilitating the manipulation of the finger, growth factorand kringle domains, and likewise should be useful in assessingthe function of these domains within the tPA molecule. We presentthis cassette gene system as a model for the analysis of proteindomain function applicable to other multi-domain proteins.     

Domain-domain interactions in hybrids of tissue-type plasminogen activator and urokinase-type plasminogen activator

Fibrin-dependent plasminogen activation by tissue-type plasminogenactivator (t-PA) is in part associated with the presence ofthe kringle 2 domain in t-PA. Within this kringle 2 domain alysyl-binding site has been described. The plasminogen to plasminconversion by urokinase-type plasminogen activator (u-PA), incontrast to that of t-PA, is not enhanced in the presence offibrin. Within the u-PA kringle domain no lysyl-binding siteis found. To study whether introduction of a lysyl-binding sitein the u-PA kringle domain will make u-PA a fibrin-dependentplasminogen activator, three stretches of amino acid residuesof the u-PA kringle domain (A²⁸-Q³³, D⁵⁵-N⁵⁷ and G⁶⁷-V⁷²) weresubstituted by three stretches of amino acids from the correspondingpositions of the kringle 2 domain of t-PA (M²⁸-K³³, D⁵⁵-D⁵⁷and N⁶⁷-W⁷²). These changes resulted in the creation of thelysyl-binding site consensus of the kringle 2 domain (K³³, D⁵⁵,D⁵⁷, W⁶² and W⁷²) in the u-PA kringle. However, the resultingu-PA mutant did not interact with lysyl-Sepharose, nor did itdisplay fibrin-enhanced plasminogen activation in the presenceof soluble fibrin mimic. When the kringle domain of u-PA wasreplaced by the kringle 2 domain of t-PA, similar results wereobtained. The hybrid protein hardly interacted with lysyl-Sepharoseand the plasminogen activation was not enhanced in the presenceof fibrin mimic However, the N-terminal fragment isolated fromthis hybrid molecule (consisting of growth factor domain andkringle 2 domain) did interact with lysyl-Sepharose, suggestingthat in the hybrid molecule a functional lysyl-binding siteis present but not operational. Indeed, lysine analogue (e-amino-caproicacid) sensitive binding of isolated t-PA kringle 2 domain tou-PA could be observed. The modified u-PA kringle, the wildtype u-PA kringle and the kringle 2 of the u-PA hybrid werealso placed N-terminal of the protease domain of t-PA. As expected,the t-PA mutant consisting of the kringle 2 domain and the proteasedomain bound to lysyl-Sepharose and showed fibrin-dependentplasminogen activation. Further, the hybrid molecule consistingof the u-PA kringle placed N-terminal of the t-PA protease domaindid not display these features. Introduction of the modifiedu-PA kringle N-terminal of the t-PA protease domain resultedin a very weak interaction with lysyl-Sepharose. Despite thehigh overall similarity in primary structure of the modifiedu-PA kringle and t-PA kringle 2 (68%), no fibrin-dependent plasminogenactivation of this hybrid molecule was observed. The above-mentionedresults question the concept that the structural auto-nomousdomains within hybrid plasminogen activators t-PA and u-PA functionas autonomous domains and suggest that interactions betweenthe kringle and the protease domain in hybrid molecules stronglyinfluences their functional features     

Expression of active domains of a human folate-dependent trifunctional enzyme in Escherichia coli

The cDNA encoding the human trifunctional enzyme methylenetetrahydrofolatedehydrogenase-methenyltetrahydrofolate cyclohydrolase-formyltetrahydrofolatesynthetase was engineered to contain a prokaryotic ribosomebinding site and was expressed under the bacteriophage T7 RNApolymerase promoter in Escherichia coli. Site-directed mutagenesiswas used to prepare constructs that encode separately the dehydrogenase/cyclohydrolase(D/C) domain as amino acid residues 1–301, and the synthetase(Syn) domain as residues 304–935. Both domains formedactive enzymes thereby demonstrating their ability to fold independently.The full-length enzyme, D/C and Syn domains were expressed atlevels 4-, 55- and 3-fold higher than the specific activitiesfound in liver. Additional mutagenesis and independent expressionof domains further defined the interdomain region to includeamino acids 292–310. The D/C domain was purified to homogeneityby a single affinity chromatographic step, and the full-lengthprotein in a twostep procedure. The kinetic properties of theD/C domain appear unaltered from those of the trifunctionalenzyme.     

Making tissue-type plasminogen activator more fibrin specific

The fibrin specificity of tissue-type plasminogen activatorcan be increased by mutagenesis within at least four sites inthe protease domain. These sites include residue I276, the newN-terminus formed by conversion to a two-chain structure, residueson either side of the active site cleft, KHRR 296–299or DDD 364–366, a charged surface involved in fibrin interactions,which includes residues H432, R434, D460, R462 and a loop structure,PQANL 466–470, near the fibrin-binding patch. Variantswith mutations at any of these sites have low fibrinogen-stimulatedactivity, whereas fibrin-stimulated activity is at least normal.Kinetic analysis reveals that mutations at these positions reducethe k_cat in the presence of fibrinogen, but leave the moleculeswith normal kinetic constants in the presence of fibrin. A significantexception is found at positions 296–299, where the presenceof fibrin manifests significant increases in both k_cat and K_m.Combinations of mutations at these sites appear to be additivewith respect to fibrin specificity.     

Synthesis and mutagenesis of an IgG-binding protein based upon protein A of Staphylococcus aureus

A novel protein able to bind with high affinity to the Fc fragmentof IgG from a variety of animals has been produced by a genesynthesis approach. The IgG binding is accomplished by the presenceof a single or two consecutive domains based upon domain B fromprotein A of Staphylo-coccus aureus. The IgG-binding moietyis fused to a peptide containing 21, 53 or 81 amino acids derivedfrom the N-terminus of bovine DNase I. The latter is presentto guide the expression of the protein in Escherichia coli intoan inclusion body. This facilitates the high expression andrecovery of the IgG-binding domains. The binding activity ofthis fusion protein is very close to that of the native proteinA. Site-directed mutagenesis of the fusion protein and subsequentidentification of changed binding interactions is reported.     

Selection and characterization of HER2/neu-binding affibody ligands

Affibody® (affibody) ligands that are specific for the extracellulardomain of human epidermal growth factor receptor 2 (HER2/neu)have been selected by phage display technology from a combinatorialprotein library based on the 58 amino acid residue staphylococcalprotein A-derived Z domain. The predominant variants from thephage selection were produced in Escherichia coli, purifiedby affinity chromatography, and characterized by biosensor analyses.Two affibody variants were shown to selectively bind to theextracellular domain of HER2/neu (HER2-ECD), but not to controlproteins. One of the variants, denoted His₆-Z_HER2/neu:4, wasdemonstrated to bind with nanomolar affinity (     

pH-sensitive interactions between IgG and a mutated IgG-binding protein based upon two B domains of Protein A from Staphylococcus aureus

A fusion protein, consisting of the N-terminal 81 amino acidsfrom an inactive bovine DNase I (Q38,E39–E38,Q39) andtwo sequential synthetic IgG-binding domains based upon domainB of Protein A from Staphylococcus aureus has been shown tobind to porcine IgG with a similar affinity and pH profile toProtein A. The same residue in each B domain (Tyr111 and Tyr169)has been mutated by cassette mutagenesis to Ser, Glu, His, Lysor Arg and the effect of the mutation on binding interactionswith porcine IgG investigated. The evidence presented suggeststhat the interactions at the B domain are highly sensitive tothe presence of a charged residue.     

Structure-function relationships in the catalytic and starch binding domains of glucoamylase

Sixteen primary sequences from five sub-families of fungal,yeast and bacterial glucoamylases were related to structuralinformation from the model of the catalytic domain of Aspergillusawamori var. X100 glucoamylase obtained by protein crystallography.This domain is composed of thirteen -belices, with five conservedregions defining the active site. Interactions between methyl-maltoside and active site residues were modelled, and the importanceof these residues on the catalytic action of different glucoamylaseswas shown by their presence in each primary sequence. The overallstructure of the starch binding domain of some fungal glucoamylaseswas determined based on homology to the Cterminal domains ofBacillus cyclodextrin glucosyltransferases. Crystallographyindicated that this domain contains 6–8 ß-strandsand homology allowed the attribution of a disulfide bridge inthe glucoamylase starch binding domain. Glucoamylase residuesThr525, Asn530 and Trp560, homologous to Bacillus stearothermophiluscyclodextrin glucosyltransferase residues binding to maltosein the Cterminal domain, could be involved in raw-starch binding.The structure and length of the linker region between the catalyticand starch binding domains in fungal glucoamylases can varysubstantially, a further indication of the functional independenceof the two domains.     

Properties of a single-chain antibody containing different linker peptides

Single-chain antibodies were constructed using six differentlinker peptides to join the V_H and V_L domains of an anti-2-phenyloxazolone(Ox) antibody. Four of the linker peptides originated from theinterdomain linker region of the fungal cellulase CBHI and consistedof 28, 11, six and two amino acid residues. The two other linkerpeptides used were the (GGGGS)₃ linker with 15 amino acid residuesand a modified IgG2b hinge peptide with 22 residues. Proteolyticstability and Ox binding properties of the six different scFvderivatives produced in Escherichia coli were investigated andcompared with those of the corresponding Fv fragment containingno joining peptide between the V domains. The hapten bindingproperties of different antibody fragments were studied by ELISAand BIAcore^TM. The interdomain linker peptide improved the haptenbinding properties of the antibody fragment when compared withFv fragment, but slightly increased its susceptibility to proteases.Single-chain antibodies with short CBHI linkers of 11, six andtwo residues had a tendency to form multimers which led to ahigher apparent affinity. The fragments with linkers longerthan 11 residues remained monomeric.     

Point mutations modifying the thrombin inhibition kinetics and antithrombotic activity in vivo of recombinant hirudin

The hirudin variant HV2 was modified by in vitro site-specificmutagenesis of HV2 cDNA to generate HV2(Asn-47 – Lys),HV2(Asn-47 – Arg) and HV2(Lys-35 – Thr, Asn-47 –Lys). Residues 35 and 47 are positioned respectively withinthe finger and prothrombin-like domains of hirudin, both ofwhich have been suggested as thrombin binding sites. The modifiedpolypeptides were synthesized in Saccharomyces cerevisiae usinga secretion vector and purified from culture supernatants. Byanalysis of the human -thrombin: hirudin inhibition reactionin steady-state conditions it was shown that the dissociationconstants for HV2(Lys-47) and HV2(Arg-47) were 5- to 14-foldlower than for unmodified HV2, whereas mutation of Lys-35 didnot significantly alter the inhibition kinetics. Furthermore,HV2(Lys-47), whose sequence is identical to a natural hirudinvariant, displayed enhanced antithrombotic activity in vivo,having a 100-fold lower ED₅₀ compared to HV2 in the rabbit Wesslervenous thrombosis model. These results support a role for theprothrombin-like domain in thrombin binding and, moreover, demonstratethat in vivo antithrombotic efficiency correlates with the dissociationconstant of the inhibition reaction.     

Affinity maturation of a Taq DNA polymerase specific affibody by helix shuffling

The possibility of increasing the affinity of a Taq DNA polymerasespecific binding protein (affibody) was investigated by an -helixshuffling strategy. The primary affibody was from a naive combinatoriallibrary of the three-helix bundle Z domain derived from staphylococcalprotein A. A hierarchical library was constructed through selectivere-randomization of six amino acid positions in one of the two-helices of the domain, making up the Taq DNA polymerase bindingsurface. After selections using monovalent phage display technology,second generation variants were identified having affinities(K_D) for Taq DNA polymerase in the range of 30–50 nM asdetermined by biosensor technology. Analysis of binding dataindicated that the increases in affinity were predominantlydue to decreased dissociation rate kinetics. Interestingly,the affinities observed for the second generation Taq DNA polymerasespecific affibodies are of similar strength as the affinitybetween the original protein A domain and the Fc domain of humanimmunoglobulin G. Further, the possibilities of increasing theapparent affinity through multimerization of affibodies wasdemonstrated for a dimeric version of one of the second generationaffibodies, constructed by head-to-tail gene fusion. As comparedwith its monomeric counterpart, the binding to sensor chip immobilizedTaq DNA polymerase was characterized by a threefold higher apparentaffinity, due to slower off-rate kinetics. The results showthat the binding specificity of the protein A domain can bere-directed to an entirely different target, without loss ofbinding strength.     

Relationship between thermal stability, degradation rate and expression yield of barnase variants in the periplasm of Escherichia coli

An advantage of exporting a recombinant protein to the periplasmof Escherichia coli is decreased proteolysis in the periplasmcompared with that in the cytoplasm. However, protein degradationin the periplasm also occurs. It has been widely accepted thatthe thermodynamic stability of a protein is an important factorfor protein degradation in the cytoplasm of E.coli. To investigatethe effect of the thermodynamic stability of an exported proteinon the extent of proteolysis in the periplasm, barnase (an extracellularribonuclease from Bacillus amyloliquefaciens) fused to alkalinephosphatase leader peptide was used as a model protein. A setof singly or doubly mutated barnase variants were constructedfor export to the E.coli periplasm. It was found that the half-lifeof the barnase variants in vivo increased with their thermodynamicstability in vitro. A dominant factor for the final yield ofexported barnase was not exportability but the turnover rateof the barnase variant. The yield of a stabilized mutant wasup to 50% higher than that of the wild type. This suggests thatexporting a protein to the periplasm and using protein engineeringto enhance the stability can be combined as a strategy to optimizethe production of recombinant proteins.     

Site-directed mutagenesis of a calcium binding site modifies specifically the different biochemical properties of annexin I

All the functions of annexins in vitro as well as in vivo aremediated and probably regulated by calcium. We have used recombinantannexin I, synthesized by Escherichia coli, and we have performedsite-directed mutagenesis. We have mutated the endonexin foldof domain 2 that binds calcium. Mutations were performed inthis domain of the molecule because it perfectly matches thecalcium binding consensus sequence. The two glycines of thisfold were mutated into glutamic acid. The helix content andthe stability of the mutants are identical to those of the wild-type,suggesting that the mutations did not drastically affect thestructure of the protein. The two mutants showed modified calciumbinding affinities. However, the calcium binding affinity ofthe G131E mutant was far more altered than that of the G129Emutant. Furthermore, other biochemical properties of these mutantswere modified to different extents. The binding to phospholipidwas not seriously affected, whereas the selfassociation waslost by the G131E mutant. In the same way, liposome aggregationis conserved, but modified, while the calcium affinity measuredby equilibrium dialysis is dramatically altered.     

The random peptide library-assisted engineering of a C-terminal affinity peptide, useful for the detection and purification of a functional Ig Fv fragment

The facile detection and purification of a recombinant proteinwithout detailed knowledge about its individual biochemicalproperties constitutes a problem of general interest in proteinengineering. The use of a novel kind of random peptide libraryfor the stepwise engineering of a C-terminal fusion peptidewhich confers binding activity towards streptavidin is describedin this study. Because of its widespread use as part of a varietyof conjugates and other affinity reagents, streptavidin constitutesthe binding partner of choice both for detection and purificationpurposes. The streptavidin-affinity tag was engineered at theC-terminus of the V_H domain as part of the D1.3 Fv fragmentwhich was functionally expressed in Escherichia coli. Irrespectiveof whether it was displayed by the V_H or the V_L domain, theoptimized version of the affinity peptide termed ‘Strep-tag’allowed the detection of the Fv fragment both on Western blotsand in ELISAs by a streptavidin–alkaline phosphatase conjugate.In addition, the one-step purification of the intact Fv fragmentcarrying a single Strep-tag at the C-terminus of only one ofits domains was achieved by affinity chromatography with streptavidin-agaroseusing very mild elution conditions.     

Effects of humanization by variable domain resurfacing on the antiviral activity of a single-chain antibody against respiratory syncytial virus

HNK20 is a mouse monoclonal IgA that binds to the F glycoproteinof respiratory syncytial virus (RSV) and neutralizes the virus,both in vitro and in vivo. The single-chain antibody fragment(scFv) derived from HNK20 is equally active and has allowedus to assess rapidly the effect of mutations on affinity andantiviral activity. Humanization by variable domain resurfacingrequires that surface residues not normally found in a humanFv be mutated to the expected human amino acid, thereby eliminatingpotentially immunogenic sites. We describe the constructionand characterization of two humanized scFvs, hu7 and hu10, bearing7 and 10 mutations, respectively. Both molecules show unalteredbinding affinities to the RSV antigen (purified F protein) asdetermined by ELISA and surface plasmon resonance measurementsof binding kinetics (K_a 1x10⁹ M^–1). A competition ELISAusing captured whole virus confirmed that the binding affinitiesof the parental scFv and also of hu7 and hu10 scFvs were identical.However, when compared with the original scFv, hu10 scFv wasshown to have significantly decreased antiviral activity bothin vitro and in a mouse model. Our observations suggest thatbinding of the scFv to the viral antigen is not sufficient forneutralization. We speculate that neutralization may involvethe inhibition or induction of conformational changes in thebound antigen, thereby interfering with the F protein-mediatedfusion of virus and cell membranes in the initial steps of infection.