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The activity of tumor necrosis factor (TNF), a cytokine involved in inflammatory pathologies, can be inhibited by antibodies or trap molecules. Herein, llama-derived variable heavy-chain domains of heavy-chain antibody (VHH, also called Nanobodies™) were generated for the engineering of bivalent constructs, which antagonize the binding of TNF to its receptors with picomolar potencies. Three monomeric VHHs (VHH#1, VHH#2, and VHH#3) were characterized in detail and found to bind TNF with sub-nanomolar affinities. The crystal structures of the TNF-VHH complexes demonstrate that VHH#1 and VHH#2 share the same epitope, at the center of the interaction area of TNF with its TNFRs, while VHH#3 binds to a different, but partially overlapping epitope. These structures rationalize our results obtained with bivalent constructs in which two VHHs were coupled via linkers of different lengths. Contrary to conventional antibodies, these bivalent Nanobody™ constructs can bind to a single trimeric TNF, thus binding with avidity and blocking two of the three receptor binding sites in the cytokine. The different mode of binding to antigen and the engineering into bivalent constructs supports the design of highly potent VHH-based therapeutic entities.

Neutralizing the interaction of the platelet receptor gpIb with VWF is an attractive strategy to treat and prevent thrombotic complications. ALX-0081 is a bivalent Nanobody which specifically targets the gpIb-binding site of VWF and interacts avidly with VWF. Nanobodies are therapeutic proteins derived from naturally occurring heavy-chain-only Abs and combine a small molecular size with a high inherent stability. ALX-0081 exerts potent activity in vitro and in vivo. Perfusion experiments with blood from patients with acute coronary syndrome on standard antithrombotics demonstrated complete inhibition of platelet adhesion after addition of ALX-0081, while in the absence of ALX-0081 residual adhesion was observed. In a baboon efficacy and safety model measuring acute thrombosis and surgical bleeding, ALX-0081 showed a superior therapeutic window compared with marketed antithrombotics. Pharmacokinetic and biodistribution experiments demonstrated target-mediated clearance of ALX-0081, which leads to a self-regulating disposition behavior. In conclusion, these preclinical data demonstrate that ALX-0081 combines a high efficacy with an improved safety profile compared with currently marketed antithrombotics. ALX-0081 has entered clinical development.

Inflammatory bowel disease (IBD) is a chronic inflammatory gastrointestinal disorder. Systemic treatment of IBD patients with anti-tumor necrosis factor (TNF)-alpha antibodies has proven to be a highly promising approach, but several drawbacks remain, including side effects related to systemic administration and high cost of treatment. Lactococcus lactis was engineered to secrete monovalent and bivalent murine (m)TNF-neutralizing Nanobodies as therapeutic proteins. These therapeutic proteins are derived from fragments of heavy-chain camelid antibodies and are more stable than conventional antibodies. L. lactis-secreted anti-mTNF Nanobodies neutralized mTNF in vitro. Daily oral administration of Nanobody-secreting L. lactis resulted in local delivery of anti-mTNF Nanobodies at the colon and significantly reduced inflammation in mice with dextran sulfate sodium (DSS)-induced chronic colitis. In addition, this approach was also successful in improving established enterocolitis in interleukin 10 (IL10)(-/-) mice. Finally, L. lactis-secreted anti-mTNF Nanobodies did not interfere with systemic Salmonella infection in colitic IL10(-/-) mice.In conclusion, this report details a new therapeutic approach for treatment of chronic colitis, involving in situ secretion of anti-mTNF Nanobodies by orally administered L. lactis bacteria. Therapeutic application of these engineered bacteria could eventually lead to more effective and safer management of IBD in humans.

Objective: The advent of tumor necrosis factor (TNF)-blocking drugs has provided rheumatologists with an effective, but highly expensive, treatment for the management of established rheumatoid arthritis (RA). Our aim was to explore preclinically the application of camelid anti-TNF VHH proteins, which are single-domain antigen binding (VHH) proteins homologous to human immunoglobulin V(H) domains, as TNF antagonists in a mouse model of RA., Methods: Llamas were immunized with human and mouse TNF, and antagonistic anti-TNF VHH proteins were isolated and cloned for bacterial production. The resulting anti-TNF VHH proteins were recombinantly linked to yield bivalent mouse and human TNF-specific molecules. To increase the serum half-life and targeting properties, an anti-serum albumin anti-TNF VHH domain was incorporated into the bivalent molecules. The TNF-neutralizing potential was analyzed in vitro. Mouse TNF-specific molecules were tested in a therapeutic protocol in murine collagen-induced arthritis (CIA). Disease progression was evaluated by clinical scoring and histologic evaluation. Targeting properties were evaluated by 99mTc labeling and gamma camera imaging., Results: The bivalent molecules were up to 500 times more potent than the monovalent molecules. The antagonistic potency of the anti-human TNF VHH proteins exceeded even that of the anti-TNF antibodies infliximab and adalimumab that are used clinically in RA. Incorporation of binding affinity for albumin into the anti-TNF VHH protein significantly prolonged its serum half-life and promoted its targeting to inflamed joints in the murine CIA model of RA. This might explain the excellent therapeutic efficacy observed in vivo., Conclusion: These data suggest that because of the flexibility of their format, camelid anti-TNF VHH proteins can be converted into potent therapeutic agents that can be produced and purified cost-effectively.

Whereas functional heavy (H)-chain antibodies devoid of light (L)- chains account for about half of the circulating immunoglobulins in Camelidae, H-chain only antibodies (HCAbs) are not produced in other healthy mammals including rodents and humans. To test the feasibility of expressing single chain antibodies in the mouse, which on account of their small size and antigen-recognition properties would have a major impact on antibody engineering strategies, we constructed a rearranged dromedary H-chain gene encoding the immunoglobulin G2a (IgG2a) isotype with specificity for hen-egg lysozyme (HEL). This IgG2a H-chain gene was introduced into mouse myeloma cells not expressing endogenous immunoglobulin H- or L-chains. Unexpectedly the mouse cells processed and expressed the introduced H-chain as naturally occurring dromedary antibody. For this the first constant (C) region exon was proficiently removed from the recombinant H-chain transcript. This resulted in specific H-chain antibodies of the correct molecular weight (2 x 50 000 MW) secreted as disulfide-linked homodimers and displayed on the mouse cell surface as glycosyl-phosphatidyl-inositol-linked B-cell receptor. The results indicate that antibody expression and maturation without immunoglobulin L-chain is feasible and paves the way for the generation of transgenic single chain antibody repertoires.

Camelids produce functional antibodies devoid of light chains and CH1 domains. The antigen-binding fragment of such heavy chain antibodies is therefore comprised in one single domain, the camelid heavy chain antibody VH (VHH). Here we report on the structures of three dromedary VHH domains in complex with porcine pancreatic alpha-amylase. Two VHHs bound outside the catalytic site and did not inhibit or inhibited only partially the amylase activity. The third one, AMD9, interacted with the active site crevice and was a strong amylase inhibitor (K(i) = 10 nm). In contrast with complexes of other proteinaceous amylase inhibitors, amylase kept its native structure. The water-accessible surface areas of VHHs covered by amylase ranged between 850 and 1150 A(2), values similar to or even larger than those observed in the complexes between proteins and classical antibodies. These values could certainly be reached because a surprisingly high extent of framework residues are involved in the interactions of VHHs with amylase. The framework residues that participate in the antigen recognition represented 25-40% of the buried surface. The inhibitory interaction of AMD9 involved mainly its complementarity-determining region (CDR) 2 loop, whereas the CDR3 loop was small and certainly did not protrude as it does in cAb-Lys3, a VHH-inhibiting lysozyme. AMD9 inhibited amylase, although it was outside the direct reach of the catalytic residues; therefore it is to be expected that inhibiting VHHs might also be elicited against proteases. These results illustrate the versatility and efficiency of VHH domains as protein binders and enzyme inhibitors and are arguments in favor of their use as drugs against diabetes.

The variable domain of functional heavy chain antibodies (VHH) devoid of light chains, found in camels, constitute the smallest intact antigen-binding domain fragment. Two camel single-domain fragments, cAb-Lys2 and cAb-Lys3, recognizing an overlapping epitope of lysozyme with a dissociation constant of 2 nM and 65 nM, respectively, and a bivalent cAb-Lys3 were investigated for their ability to target transgenic tumors expressing lysozyme on their membrane. Biodistribution studies revealed that these non-immunogenic monomeric and bivalent camel single-domain antigen binders specifically target lysozyme-expressing tumors and metastatic lesions. The excess of antibody is rapidly eliminated from the blood circulation and no cAb retention was observed in normal organs. The tumor to organ cAb-ratios at 2 and 8 hr were in the (2.1-10.8):1 and (6.2-23.7):1 range, respectively. The degree and specificity of tumor retention is independent of the affinity of the recombinant camel single-domain fragments for their antigen and from their univalent monomeric (15 kDa) or bivalent format (33 kDa). This study demonstrates the successful and specific in vivo targeting of tumors by camel single-domain fragments. It may open perspectives for their future use as tumor-targeting vehicle, due to their small size, soluble behaviour and because they are non-immunogenic and interact with epitopes that are less antigenic for conventional antibodies., (Copyright 2002 Wiley-Liss, Inc.)

Small, soluble single-domain fragments derived from the unique variable region of dromedary heavy-chain antibodies (VHHs) against enzymes are known to be potent inhibitors. The immunization of dromedaries with the TEM-1 and BcII beta-lactamases has lead to the isolation of such single-domain antibody fragments specifically recognizing and inhibiting those beta-lactamases. Two VHHs were isolated that inhibit TEM-1 and one BcII inhibiting VHH was identified. All inhibitory VHHs were tight-binding inhibitors. The 50% inhibitory concentrations were determined for all inhibitors and they were all in the same range as the enzyme concentration used in the assay. Addition of the VHHs to the TEM-1 beta-lactamase, expressed on the surface of bacteria, leads to a higher ampicillin sensitivity of the bacteria. This innovative strategy could generate multiple potent inhibitors for all types of beta-lactamases.

Single-domain antibodies against various antigens are isolated from the unique heavy-chain antibodies of immunized camels and llamas. These minimal sized binders are very robust and bind the antigen with high affinity in a monomeric state. We evaluated the feasibility to produce soluble, functional bispecific and bivalent antibodies in Escherichia coli with camel single-domain antibody fragments as building blocks. Two single-domain antibody fragments were tethered by the structural upper hinge of a natural antibody to generate bispecific molecules. This linker was chosen for its protease resistance in serum and its natural flexibility to reorient the upstream and downstream located domains. The expression levels, ease of purification, and the solubility of the recombinant proteins were comparable with those of the constituent monomers. The individual moieties fully retain the binding capacity and the binding characteristics within the recombinant bispecific constructs. The easy generation steps and the biophysical properties of these bispecific and bivalent constructs based on camel single-domain antibody fragments makes them particularly attractive for use in therapeutic or diagnostic programs.

Background: Camelid serum contains a large fraction of functional heavy-chain antibodies - homodimers of heavy chains without light chains. The variable domains of these heavy-chain antibodies (VHH) have a long complementarity determining region 3 (CDR3) loop that compensates for the absence of the antigen-binding loops of the variable light chains (VL). In the case of the VHH fragment cAb-Lys3, part of the 24 amino acid long CDR3 loop protrudes from the antigen-binding surface and inserts into the active-site cleft of its antigen, rendering cAb-Lys3 a competitive enzyme inhibitor., Results: A dromedary VHH with specificity for bovine RNase A, cAb-RN05, has a short CDR3 loop of 12 amino acids and is not a competitive enzyme inhibitor. The structure of the cAb-RN05-RNase A complex has been solved at 2.8 A. The VHH scaffold architecture is close to that of a human VH (variable heavy chain). The structure of the antigen-binding hypervariable 1 loop (H1) of both cAb-RN05 and cAb-Lys3 differ from the known canonical structures; in addition these H1 loops resemble each other. The CDR3 provides an antigen-binding surface and shields the face of the domain that interacts with VL in conventional antibodies., Conclusions: VHHs adopt the common immunoglobulin fold of variable domains, but the antigen-binding loops deviate from the predicted canonical structure. We define a new canonical structure for the H1 loop of immunoglobulins, with cAb-RN05 and cAb-Lys3 as reference structures. This new loop structure might also occur in human or mouse VH domains. Surprisingly, only two loops are involved in antigen recognition; the CDR2 does not participate. Nevertheless, the antigen binding occurs with nanomolar affinities because of a preferential usage of mainchain atoms for antigen interaction.

The humoral immune response of camels, dromedaries and llamas includes functional antibodies formed by two heavy chains and no light chains. The amino acid sequence of the variable domain of the naturally occurring heavy-chain antibodies reveals the necessary adaptations to compensate for the absence of the light chain. In contrast to the conventional antibodies, a large proportion of the heavy-chain antibodies acts as competitive enzyme inhibitors. Studies on the dromedary immunoglobulin genes start to shed light on the ontogeny of these heavy-chain antibodies. The presence of the heavy-chain antibodies and the possibility of immunizing a dromedary allows for the production of antigen binders consisting of a single domain only. These minimal antigen-binding fragments are well expressed in bacteria, bind the antigen with affinity in the nM range and are very stable. We expect that such camelid single domain antibodies will find their way into a number of biotechnological or medical applications. The structure of the camelid single domain is homologous to the human VH, however, the antigen-binding loop structures deviate fundamentally from the canonical structures described for human or mouse VHs. This has two additional advantages: (1) the camel or llama derived single domain antibodies might be an ideal scaffold for anti-idiotypic vaccinations; and (2) the development of smaller peptides or peptide mimetic drugs derived from of the antigen binding loops might be facilitated due to their less complex antigen binding site., (Copyright 1999 John Wiley & Sons, Ltd.)

Evidence is provided that dromedary heavy-chain antibodies, in vivo-matured in the absence of light chains, are a unique source of inhibitory antibodies. After immunization of a dromedary with bovine erythrocyte carbonic anhydrase and porcine pancreatic alpha-amylase, it was demonstrated that a considerable amount of heavy-chain antibodies, acting as true competitive inhibitors, circulate in the bloodstream. In contrast, the conventional antibodies apparently do not interact with the enzyme's active site. Next we illustrated that peripheral blood lymphocytes are suitable for one-step cloning of the variable domain fragments in a phage-display vector. By bio-panning, several antigen-specific single-domain fragments are readily isolated for both enzymes. In addition we show that among those isolated fragments active site binders are well represented. When produced as recombinant protein in Escherichia coli, these active site binders appear to be potent enzyme inhibitors when tested in chromogenic assays. The low complexity of the antigen-binding site of these single-domain antibodies composed of only three loops could be valuable for designing smaller synthetic inhibitors.

The integrin alpha 2 beta 1 is a major cellular receptor for collagen. The alpha 2 subunit contains an +/- 200 amino acids inserted domain (I-domain) in the N-terminal region. A certain degree of homology exists between the I-domains found in integrins, collagen and the A-domains of vWF. The alpha 2-I-domain encoding region (aa residues D145 to S334) was obtained by RT-PCR from mRNA of non stimulated human PBL's. The primers were designed to introduce the necessary restriction sites for cloning of the DNA fragment in frame downstream of the malE gene, as well as a stop codon after the last triplet. The resulting construct pMAL-c2-alpha 2-I allows the expression of the I-domain, fused to the C-terminus of maltose binding protein (mal). The alpha 2-I-mal is purified from the bacterial extract by affinity chromatography on an amylose column. The purified alpha 2-I-mal has been characterized by ELISA's. The alpha 2-I-mal bound to immobilised collagen type I in a concentration dependent manner and could be blocked by the functional monoclonal anti-alpha 2 beta 1 antibody 6F1. The interaction of alpha 2-I-mal with collagen furthermore is Mg(2+)-dependent since the binding was inhibited in the presence of 10 mM EDTA or 10 mM Ca2+ but sustained in the presence of 10 mM Mg2+. Finally, alpha 2-I-mal itself was able to inhibit adhesion of washed platelets to collagen immobilised on a microtiterplate in a dose-dependent manner (alpha 2-I-mal IC50:0.7 microM) as well as platelet aggregation induced by collagen type I (alpha 2-I-mal IC50:0.7 microM). With these results we could confirm that the alpha 2-I-domain represents the collagen-binding site of alpha 2 beta 1 and we furthermore could indicate that this domain is able to prevent platelet adhesion to collagen and collagen-induced platelet aggregation, pointing to the primordial role of alpha 2-I-mal and hence of alpha 2 beta 1 in platelet-collagen interaction.

Hookworms are hematophagous nematodes that infect a wide range of mammalian hosts, including humans. There has been speculation for nearly a century as to the identity of the anticoagulant substances) used by these organisms to subvert host hemostasis. Using molecular cloning, we describe a family of potent small protein (75-84 amino acids) anticoagulants from the hookworm Ancylostoma caninum termed AcAP (A. caninum anticoagulant protein). Two recombinant AcAP members (AcAP5 and AcAP6) directly inhibited the catalytic activity of blood coagulation factor Xa (fXa), while a third form (AcAPc2) predominantly inhibited the catalytic activity of a complex composed of blood coagulation factor VIIa and tissue factor (fVIIa/TF). The inhibition of fVIIa/TF was by a unique mechanism that required the initial formation of a binary complex of the inhibitor with fXa at a site on the enzyme that is distinct from the catalytic center (exo-site). The sequence of AcAPc2 as well as the utilization of an exo-site on fXa distinguishes this inhibitor from the mammalian anticoagulant TFPI (tissue factor pathway inhibitor), which is functionally equivalent with respect to fXa-dependent inhibition of fIIa/TF. The relative sequence positions of the reactive site residues determined for AcAP5 with the homologous regions in AcAP6 and AcAPc2 as well as the pattern of 10 cysteine residues present in each of the inhibitors suggest that the AcAPs are distantly related to the family of small protein serine protease inhibitors found in the nonhematophagous nematode Ascaris lumbricoides var. suum.

Tick anticoagulant peptide (TAP) is a potent and specific inhibitor of the blood coagulation protease Factor Xa. We designed and assembled a synthetic TAP-encoding gene (tapo) based on codons preferentially observed in the highly expressed Pichia pastoris alcohol oxidase 1 gene (AOX1), and fused it to a novel hybrid secretory prepro leader sequence. Expression from this gene yielded biologically active rTAP, which was correctly processed at the amino-terminal fusion site, and accumulated in the medium to approximately 1.7 g/l. This corresponds to a molar concentration of 0.24 mM, and is the highest yet described for a recombinant product secreted from P. pastoris. It also represents a seven-fold improvement in productivity compared to rTAP secretion from Saccharomyces cerevisiae, making P. pastoris an attractive host for the industrial-scale production of this potential therapeutic agent. This system was also used to prepare 21 mg 15N-rTAP, 11 mg 13C-rTAP and 27 mg 15N/13C-rTAP, with isotope incorporation levels higher than 98%, and purities sufficient to allow their use in determining the solution structure of the tick anticoagulant peptide using high field NMR.

We describe the production and biochemical characterization of the first GPIIb/IIIa-inhibiting monoclonal antibody that contains an RGD sequence in the CDR3 region of the heavy chain. Monoclonal antibodies obtained by immunizing mice with human platelets were screened using consecutive ELISAs based on human platelets and immuno-affinity-purified glycoprotein (GP) IIb/IIIa coated on microtitre plates. Out of 30 monoclonal antibodies reacting with GPIIb/IIIa, one, MA-16N7C2, potently inhibited platelet aggregation induced by ADP, thrombin, arachidonic acid, collagen, U46619, adrenaline and platelet-activating factor, whereas ristocetin-induced aggregation was unaffected. MA-16N7C2 (IgG2a) bound approximately 4 times faster to activated than to resting platelets, with a Kdcalc of 6.6nM and of 17.5nM, respectively. Equilibrium binding studies to non-activated platelets showed a Kd of 18.2nM with 41 x 10(3) binding sites per platelet. The antibody recognized GPIIb/IIIa only as a Ca(2+)-dependent complex. MA-16N7C2 blocked fibrinogen and von Willebrand factor binding to GPIIb/IIIa in a competitive manner with a Ki of 8.5nM and 13.2nM, respectively. Sequence analysis revealed a RGD-containing sequence with homology to disintegrins, in the CDR3 region of the heavy chain. That this RGD-containing sequence could be involved in the interaction of the antibody to GPIIb/IIIa was finally indicated by showing that the binding is completely and competitively inhibited by echistatin.

Cutinases are extracellular enzymes involved in the disruption of cutine, an insoluble polyester which covers the surface of plants. They belong to a class of serine esterases that are able to hydrolyse fatty acid esters and emulsified triglycerides as efficiently as lipases, but without displaying interfacial activation. Classical crystallographic methods for obtaining heavy-atom derivatives failed, so the cutinase structure has been solved exclusively by the multiple isomorphous replacement method using four Hg derivatives obtained from mutants S4C, S92C, S120C and S129C. Two of these derivatives behaved as expected: (i) the cys mutant of the catalytic Ser S120C, located at the surface of the active site pocket, leads to a good derivative; and (ii) the Hg atom of the derivative obtained with the S92C mutant is completely accessible to the solvent and occupies two alternative positions--consequently a poor derivative results. In contrast, two mutants show an unexpected behaviour: (i) the Hg atom in the S129C mutant was completely buried 10 A below the protein surface and yielded the best derivative; and (ii) a poor quality derivative was obtained with the S4C mutant. Cys 4 belongs to the disordered propeptide 1-16. The Cys 4 bound Hg atom is located in front of the Asp58 side chain, but neither Cys4 nor parts of the propeptide are clearly visible in the electron density maps of the derivative structure.

K12G0S32 is a 57-kDa recombinant single-chain chimeric plasminogen activator consisting of scFv-K12Go, a single-chain variable-region antigen-binding fragment (Fv) of the monoclonal antibody MA-15C5, which is specific for fragment D-dimer of human cross-linked fibrin, and a low-molecular-mass (33 kDa) urokinase-type plasminogen activator (u-PA-33k) containing amino acids Ala132-Leu411 (Holvoet, P., Laroche, Y., Lijnen, H. R., Van Cauwenberghe, R., Demarsin, E., Brouwers, E., Matthyssens, G. & Collen D. (1991) J. Biol. Chem. 266, 19717-19724). In addition, the Arg156-Phe157 thrombin-cleavage site in the u-PA moiety of K12G0S32 is removed by substitution of Phe157 with Asp. In the present study, the fibrinolytic potency of K12G0S32, determined in a system composed of a 125I-fibrin-labeled human plasma clot submerged in citrated plasma, was found to be only twofold higher than that of intact single-chain u-Pa (rscu-PA), but 17-fold higher than that of rscu-PA(M), a variant of rscu-PA in which the thrombin-cleavage site was removed by substitution of Phe157 with Asp. The fibrinolytic potency of K12G0S32T, with an intact thrombin-cleavage site, was 6-15-fold higher than that of rscu-PA. Conversion of 1 microM single-chain K12G0S32 or rscu-PA(M) into their two-chain derivatives with plasmin occurred at a rate of 1.0 +/- 0.15 nmol.min-1.nmol plasmin-1 and 0.85 +/- 0.074 nmol.min-1.nmol plasmin-1, compared to 14 +/- 2.3 nmol.min-1.nmol plasmin-1 and 18 +/- 2.6 nM.min-1.nmol plasmin-1 for K12G0S32T and rscu-PA, respectively. Purified fragment D-dimer of human cross-linked fibrin inhibited the fibrinolytic potency of single-chain K12G0S32T, but not of two-chain K12G0S32T, in a dose-dependent manner. Furthermore, the fibrinolytic potencies of two-chain K12G0S32 and K12G0S32T were not significantly higher than those of recombinant two-chain u-PA (rtcu-PA) or of rtcu-PA(M). These findings suggest that the 59-fold increase in fibrinolytic potency of K12G0S32T, relative to that of rscu-PA(M), is due both to targeting of the activator to the clot via the single-chain Fv fragment (sixfold increase) and to a more efficient conversion of single-chain K12G0S32T to its two-chain derivative (eightfold increase). Thus, targeting to clots by means of fibrin-specific antibodies results in a significant increase of the fibrinolytic potency of single-chain but not of two-chain u-PA.(ABSTRACT TRUNCATED AT 400 WORDS)

The structure and function of the xylose (glucose) isomerase from Actinoplanes missouriensis have been analyzed by X-ray crystallography and site-directed mutagenesis after cloning and overexpression in Escherichia coli. The crystal structure of wild-type enzyme has been refined to an R factor of 15.2% against diffraction data to 2.2-A resolution. The structures of a number of binary and ternary complexes involving wild-type and mutant enzymes, the divalent cations Mg2+, Co2+, or Mn2+, and either the substrate xylose or substrate analogs have also been determined and refined to comparable R factors. Two metal sites are identified. Metal site 1 is four-coordinated and tetrahedral in the absence of substrate and is six-coordinated and octahedral in its presence; the O2 and O4 atoms of linear inhibitors and substrate bind to metal 1. Metal site 2 is octahedral in all cases; its position changes by 0.7 A when it binds O1 of the substrate and by more than 1 A when it also binds O2; these bonds replace bonds to carboxylate ligands from the protein. Side chains involved in metal binding have been substituted by site-directed mutagenesis. The biochemical properties of the mutant enzymes are presented. Together with structural data, they demonstrate that the two metal ions play an essential part in binding substrates, in stabilizing their open form, and in catalyzing hydride transfer between the C1 and C2 positions.

Site-directed mutagenesis in the active site of xylose isomerase derived from Actinoplanes missouriensis is used to investigate the structural and functional role of specific residues. The mutagenesis work together with the crystallographic studies presented in detail in two accompanying papers adds significantly to the understanding of the catalytic mechanism of this enzyme. Changes caused by introduced mutations emphasize the correlation between substrate specificity and cation preference. Mutations in both His 220 and His 54 mainly affect the catalytic rate constant, with catalysis being severely reduced but not abolished, suggesting that both histidines are important, but not essential, for catalysis. Our results thus challenge the hypothesis that His 54 acts as an obligatory catalytic base for ring opening; this residue appears instead to be implicated in governing the anomeric specificity. With none of the active site histidines acting as a catalytic base, the role of the cations in catalyzing proton transfer is confirmed. In addition, Lys 183 appears to play a crucial part in the isomerization step, by assisting the proton shuttle. Other residues also are important but to a lesser extent. The conserved Lys 294 is indirectly involved in binding the activating cations. Among the active site aromatic residues, the tryptophans (16 and 137) play a role in maintaining the general architecture of the substrate binding site while the role of Phe 26 seems to be purely structural.

Lipases belong to a class of esterases whose activity on triglycerides is greatly enhanced at lipid-water interfaces. This phenomenon, called interfacial activation, has a structural explanation: a hydrophobic lid, which at rest covers the catalytic site, is displaced on substrate or inhibitor binding and probably interacts with the lipid matrix. Fusarium solani pisi cutinase belongs to a group of homologous enzymes of relative molecular mass 22-25K (ref. 7) capable of degrading cutin, the insoluble lipid-polyester matrix covering the surface of plants, and hydrolysing triglycerides. Cutinases differ from classical lipases in that they do not exhibit interfacial activation; they are active on soluble as well as on emulsified triglycerides. Cutinases therefore establish a bridge between esterases and lipases. We report here the three-dimensional structure of a recombinant cutinase from F. solani pisi, expressed in Escherichia coli. Cutinase is an alpha-beta protein; the active site is composed of the triad Ser 120, His 188 and Asp 175. Unlike other lipases, the catalytic serine is not buried under surface loops, but is accessible to solvent. This could explain why cutinase does not display interfacial activation.

Enhanced expression of the bialaphos resistance (bar) from Streptomyces hygroscopicus, which confers resistance to the herbicides bialaphos and phosphinothricin (PPT), has been obtained in Escherichia coli using a vector system based on translational coupling. The gene product, PPT acetyltransferase, was purified to homogeneity and its enzymatic properties were analyzed. Hybrid gene constructs with gene fragments fused to the 3'-terminus of bar yield fusion proteins having acetyltransferase activity, with a Michaelis constant for the PPT substrate comparable to the unmodified enzyme. The bar gene represents a selectable and assayable reporter gene especially suitable for 3'-terminal gene fusions.

The unfolding by guanidine hydrochloride of the toxic fragment of a Bacillus thuringiensis toxin belonging to the CryIC class reveals a two-step denaturation under both acid and alkaline conditions. This demonstrates the existence of two structural domains as building blocks for this toxin. Protease digests performed on a CryIA(b) and CryIC B. thuringiensis toxin, under native and partially denatured conditions, confirm this conclusion. Whereas the native CryIC toxin is completely protease resistant, the CryIA(b) toxin, earlier described as consisting of two structural domains [Convents, D., Houssier, C., Lasters, I. & Lauwereys, M. (1990) J. Biol. Chem. 265, 1369-1375], is cleaved by three proteases, resulting in at least two common fragments. This suggests that this toxin is built up of two globular units linked by a protease-susceptible linker. The detection of a stable intermediate along the denaturation curve allows us to study and compare the consecutive unfolding of the structural domains for both toxins. By addition of a protease, under conditions where such an unfolding intermediate exists, a single denaturation phase can be assigned to a specific part of the protein. These experiments lead to the conclusion that the domain whose stability is highly dependent on pH corresponds to the N-terminal half of both toxins.

Recombinant cutinase from Fusarium solani pisi is expressed and excreted with very high yields in Escherichia coli cultures. Cutinase was crystallized at 20 degrees C using the vapour diffusion technique, with polyethylene glycol 6000 as precipitant. Best crystals were obtained at pH 7.0 with polyethylene glycol 6000 as precipitant. Best crystals were obtained at pH 7.0 with polyethylene glycol at 15 to 20%. They are monoclinic, with space group P2(1) and cell dimensions a = 35.1 A, b = 67.4 A, c = 37.05 A and beta = 94.0 degrees; they diffract beyond 1.5 A resolution. The asymmetric unit contains one molecule of 22,000 Da (Vm = 1.98 A3/Da; 38% water).

The conformational characteristics of the minimal toxic fragment of the delta-endotoxin from Bacillus thuringiensis berliner 1715 were examined by fluorescence and circular dichroism spectroscopy. This insecticidal protein, specifically toxic to lepidopteran species, was found to consist of two structural domains. Experimental evidence for this conclusion was provided by biphasic guanidine hydrochloride unfolding curves at different pH values and electrophoretic patterns of protease digests. Two stable fragments of comparable molecular weight were obtained using four different broad specificity proteolytic enzymes. A secondary structure model was constructed using seven B. thuringiensis toxin sequences. These toxins were selected on the basis of their limited sequence homology and represent all known insecticidal specificities. Despite this divergence, a consensus secondary structure pattern was obtained, confirming the structural homology among the toxins. The N-terminal halves of all toxins are predicted to be relatively rich in alpha-helix structure and the C-terminal parts to contain alternating beta-strand and coil structures. The latter seems characteristic for a beta-sheet conformation. Comparing this model to the unfolding data obtained by circular dichroism, whose far UV signal gives a measure of the alpha-helix content, allowed us to delineate the structural domains into the primary structure.

A gene which confers resistance to the herbicide bialaphos (bar) has been characterized. The bar gene was originally cloned from Streptomyces hygroscopicus, an organism which produces the tripeptide bialaphos as a secondary metabolite. Bialaphos contains phosphinothricin, an analogue of glutamate which is an inhibitor of glutamine synthetase. The bar gene product was purified and shown to be a modifying enzyme which acetylates phosphinothricin or demethylphosphinothricin but not bialaphos or glutamate. The bar gene was subcloned and its nucleotide sequence was determined. Interspecific transfer of this Streptomyces gene into Escherichia coli showed that it could be used as a selectable marker in other bacteria. In the accompanying paper, bar has been used to engineer herbicide-resistant plants.

Recently (1) we reported the isolation and partial biochemical characterization of a novel polypeptide from human brain. Now we report the isolation of an almost identical polypeptide from human liver tissue and further biochemical characterization of both polypeptides. Thorough analytical investigation with several methods, fully described hereafter, of both polypeptides, leads on the one hand to a more complete picture of the polypeptide and on the other hand to the correction of earlier assumptions. The monomeric polypeptide, now identified as a glycopolypeptide, is indeed an independent chain of approximately 220 amino-acid residues and possesses 2 free sulphydryl groups. From the almost complete identity between the CNS and liver polypeptide it is concluded that they are most probably one and the same polypeptide and consequently, that h3 is not brain specific.

Muscarinic receptors have been purified from calf forebrain plasma cell membranes by affinity chromatography on a dexetimide-agarose gel. SDS-PAGE analysis showed a single 70 kDa band. Monoclonal antibodies have been prepared against these affinity purified 70 kDa protein(s). One antibody, M-35, immunoprecipitated up to 80% of digitonin-solubilized muscarinic receptors. M-35 had agonist-like effects on guinea-pig myometrium: it increased the intracellular cyclic GMP content, decreased prostaglandin-induced cyclic AMP accumulation and caused muscle contractions. The two first effects were inhibited by atropine. M-35 was used to visualize muscarinic receptors at the surface of human fibroblastic cells. In the particular cell line used, the receptors have a low affinity for pirenzepine, were negatively coupled to adenylate cyclase and mediated increase in the phosphatidyl-inositol breakdown.