Development of monoclonal antibodies to human kallikrein-related peptidase 6 (KLK6) and their use in an immunofluorometric assay for free KLK6.

We have raised monoclonal antibodies against KLK6 and constructed a 'sandwich' type immunoassay using 8A8G3 as capture and 3H3E9 as tracer antibodies. 8A8G3 bound to one side of KLK6, whereas 3H3E9 probably bound near its catalytic site. The assay did not detect complexed forms of KLK6, indicating that it quantifies only free KLK6 (fKLK6). fKLK6 was higher in serum of patients with ovarian cancer (11.34 µg/l±1.37) than in controls (3.39 µg/l±0.42). The cerebrospinal fluid contained high concentrations of fKLK6 (257-965 µg/l). This assay could facilitate the evaluation of fKLK6 in human diseases.

Crystal structure of human prostate-specific antigen in a sandwich antibody complex.

Human prostate-specific antigen (PSA or human kallikrein-related peptidase 3) present in small quantities in the sera of healthy men becomes elevated in prostate cancer (PCa) and other prostate disorders. The ability to identify the free PSA fraction associated with PCa could increase the reliability of the PSA diagnostic test. Here we present the crystal structure of human PSA from seminal fluid in a sandwich complex with two monoclonal antibodies (mAbs). MAb 5D5A5 captures total PSA with exceptionally high affinity, and mAb 5D3D11 selectively discriminates between free PSA subforms that are more abundant in sera from patients with PCa. Although the antigen is not of seric origin, several insights into cancer diagnosis can be discerned from this complex. MAb 5D3D11 recognizes a PSA conformation different from that previously reported. Interacting with the kallikrein loop, the PSA N-linked glycan attached to asparagine 61 is an uncommonly complex sialated triantennary chain. O-linked glycosylation is observed at threonine 125. The description of how PSA subforms in prostatic fluid can be discriminated using pairs of antibodies is a first step in the design of new strategies that are capable of real discrimination among PSA subforms, which will lead to the formulation of more reliable diagnostic tests. In a companion article [Muller, B. H., Savatier, A., L'Hostis, G., Costa, N., Bossus, M., Michel, S., et al. (2011). In vitro affinity maturation of an anti-PSA antibody for prostate cancer diagnostic assay. J. Mol. Biol.], we describe engineering efforts to improve the affinity of mAb 5D3D11, a first step towards such goal.

Clostridium perfringens delta toxin is sequence related to beta toxin, NetB, and Staphylococcus pore-forming toxins, but shows functional differences.

Clostridium perfringens produces numerous toxins, which are responsible for severe diseases in man and animals. Delta toxin is one of the three hemolysins released by a number of C. perfringens type C and possibly type B strains. Delta toxin was characterized to be cytotoxic for cells expressing the ganglioside G(M2) in their membrane. Here we report the genetic characterization of Delta toxin and its pore forming activity in lipid bilayers. Delta toxin consists of 318 amino acids, its 28 N-terminal amino acids corresponding to a signal peptide. The secreted Delta toxin (290 amino acids; 32619 Da) is a basic protein (pI 9.1) which shows a significant homology with C. perfringens Beta toxin (43% identity), with C. perfringens NetB (40% identity) and, to a lesser extent, with Staphylococcus aureus alpha toxin and leukotoxins. Recombinant Delta toxin showed a preference for binding to G(M2), in contrast to Beta toxin, which did not bind to gangliosides. It is hemolytic for sheep red blood cells and cytotoxic for HeLa cells. In artificial diphytanoyl phosphatidylcholine membranes, Delta and Beta toxin formed channels. Conductance of the channels formed by Delta toxin, with a value of about 100 pS to more than 1 nS in 1 M KCl and a membrane potential of 20 mV, was higher than those formed by Beta toxin and their distribution was broader. The results of zero-current membrane potential measurements and single channel experiments suggest that Delta toxin forms slightly anion-selective channels, whereas the Beta toxin channels showed a preference for cations under the same conditions. C. perfringens Delta toxin shows a significant sequence homolgy with C. perfringens Beta and NetB toxins, as well as with S. aureus alpha hemolysin and leukotoxins, but exhibits different channel properties in lipid bilayers. In contrast to Beta toxin, Delta toxin recognizes G(M2) as receptor and forms anion-selective channels.

Crystal structure of a ternary complex between human prostate-specific antigen, its substrate acyl intermediate and an activating antibody.

Human prostate-specific antigen (PSA or KLK3) is an important marker for the diagnosis and management of prostate cancer. This is an androgen-regulated glycoprotein of the kallikrein-related protease family secreted by prostatic epithelial cells. Its physiological function is to cleave semenogelins in the seminal coagulum and its enzymatic activity is strongly modulated by zinc ions. Here we present the first crystal structure of human PSA in complex with monoclonal antibody (mAb) 8G8F5 that enhances its enzymatic activity. The mAb recognizes an epitope composed of five discontinuous segments including residues from the kallikrein loop and stabilizes PSA in an "open and active conformation" that accelerates catalysis. We also present the crystal structure of PSA in complex with both the mAb 8G8F5 and a fluorogenic substrate Mu-KGISSQY-AFC, derived from semenogelin I. By exploiting the inhibition of PSA by zinc ions, we were able to obtain a substrate acyl intermediate covalently linked to the catalytic serine, at pH 7.3 but not at pH 5.5. Moreover, the inhibition of PSA activity by zinc was found to be modulated by pH variations but not by the antibody binding. The correlation of the different data with the physiological conditions under which PSA can cleave semenogelins is discussed.

Isoelectric point determination of cardiac troponin I forms present in plasma from patients with myocardial infarction.

BACKGROUND: Cardiac troponin I (cTnI) is a specific marker of myocardial injury. In blood of patients with cardiovascular diseases, cTnI is released as a mixture of free, complexed and post-translationally modified forms. METHODS: The cTnI forms present in the plasma from 8 patients with acute myocardial infarction (AMI) have been analysed by two-dimensional gel electrophoresis (2-DE) and Western Blot using anti-cTnI mAb 19C7 and anti-phosphorylated cTnI (Serines 22-23) mAb 5E6. RESULTS: After immunoextraction of cTnI in plasma samples by 19C7 and 2-DE separation, 4 different forms were detected by 19C7 in 7 out the 8 AMI plasma samples. Two 29 kDa spots corresponding to intact free cTnI forms were detected at pIs 5.2 and 5.4. However, spot with pI 5.4 was also recognized by mAb 5E6, and should be bis-phosphorylated cTnI. Two 55 kDa spots with pIs 6.6 and 6.7 could be IC complexes. CTnI forms with pIs lower than the theoretical pI were also found in free cTnI and phosphorylated cTnI purified materials. CONCLUSIONS: 2-DE analysis of AMI plasma showed the presence of acidic cTnI forms, one of them being phosphorylated. The clinical significance of these forms has to be further investigated.

SELDI-TOF MS analysis of the Cardiac Troponin I forms present in plasma from patients with myocardial infarction.

The troponin (Tn) complex is composed of troponin T, troponin C and troponin I. The cardiac isoform of TnI (cTnI) is modified and released in blood of patients with cardiovascular diseases as a heterogeneous mixture of free, complexed and posttranslationally modified forms. With the aim to determine later, whether specific forms of cTnI could be associated with the different pathologies leading to cTnI release, the cTnI forms present in the plasma from 64 patients with acute myocardial infarction (AMI) have been analysed by SELDI-TOF MS using anti-TnI mAbs coupled to PS20 ProteinChips arrays. Upfront immunoaffinity enrichment using anti-cTnI 19C7 mAb allowed us to detect cTnI and bis-phosphorylated cTnI in 11/12 and 9/12 analyses respectively, as well as truncated cTnI in plasma with concentration of cTnI as low as 8 ng/mL. Cardiac troponin C (cTnC) and covalent TnIC complex were also found in pools of plasma with higher concentrations of cTnI. MAb 19C7-affinity SELDI-TOF MS analysis performed after immunopurification of one pool of AMI plasma with anti-free cTnI, anti-cTnC, and anti-phosphorylated cTnI mAbs indicated that intact and bis-phosphorylated cTnI were mostly under the free form. Besides, a 18 718 m/z peak could correspond to a truncated phosphorylated form initially complexed with cTnC.

Recombinant kallikrein expression: site-specific integration for hK6 production in human cells.

Kallikreins have been implicated in carcinogenesis and are promising biomarkers for the diagnosis and follow-up of various cancers. To evaluate the functions and clinical interest of kallikreins, it is important to be able to produce them as recombinant proteins. Here we summarize the various strategies used to produce kallikreins, emphasizing their advantages and limitations. We also describe an approach to achieve high-level production of kallikreins, such as hK6, with correct folding and activity, combining an expression system with targeted transgene integration and an efficient cultivation device to increase yield, the CELLine bioreactor. This novel method for recombinant kallikrein production will be useful to study their bio-pathological functions and to develop anti-bodies.

Characterization of prostate-specific antigen binding peptides selected by phage display technology.

Prostate-specific antigen (PSA) is an important marker for the diagnosis and management of prostate cancer. Free PSA has been shown to be more extensively cleaved in sera from benign prostatic hyperplasia patients than in sera from prostate cancer patients. Moreover, the presence of enzymatically activatable PSA was characterized previously in sera from patients with prostate cancer by the use of the specific anti-free PSA monoclonal antibody (mAb) 5D3D11. As an attempt to obtain ligands for the specific recognition of different PSA forms including active PSA, phage-displayed linear and cyclic peptide libraries were screened with PSA coated directly into microplate wells or presented by two different anti-total PSA mAbs. Four different phage clones were selected for their ability to recognize PSA and the inserted peptides were produced as synthetic peptides. These peptides were found to capture and to detect specifically free PSA, even in complex biological media such as sera or tumour cell culture supernatants. Alanine scanning of peptide sequences showed the involvement of aromatic and hydrophobic residues in the interaction of the peptides with PSA whereas Spotscan analysis of overlapping peptides covering the PSA sequence identified a peptide binding to the kallikrein loop at residues 82-87, suggesting that the peptides could recognize a non-clipped form of PSA. Moreover, the PSA-specific peptides enhance the enzymatic activity of PSA immobilized into microplate wells whereas the capture of PSA by the peptides inhibited totally its enzymatic activity while the peptide binding to PSA had no effect in solution. These PSA-specific peptides could be potential tools for the recognition of PSA forms more specifically associated to prostate cancer.

Usefulness of the phage display technology for the identification of a hepatitis C virus NS4A epitope recognized early in the course of the disease.

A dodecapeptide phage-displayed library was screened with the mouse monoclonal antibody (mAb) 2E3C2 which competed with human antibodies for the binding to the HCV c100 recombinant protein. Four mimotopes shared a consensus motif with the HCV 1701-1707 sequence corresponding to the carboxyl-terminal domain of the non-structural protein NS4A. However, these mimotopes reacted with 2E3C2 only, whereas the corresponding NS4 epitope defined at the sequence 1698-1709 and displayed on phage was recognized by both 2E3C2 and sera from HCV infected patients. Using the Spot method of multiple peptide synthesis and alanine replacement analysis, the respective reactivities of mAb 2E3C2 and anti-NS4A human antibodies against NS4 were shown to be directed against two slightly different overlapping minimal linear sequences and to involve different critical residues. The phage clone displaying the NS4 epitope was used to study the specific recognition of this epitope by different individual HCV positive sera as well as by two seroconversion panels of sera from HCV infected patients. Compared with the detection by RIBA of the different HCV antigens and c100 particularly, these results indicated that the antibodies directed against the NS4 (1698-1709) epitope were produced early during the course of the disease and decreased later.

Comparison of the crystallization and crystal packing of two Fab single-site mutant protein L complexes.

Protein L from Peptostreptococcus magnus (PpL) is a multidomain protein composed of four or five immunoglobulin-binding domains that target the kappa light chain of a large repertoire of human and murine antibodies. Thus, a single domain of this protein can be used to aid the crystallization of Fab, free or complexed to their antigen when it is not possible to obtain crystals without it. Each wild-type PpL domain has two light-chain binding sites that target the same region of the light chain and can thus bring together two Fab-antigen complexes within the crystal lattice. In this context the small PpL domain is sandwiched between two Fab and cannot participate in crystal contacts, thus mutants are unlikely to increase the chances of crystallizing a particular complex. However, it is possible to design mutants that can bind at only one site by making use of the crystal structures obtained so far. Such mutants will have a free surface that can participate in crystal contacts and that can be modified to improve its crystal contact-forming properties. Here, a comparison of two single-site mutants that differ at three different positions is reported. In both mutants two different tryptophan residues participate in crystal-packing interactions, suggesting that this residue may be particularly interesting for enhancing crystal-contact formation.

Different crystal packing in Fab-protein L semi-disordered peptide complex.

Proteins and peptides with variable degrees of disorder are a challenge for protein crystallization. These may be completely disordered or just contain regions with a high degree of mobility that may be represented by a multitude of discretely defined conformations. These difficulties are not insurmountable, but it may be unreasonable to expect a clean result from a structural point of view. The complex between a murine monoclonal antibody (19D9D6) and a synthetic peptide that encompasses the first 45 residues of the core protein of Hepatitis C virus that is poorly structured in solution has been crystallized. In order to make the crystallization possible, use was made of a single immunoglobulin-binding domain of protein L from Peptostreptococcus magnus (PpL), a bacterial protein that can bind the variable region (Fv) of a large population of antibodies through its light chain with no interference with antibody-antigen recognition. Crystals were obtained in different space groups where the size of the cavity that accommodates the peptide is different, although many of the crystal contacts and the overall lattice are preserved. The peptide can be considered to be semi-disordered and the larger cavity accommodates a better ordered peptide than the smaller one. The lattice is of interest for the design of a scaffold system for the crystallization of peptide-tagged proteins since a cavity that accommodates a disordered entity might be able to host ordered proteins of the same size and shape as the cavity. Here, the differences between the lattices formed by this trimolecular complex are described and it is discussed how such a system may be adapted to the crystallization of peptide-tagged proteins.

Selective recognition of enzymatically active prostate-specific antigen (PSA) by anti-PSA monoclonal antibodies.

Prostate-specific antigen (PSA) is widely used as a serum marker for the diagnosis of prostate cancer. To evaluate two anti-free PSA monoclonal antibodies (mAbs) as potential tools in new generations of more relevant PSA assays, we report here their properties towards the recognition of specific forms of free PSA in seminal fluids, LNCaP supernatants, 'non-binding' PSA and sera from cancer patients. PSA from these different origins was immunopurified by the two anti-free PSA mAbs (5D3D11 and 6C8D8) as well as by an anti-total PSA mAb. The composition of the different immunopurified PSA fractions was analysed and their respective enzymatic activities were determined. In seminal fluid, enzymatically active PSA was equally purified with the three mAbs. In LNCaP supernatants and human sera, 5D3D11 immunopurified active PSA mainly, whereas 6C8D8 immunopurified PSA with residual activity. In sera of prostate cancer patients, we identified the presence of a mature inactive PSA form which can be activated into active PSA by use of high saline concentration or capture by an anti-total PSA mAb capable of enhancing PSA activity. According to PSA models built by comparative modelling with the crystal structure of horse prostate kallikrein described previously, we assume that active and activable PSA could correspond to mature intact PSA with open and closed conformations of the kallikrein loop. The specificity of 5D3D11 was restricted to both active and activable PSA, whereas 6C8D8 recognized all free PSA including intact PSA, proforms and internally cleaved PSA.

Immunization with hepatitis C virus core gene triggers potent T-cell response, but affects CD4+ T-cells.

Numerous attempts to induce immunity against HCV core (HCV-C) by DNA immunization met serious difficulties in optimizing T-helper cell and antibody responses. Immunomodulatory properties of HCV-C could be blamed that seem to be dependent on the genotype of HCV source. Here, we characterized HCV-C gene from HCV 1b isolate 274933RU. Eukaryotic expression of HCV-C was effectively driven by CMVIE, while human elongation factor 1 alpha promoter directed low levels of HCV-C expression. C57BL/6 mice were immunized with CMVIE-driven HCV-C gene, and assessed for specific antibody production, T-cell proliferation and cytokine secretion. The number and proportion of CD19+, CD3+, CD3+/CD4+, and CD3+/CD8+ splenocytes in HCV-C gene recipients was evaluated by flow cytometry. A significant mounting drop in CD3+/CD4+ T-cell counts occurred in HCV-C gene-recipients as compared to the controls. Despite that, 75% of mice exhibited core-specific cellular reactivity revealed as high proliferative responses to HCV-C and HCV-C peptides. Stimulated T-cells secreted predominantly IFN-gamma and IL-2. A shift of epitope specificity was observed with the early response being broad, and the late limited to the HCV-C C-terminus. Thus, we demonstrate both T-cell immunogenicity and T-cell modulation by core of HCV 1b. Immune modulation by HCV core may affect host ability to mount long-lasting cellular and antibody response and should be dealt with in designing core-based HCV vaccines.

Characterization of mimotopes mimicking an immunodominant conformational epitope on the hepatitis C virus NS3 helicase.

The hepatitis C virus (HCV) nonstructural 3 (NS3) protein is composed of an amino terminal protease and a carboxyl terminal RNA helicase. NS3 contains major antigenic epitopes. The antibody response to NS3 appears early in the course of infection and is focused on the helicase region. However, this response cannot be defined by short synthetic peptides indicating the recognition of conformation-dependent epitopes. In this study, we have screened a dodecapeptide library displayed on phage with anti-NS3 mouse monoclonal antibodies (mAbs) that compete with each other and human anti-HCV NS3 positive sera. Two peptides (mimotopes) were selected that appeared to mimic an immunodominant epitope since they were recognized specifically by the different anti-NS3 mAbs of the study and by human sera from HCV infected patients. Homology search between the two mimotopes and the NS3 sequence showed that one of the two peptides shared amino acid similarities with NS3 at residues 1396-1398 on a very accessible loop as visualized on the three-dimensional structure of the helicase domain whereas the other one had two amino acids similar to nearby residues 1376 and 1378. Reproduced as synthetic dodecapeptides, the two mimotopes were recognized specifically by 19 and 22, respectively, out of 49 sera from HCV infected patients. These mimotopes allowed also the detection of anti-NS3 antibodies in sera of HCV patients at the seroconversion stage. These results suggest that the two NS3 mimotopes are potential tools for the diagnosis of HCV infection.

Mapping of a conformational epitope shared between E1 and E2 on the serum-derived human hepatitis C virus envelope.

Monoclonal antibody D32.10 produced by immunizing mice with a hepatitis C virus (HCV)-enriched pellet obtained from plasmapheresis of a chronically HCV1b-infected patient binds HCV particles derived from serum of different HCV1a- and HCV1b-infected patients. Moreover, this monoclonal has been shown to recognize both HCV envelope proteins E1 and E2. In an attempt to provide novel insight into the membrane topology of HCV envelope glycoproteins E1 and E2, we localized the epitope recognized by D32.10 on the E1 and/or E2 sequence using Ph.D.-12 phage display peptide library technology. Mimotopes selected from the phage display dodecapeptide library by D32.10 shared partial similarities with 297RHWTTQGCNC306 of the HCV E1 glycoprotein and with both 613YRLWHYPCT621 and 480PDQRPYCWHYPPKPC494 of the HCV E2 glycoprotein. Immunoreactivity of D32.10 with overlapping peptides corresponding to these three HCV regions confirmed these localizations and suggested that the three regions identified are likely closely juxtaposed on the surface of serum-derived particles as predicted by the secondary model structure of HCV E2 derived from the tick-borne encephalitis virus E protein. This assertion was supported by the detection of specific antibodies directed against these three E1E2 regions in sera from HCV-infected patients.

Crystal structure of a hydrophobic immunodominant antigenic site on hepatitis C virus core protein complexed to monoclonal antibody 19D9D6.

The first crystal structure of a complex between a hepatitis C virus (HCV) core protein-derived peptide (residues 13-40) and the Ab fragment of a murine mAb (19D9D6) has been solved, allowing determination of the recognized epitope and elucidation of its conformation. This Ab, raised against the first 120 residues of the core protein, recognizes core particles and strongly competes with anticore human Abs, suggesting that it is highly representative of the human anti-HCV core response. Its epitope lies within the first 45 aa of the protein, the major antigenic segment of core recognized both by murine and human Abs. Surprisingly, the recognized epitope (29-37: QIVGGVYLL) has an unusual preponderance of hydrophobic residues, some of which are buried in a small hydrophobic core in the nuclear magnetic resonance structure of the peptide (2-45) in solution, suggesting that the Ab may induce a structural rearrangement upon recognition. The flexibility may reside entirely within the Ag, since the Fab'-peptide complex structure at 2.34 A shows that the Ab binding site is hardly perturbed by complexation. Given that the recognized residues are unlikely to be solvent exposed, we are left with the interesting possibility that Ab-core interactions may take place in a nonaqueous environment.

Evidence for plasticity and structural mimicry at the immunoglobulin light chain-protein L interface.

The multidomain bacterial surface protein L (PpL) is a virulence factor expressed by only 10% of Peptostreptococcus magnus strains, and its expression is correlated with bacterial vaginosis. The molecular basis for its ability to recognize 60% of mammalian immunoglobulin light chain variable regions (V(L)) has been described recently by x-ray crystallography, which suggested the presence of two V(L) binding sites on each protein L domain (Graille, M., Stura, E. A., Housden, N. G., Beckingham, J. A., Bottomley, S. P., Beale, D., Taussig, M. J., Sutton, B. J., Gore, M. G., and Charbonnier, J. (2001) Structure 9, 679-687). Here, we report the crystal structure at 2.1 A resolution of a protein L mutant complexed to an Fab' fragment with only 50% of the V(L) residues interacting with PpL site 1 conserved. Comparison of the site 1 interface from both structures shows how protein L is able to accommodate these sequence differences and therefore bind to a large repertoire of Ig. The x-ray structure and NMR results confirm the existence of two V(L) binding sites on a single protein L domain. These sites exhibit a remarkable structural mimicry of growth factors binding to their receptors. This could explain the protein L superantigenic activity on human B lymphocytes.