Antiamylase-pullulanase enzyme monoclonals which specifically inhibit amylase or pullulanase activity

Monoclonal antibodies against amylase-pullulanase enzyme from Bacillus circulans F-2 have been produced to locate and characterize the catalytic sites of the enzyme. The antibodies have been examined for inhibition of both enzyme activities of amylase and pullulanase and then classified into four types: Type I which inhibited amylase activity, Type II which inhibited pullulanase activity, Type III which inhibited both enzyme activities, and Type IV which had no effect on either enzyme activity. Only two monoclonal antibodies (MAP-12 and MAP-17) as Type I and two antibodies (MAP-3 and MAP-5) as Type II were isolated. The inhibitory activities of the antibodies were characterized and compared. In Type II antibodies, the maximal demonstrated inhibition on the pullulanase activity was 88% for MAP-3 with 1 microg of antibody and 90% for MAP-5 with 2 microg of antibody, but did not inhibit the amylase activity. In Type I antibodies, in contrast, the maximal demonstrated inhibition on the amylase activity was 94% for MAP-12 and 97% for MAP-17 with 1 microg of antibody, respectively, but no inhibition of the pullulanase was noted. MAP-12 recognized sequential epitope, while MAP-17 recognized conformation-dependent epitope of amylase activity-related regions. However, both MAP-3 and MAP-5 recognized the conformation-dependent epitope of the pullulanase activity-related region. Furthermore, the antibodies of MAP-3, MAP-5, MAP-12, and MAP-17 did not compete with one another for binding to the enzyme, indicating that they have different target epitopes on the enzyme. Antibody binding of MAP-12 and MAP-17 to the enzyme was not specifically affected by any of the antiamylase compounds tested: (a) nojirimycin; and (b) 1-deoxynojirimycin. Kinetic analysis of their effects provides evidence that both antibodies of MAP-12 and MAP-17 decrease the catalytic rate of enzyme activity and have little or no effect on substrate binding.     

Molecular Analysis of the Gal/GalNAc Adhesin of Entamoeba histolytica1

Attachment of Entamoeba histolytica to colonic epithelium and a variety of other target cells is mediated by a galactosc/N-acetyl D-galactosamine (Gal/GalNAc) inhibitable adhesin. Seven monoclonal antibodies specific for nonoverlapping epitopes of the 170 kDa subunit have been shown to have distinct effects on adherence. Four of these monoclonal antibodies inhibit or have no effect on amebic adherence while two others enhance amebic adherence. The epitopes recognized by these seven monoclonal antibodies have been mapped to the extracellular cysteine rich region of the 170 kDa subunit. The conformational nature of the epitopes was examined by testing monoclonal antibody reactivity with isolated regions of the 170 kDa subunit expressed as fusion proteins in E. coli and also with denatured native adhesin. These analyses suggested that three of monoclonal antibodies recognized conformational epitopes while the remaining four recognized linear epitopes. The mapping of these monoclonal antibodies have identified functionally important regions of the Gal/GalNAc adhesin and have also shown that recombinant Gal/GalNAc adhesin, when expressed in E. coli, retained at least some of its native conformation.     

Monoclonal antibodies to gonococcal outer membrane protein I: location of a conserved epitope on protein IB

Hybrid cell lines have been derived which produce monoclonal antibodies reacting with outer membrane protein I from Neisseria gonorrhoeae strain P9. The antibodies obtained showed variable reactivity with other strains but one antibody recognized an epitope present on all of the strains tested which expressed the protease sensitive protein IB. Purified IgG labelled with 125I was used in competitive radioimmunoassays with unlabelled antibody to investigate the spacial distribution of the epitopes recognized. Each pair of antibodies showed some degree of inhibition. The relative magnitude of inhibition suggested that the conserved epitope lies within a variable region containing other epitopes which determine the antigenic specificity of the protein. Western blotting of peptides derived by proteolytic digestion of protein IB revealed that the conserved epitope is located close to the chymotrypsin cleavage site within a 7000 Mr surface exposed region of the molecule.     

Comparison of the antibody response to bee venom phospholipase A2 induced by natural exposure in humans or by immunization in mice

Two human and twelve murine monoclonal antibodies directed against the main bee venom allergen phospholipase A₂ (PLA) were evaluated for their fine specificity of binding to antigen and their ability to inhibit the enzymatic activity of the antigen. Antibodies were induced by natural exposure of beekeepers to bee venom or immunization of mice via different methods. Both human monoclonal antibodies (hmAbs) were previously shown to recognize the native three-dimensional conformation of PLA and are directed against discontinuous epitopes which include lysine residue at position 25 as a contact residue. In contrast, six of the murine monoclonal antibodies (mmAbs) bind to the denatured structure of the protein as determined by enzyme-linked immunosorbent assay. The epitopes recognized are located near the C-terminal end (n=8), in the centre of the polypeptide (n=1), near the N-terminal end (n=1) or include the carbohydrate part (n=2) of the PLA molecule. The capacity of the antibodies to modify the enzymatic activity was also determined. The hmAbs significantly inhibit the enzyme (70–79%), whereas the mmAbs produced various degrees of inhibition (39–100%). Since the X-ray structure of PLA is known, the epitopes can be visualized in the context of the three-dimensional structure of the antigen. A qualitative correlation was found between the location of epitopes and the inhibition pattern. Strong inhibition was seen with those antibodies that recognize epitopes that lie on the surface of the enzyme that is thought to contact the phospholipid bilayer. The results show that even though both hmAbs and most mmAbs inhibit the enzymatic activity of PLA, the antigen-binding properties of antibodies from different species raised after different routes of immunization differ significantly. Thus, detailed epitope mapping studies using murine antibodies prepared by artificial immunization may have limited value in predicting epitope patterns relevant to an antibody response to allergens in humans naturally exposed to antigen/allergen. © 1997 John Wiley & Sons, Ltd.     

The binding of monoclonal and polyclonal antibodies to the Ca2(+)-ATPase of sarcoplasmic reticulum: effects on interactions between ATPase molecules.

We analyzed the interaction of 14 monoclonal and 5 polyclonal anti-ATPase antibodies with the Ca2(+)-ATPase of rabbit sarcoplasmic reticulum and correlated the location of their epitopes with their effects on ATPase-ATPase interactions and Ca2+ transport activity. All antibodies were found to bind with high affinity to the denatured Ca2(+)-ATPase, but the binding to the native enzyme showed significant differences, depending on the location of antigenic sites within the ATPase molecule. Of the seven monoclonal antibodies directed against epitopes on the B tryptic fragment of the Ca2(+)-ATPase, all except one (VIE8) reacted with the enzyme in native sarcoplasmic reticulum vesicles in both the E1 and E2V conformations. Therefore these regions of the Ca2(+)-ATPase molecule are freely accessible in the native enzyme. The monoclonal antibody VIE8 bound with high affinity to the Ca2(+)-ATPase only in the E1 conformation stabilized by 0.5 mM Ca2+ but not in the E2V conformation stabilized by 0.5 mM EGTA and 5 mM vanadate. Several antibodies that reacted with the B fragment interfered with the crystallization of Ca2(+)-ATPase in the presence of EGTA and vanadate and at least two of them destabilized preformed Ca2(+)-ATPase crystals, suggesting inhibition of interactions between ATPase molecules. Of five monoclonal antibodies with epitopes on the A1 tryptic fragment of the Ca2(+)-ATPase only one gave strong reaction with the native enzyme, and none interfered with ATPase-ATPase interactions as measured by the polarization of fluorescence of FITC-labeled Ca2(+)-ATPase. Therefore the regions of the molecule containing these epitopes are relatively inaccessible in the native structure. Partial tryptic cleavage of the Ca2(+)-ATPase into the A1, A2 and B fragments did not promote the reaction of anti-A1 antibodies with sarcoplasmic reticulum vesicles, but solubilization of the membrane with C12E8 rendered the antigenic site fully accessible to several of them, suggesting that their epitopes are located in areas of contacts between ATPase molecules. Two monoclonal anti-B antibodies that interfered with ATPase-ATPase interactions, produced close to 50% inhibition of the rate of ATP-dependent Ca2+ transport, with significant inhibition of ATPase; this may suggest a role for ATPase oligomers in the regulation of Ca2+ transport. The other antibodies that interact with the native Ca2(+)-ATPase produced no significant inhibition of ATPase activity even at saturating concentrations; therefore their antigenic sites do not undergo major movements during Ca2+ transport.     

Disposition of mitochondrially bound hexokinase at the membrane surface, deduced from reactivity with monoclonal antibodies recognizing epitopes of defined location.

A panel of monoclonal antibodies against rat brain hexokinase (ATP: D-hexose 6-phosphotransferase, EC 2.7.1.1) has been employed to investigate the orientation of the mitochondrially bound enzyme on the mitochondrial surface. Based on their ability to immunoprecipitate truncated forms of the protein, obtained by in vitro translation of truncated versions of the mRNA, the epitopes for seven monoclonal antibodies were mapped to regions consisting of 20-50 amino acid residues within the sequence of the N-terminal half of the enzyme. There is extensive sequence similarity between the N- and C-terminal halves of this enzyme, which is thought to have evolved by a process of gene duplication and fusion. However, these antibodies react selectively with epitopes in the N-terminal half, and thus epitopic regions for several of these antibodies could be further defined by eliminating from consideration regions showing substantial sequence similarity with the C-terminal half. The epitope for one of the monoclonal antibodies, designated 4D4, was shown to involve the extreme N-terminus of the enzyme; selective proteolytic modification of this region resulted in loss of immunoreactivity. Relative location of epitopes for three other antibodies, designated 2B, 1C5, and 4C5, within a 20-residue segment was deduced from effects of modifying sulfhydryl residues within this segment on immunoreactivity. Thus, by a combination of sequence analysis and experimental methods, the epitopes for these seven antibodies could be localized to defined regions within the overall sequence. The ability of these antibodies to prevent binding of hexokinase to mitochondria, and their ability to recognize the mitochondrially bound enzyme, provided a basis for assessing the relative proximity of the corresponding epitopes to the mitochondrial surface when the enzyme was bound. The disposition of the bound enzyme on the mitochondrial surface was deduced by relating these results to the proposed structure for brain hexokinase.     

Syngeneic anti-idiotypic monoclonal antibodies against an anti-human chorionic gonadotrophin antibody

Monoclonal antibody designated 1B10 (Mab 1B10) has been shown to be highly specific for the beta-chain of human chorionic gonadotrophin (HCG). We used this antibody to investigate its paratope using anti-idiotypic antibodies. Purified Mab 1B10 has been used to immunize syngeneic BALB/c mice to produce anti-idiotypic monoclonal antibodies. An enzyme immunoassay (ELISA) on Mab 1B10 coated plate was employed to screen the supernatants of growing hybridomas. The specificity of each antibody selected was assessed using an inhibition ELISA and immunoblotting. Monoclonal antibodies belonging to two categories were selected. (a) Those (designated Mab 4F8 and Mab 7G9) recognizing epitopes of the Ig molecule located in/or near the antigen-binding site of Mab 1B10. In ELISA these antibodies were shown to inhibit in a dose-dependent manner, the reaction of Mab 1B10 with its specific antigen; (b) those (Mab 2B8, Mab 3B8) reacting with epitopes located outside of the antigen binding site of the antiHCG antibody molecule and did not influence the reactions of Mab 1B10 and its antigen. Following immunization of syngeneic BALB/c mice monoclonal antibodies (Mab 4F8, Mab 7G9) were produced which recognized epitopes located on the variable region of Mab 1B10 since they did not react with other marine monoclonal antibodies of the same isotype. These antibodies inhibited the binding of Mab 1B10 to its corresponding epitope on the molecule of HCG and they can be defined as syngeneic anti-idiotypic antibodies.     

Characterization of T8 epitopes by enzymatic digestion, crossblocking, and involvement in cell-mediated lympholysis

Eleven monoclonal antibodies, directed versus the T8 glycoprotein, were compared using enzyme digestion, phylogenetic comparisons, cross-blocking of antibody binding, and blocking of specific cell-mediated lympholysis (CML). It was found that none of the 11 anti-T8 antibodies tested define the same epitope on the T8 glycoprotein. Some of these antibodies react, however, with closely related structures, as shown by cross-blocking of antibody binding and similar enzyme sensitivity of the epitopes. Moreover, these structural related epitopes show a similar involvement in the effector phase of CML reactions, since the antibodies to these neighboring epitopes inhibit the same CML reactions. Thus, it is possible to apply structural and functional criteria to define "regions" on the T8 glycoprotein, some of which are consistently involved in CML reactions, some never, and some of these regions appear to be involved in specific effector-target cell combinations only.     

Epitope mapping of monoclonal antibodies toEscherichia coli ribosomal protein S3

The antigenic structure ofEscherichia coli ribosomal protein S3 has been investigated by use of monoclonal antibodies. Six S3-specific monoclonal antibodies secreted by mouse hybridomas have been identified by immunoblotting of two-dimensional ribosomal protein separation gels. By using a competitive enzyme-linked immunosorbent assay, we have divided these monoclonal antibodies into three mutual inhibition groups, members of which are directed to three distinct regions of the S3 molecule. The independence of these monoclonal antibody-defined regions was confirmed by the failure of pairs of monoclonal antibodies from two inhibition groups to block the binding of biotinylated monoclonal antibodies of the third group. To determine the regions recognized by these monoclonal antibodies, chemically cleaved S3 peptides were fractionated by gel filtration and reverse-phase high-performance liquid chromatography. The fractionated peptides were coated on plates and examined for specific interaction with monoclonal antibody by enzyme immunoassay. In this manner, two epitopes have been mapped at the ends of the S3 molecule: one, in the last 22 residues, is recognized by three monoclonal antibodies; and the second, in the first 21 residues, is defined by two monoclonal antibodies. The third S3 epitope, recognized by a single monoclonal antibody, has been localized in a central segment of about 90 residues by gel electrophoresis and immunoblotting. These epitope-mapped monoclonal antibodies are valuable probes for studying S3 structurein situ.     

Epitope mapping of monoclonal antibodies toEscherichia coli ribosomal protein S3

The antigenic structure ofEscherichia coli ribosomal protein S3 has been investigated by use of monoclonal antibodies. Six S3-specific monoclonal antibodies secreted by mouse hybridomas have been identified by immunoblotting of two-dimensional ribosomal protein separation gels. By using a competitive enzyme-linked immunosorbent assay, we have divided these monoclonal antibodies into three mutual inhibition groups, members of which are directed to three distinct regions of the S3 molecule. The independence of these monoclonal antibody-defined regions was confirmed by the failure of pairs of monoclonal antibodies from two inhibition groups to block the binding of biotinylated monoclonal antibodies of the third group. To determine the regions recognized by these monoclonal antibodies, chemically cleaved S3 peptides were fractionated by gel filtration and reverse-phase high-performance liquid chromatography. The fractionated peptides were coated on plates and examined for specific interaction with monoclonal antibody by enzyme immunoassay. In this manner, two epitopes have been mapped at the ends of the S3 molecule: one, in the last 22 residues, is recognized by three monoclonal antibodies; and the second, in the first 21 residues, is defined by two monoclonal antibodies. The third S3 epitope, recognized by a single monoclonal antibody, has been localized in a central segment of about 90 residues by gel electrophoresis and immunoblotting. These epitope-mapped monoclonal antibodies are valuable probes for studying S3 structurein situ.     

Monoclonal antibodies against rat brain hexokinase. Utilization in epitope mapping studies and establishment of structure-function relationships

The effects of seven monoclonal antibodies on various functions of rat brain hexokinase (ATP:D-hexose-6-phosphotransferase, EC 2.7.1.1) have been assessed. Specifically, effects on catalytic properties (Km values for substrates, glucose and ATP X Mg2+; Ki for inhibition by glucose 6-phosphate), binding to the outer mitochondrial membrane, and glucose 6-phosphate-induced solubilization of mitochondrially bound hexokinase were examined. Epitope mapping studies with the native enzyme provided information about the relative spatial distribution of the epitopes on the surface of the native molecule. Binding of nucleotides (ATP or ATP X Mg2+) was shown to perturb the epitopes recognized by two of these antibodies. Neither nucleotides nor other ligands (glucose, glucose 6-phosphate, Pi) had detectable effect on epitopes recognized by the other five antibodies. Peptide mapping techniques in conjunction with immunoblotting permitted assignment of the epitopes recognized by several of the antibodies to specific segments within the overall primary structure. These results, together with previous work relating to the organization of structural domains within the molecule, permitted development of a three-dimensional model which provides a useful representation of major structural and immunological features of the enzyme, and depicts the association of those features with specific functions.     

Cross-linking of rotavirus outer capsid protein VP7 by antibodies or disulfides inhibits viral entry

Antibodies that neutralize rotavirus infection target outer coat proteins VP4 and VP7 and inhibit viral entry. The structure of a VP7-Fab complex (S. T. Aoki, et al., Science 324:1444-1447, 2009) led us to reclassify epitopes into two binding regions at inter- and intrasubunit boundaries of the calcium-dependent trimer. It further led us to show that antibodies binding at the intersubunit boundary inhibit uncoating of the virion outer layer. We have now tested representative antibodies for each of the defined structural epitope regions and find that antibodies recognizing epitopes in either binding region neutralize by cross-linking VP7 trimers. Antibodies that bind at the intersubunit junction neutralize as monovalent Fabs, while those that bind at the intrasubunit region require divalency. The VP7 structure has also allowed us to design a disulfide cross-linked VP7 mutant which recoats double-layered particles (DLPs) as efficiently as does wild-type VP7 but which yields particles defective in cell entry as determined both by lack of infectivity and by loss of α-sarcin toxicity in the presence of recoated particles. We conclude that dissociation of the VP7 trimer is an essential step in viral penetration into cells.     

Human thyroid peroxidase: mapping of autoantibodies, conformational epitopes to the enzyme surface

The enzyme, thyroid peroxidase (TPO), is a dominant antigen in thyroid autoimmune diseases. Autoantibodies recognised two major dominant conformational epitopes termed A and B. The epitopes have been defined by mAbs, but the amino acid residues which constitute these determinants remain unknown. Using a model of TPO, built from the structure of myeloperoxidase (MPO), we have synthesised peptides corresponding to exposed loops and generated rabbit antibodies to the peptides. Antisera to peptide sequence 599-617 (peptide 14) representing a highly protrusive loop on the TPO, showed the highest inhibition in 65 sera from patients positive with anti-TPO antibodies. The inhibition was by 15-80% (mean 41%), and no other antibody showed any inhibition. Binding of hFabs to the B determinant on TPO was inhibited by anti-peptide 14 antibodies more then 85%, but not Fabs to the A determinant. In conclusion, the peptide 14 defines a sequence taking part in building up the B major conformational epitope. None of generated anti-peptide antibodies alone inhibited the binding of human Fabs to the A epitope, however a combination of four anti-peptide antibodies (P1, P12, P14 and P18) inhibits Fabs binding to the A determinant by more then 60% and autoantibodies binding from 65% to 94%. Combination of antibodies reacting with peptides outside the surface defined by those four antipeptide antibodies did not give any inhibition of Fabs to TPO. The inhibition of Fabs and auto Abs to TPO by this combination of anti-peptide Abs is the result of steric hindrance as none of these Abs individually inhibited auto Abs' or Fabs' binding to TPO. The four peptides define an area on the enzyme surface where the A and B major conformational epitopes are localised.     

Microinjection of an antibody against the cysteine-protease involved in male chromatin remodeling blocks the development of sea urchin embryos at the initial cell cycle

We reported recently that the inhibition of cysteine-proteases with E-64-d disturbs DNA replication and prevents mitosis of the early sea urchin embryo. Since E-64-d is a rather general inhibitor of thiol-proteases, to specifically target the cysteine-protease previously identified in our laboratory as the enzyme involved in male chromatin remodeling after fertilization, we injected antibodies against the N-terminal sequence of this protease that were able to inhibit the activity of this enzyme in vitro. We found that injection of these antibodies disrupts the initial zygotic cell cycle. As shown in this report in injected zygotes a severe inhibition of DNA replication was observed, the mitotic spindle was not correctly bipolarized the embryonic development was aborted at the initial cleavage division. Consequently, the injection of these antibodies mimics perfectly the effects previously described for E-64-d, indicating that the effects of this inhibitor rely mainly on the inhibition of the cysteine-protease involved in male chromatin remodeling after fertilization. These results further support the crucial role of this protease in early embryonic development.     

Monoclonal antibody studies of ferredoxin:NADP+ oxidoreductase.

Eleven independent monoclonal antibodies, all IgG's, have been raised against the ferredoxin:NADP+ oxidoreductase of spinach leaves. All 11 monoclonal antibodies were able to produce substantial inhibition of the NADPH to 2,6-dichlorophenol indophenol (DCPIP) diaphorase activity of the enzyme, but none of the antibodies produced any significant inhibition of electron flow from NADPH to ferredoxin catalyzed by the enzyme. Spectral perturbation assays were used to demonstrate that antibody interaction with NADP+ reductase did not interfere significantly with the binding of either ferredoxin or NADP+ to the enzyme. Ultrafiltration binding assays were used to confirm that the monoclonal antibodies did not interfere with complex formation between ferredoxin and the enzyme. These results have been interpreted in terms of the likely presence of one or more highly antigenic epitopes at the site where the nonphysiological electron acceptor, DCPIP, binds to the enzyme. Furthermore, the results suggest that the site where DCPIP is reduced differs from both of the two separate sites at which the two physiological substrates, ferredoxin and NADP+/NADPH, are bound.     

Monoclonal antibodies to three phospholipase C isozymes from bovine brain

Murine hybridoma cell lines secreting antibodies against the three bovine isozymes of phosphoinositide-specific phospholipase C (PLC) were established: 6, 23, and 12 lines were obtained for PLC-I (150 kDa), PLC-II (145 kDa), and PLC-III (85 kDa), respectively. The antibodies were purified from ascites fluid, and their properties were studied in detail. All the antibodies cross-reacted with their corresponding PLC enzymes, but not with the other two isozymes, suggesting that the three enzymes contain very different antigenic determinants. The six antibodies elicited by bovine PLC-I also cross-reacted with human and rat enzyme, whereas three each from anti-PLC-II antibodies and anti-PLC-III antibodies did not react with the enzymes from different species. Each antibody exerts different effects on the phosphatidylinositol-hydrolyzing activity of PLC. The most inhibitory antibody for either isozyme PLC-I or PLC-II exhibits 80% inhibition, whereas no more than 20% inhibition was observed for the anti-PLC-III antibodies. Purified PLC-I frequently contains catalytically active 140- and 100-kDa forms and an inactive 41-kDa protein in addition to the intact 150-kDa form, probably due to its high sensitivity to an unidentified endogenous protease. The five anti-PLC-I antibodies which bind to the denatured 150-kDa polypeptide also recognized the 140-kDa form, whereas only three cross-reacted with the 100-kDa form, and the remaining two bound to the 41-kDa protein. Competitive binding studies with intact PLC enzymes and Western blot experiments with proteolytic digests revealed that the 6 anti-PLC-I, 23 anti-PLC-II, and 12 anti-PLC-III antibodies bind at least five, six, and seven different epitopes on PLC-I, PLC-II, and PLC-III, respectively. The fact that these monoclonal antibodies bind to different epitopes on the same enzyme allowed one to develop a highly specific and sensitive tandem radioimmunoassay for quantitating PLC-I, PLC-II, and PLC-III. The principle of the assay is that binding of an 125I-labeled antibody to the antigen immobilized by another antibody at a distinctive binding site is proportional to the amount of antigen present. By using this method, PLC-I, PLC-II, and PLC-III could be measured quantitatively in the presence of other proteins, detergents, lipids, polyanions, and metal ions, all of which greatly affect the activity of PLC enzymes.     

Identification of a functional domain of human granulocyte colony-stimulating factor using neutralizing monoclonal antibodies.

Human granulocyte colony-stimulating factor (G-CSF) is a hemopoietic growth factor that is being used successfully to treat various forms of neutropenia. To define functionally important regions of G-CSF, we have prepared 37 monoclonal anti-G-CSF antibodies and mapped the regions of G-CSF recognized by different antibody groups. Antibodies recognizing similar epitopes were identified by competition assays, neutralization assays, conformation dependence and cross-reactivity with canine G-CSF. Seven of eight neutralizing antibodies fell into two related epitope groups and were conformation-dependent. The eighth was unrelated and conformation-independent. Peptides of G-CSF were generated by chemical or enzymatic digestion and tested for antibody reactivity. One of the neutralizing antibodies (LMM351) recognized a small, disulfide-bonded peptide from the V8 protease digest (residues 34-46). A synthetic peptide (residues 20-58) was recognized by all the neutralizing antibodies, implicating this disulfide-bonded loop in receptor binding. The epitopes recognized by nonneutralizing antibodies were found throughout G-CSF. Thus, regions of G-CSF that are not involved in receptor binding have also been defined. A CNBr peptide (residues 1-121) had greatly reduced biological activity, indicating that the COOH terminus is required for receptor binding. We predict that residues 20-46 and the COOH terminus bind to the G-CSF receptor.     

High-resolution glycosylation site-engineering method identifies MICA epitope critical for shedding inhibition activity of anti-MICA antibodies

As an immune evasion strategy, MICA and MICB, the major histocompatibility complex class I homologs, are proteolytically cleaved from the surface of cancer cells leading to impairment of CD8 + T cell- and natural killer cell-mediated immune responses. Antibodies that inhibit MICA/B shedding from tumors have therapeutic potential, but the optimal epitopes are unknown. Therefore, we developed a high-resolution, high-throughput glycosylation-engineered epitope mapping (GEM) method, which utilizes site-specific insertion of N-linked glycans onto the antigen surface to mask local regions. We apply GEM to the discovery of epitopes important for shedding inhibition of MICA/B and validate the epitopes at the residue level by alanine scanning and X-ray crystallography (Protein Data Bank accession numbers 6DDM (1D5 Fab-MICA*008), 6DDR (13A9 Fab-MICA*008), 6DDV (6E1 Fab-MICA*008). Furthermore, we show that potent inhibition of MICA shedding can be achieved by antibodies that bind GEM epitopes adjacent to previously reported cleavage sites, and that these anti-MICA/B antibodies can prevent tumor growth in vivo.     

Monoclonal antibodies neutralizing mammalian DNA topoisomerase I activity

We have isolated three different monoclonal antibodies specific for mammalian type-I DNA topoisomerase. The antibodies react with three closely adjacent epitopes located in a central section of the enzyme (between amino acid residues 344 and 483). Two of the antibodies inhibit an early step of the nicking/closing pathway. We provide evidence showing that the antibodies do not block the association of the enzyme with DNA. The antibodies are useful for immunocytochemical investigation and for further exploration of the biochemical function of mammalian type-I DNA topoisomerase.